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Molecular Recognition of GalNAc in Mucin-Type O-Glycosylation
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Molecular Recognition of GalNAc in Mucin-Type O-Glycosylation
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  • Ignacio Sanz-Martinez
    Ignacio Sanz-Martinez
    Institute of Biocomputation and Physics of Complex Systems (BIFI), Glycobiology Unit, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, 50018 Zaragoza, Spain
    Department of Organic Chemistry, Faculty of Sciences, University of Zaragoza, Campus San Francisco, 50009 Zaragoza, Spain
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  • Sandra Pereira
    Sandra Pereira
    Institute of Biocomputation and Physics of Complex Systems (BIFI), Glycobiology Unit, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, 50018 Zaragoza, Spain
    Department of Organic Chemistry, Faculty of Sciences, University of Zaragoza, Campus San Francisco, 50009 Zaragoza, Spain
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  • Pedro Merino*
    Pedro Merino
    Institute of Biocomputation and Physics of Complex Systems (BIFI), Glycobiology Unit, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, 50018 Zaragoza, Spain
    Department of Organic Chemistry, Faculty of Sciences, University of Zaragoza, Campus San Francisco, 50009 Zaragoza, Spain
    *E-mail: [email protected]
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    Orcidhttps://orcid.org/0000-0002-2202-3460
  • Francisco Corzana*
    Francisco Corzana
    Department of Chemistry, Centro de Investigación en Síntesis Química, University of La Rioja, Madre de Dios 53, 26006 Logroño, Spain
    *E-mail: [email protected]
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    Orcidhttps://orcid.org/0000-0001-5597-8127
  • Ramon Hurtado-Guerrero*
    Ramon Hurtado-Guerrero
    Institute of Biocomputation and Physics of Complex Systems (BIFI), Glycobiology Unit, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, 50018 Zaragoza, Spain
    Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen DK-2200, Denmark
    Fundación ARAID, 50018 Zaragoza, Spain
    *E-mail: [email protected]
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    Orcidhttps://orcid.org/0000-0002-3122-9401
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Cite this: Acc. Chem. Res. 2023, 56, 5, 548–560
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https://doi.org/10.1021/acs.accounts.2c00723
Published February 23, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Conspectus

N-Acetylgalactosamine (GalNAc)-type O-glycosylation is an essential posttranslational modification (PTM) that plays fundamental roles in biology. Malfunction of this PTM is exemplified by the presence of truncated O-glycans in cancer. For instance, the glycoprotein MUC1 is overexpressed in many tumor tissues and tends to carry simple oligosaccharides that allow for the presentation of different tumor-associated antigens, such as the Tn or sTn antigens (GalNAc-α-1-O-Thr/Ser and Neu5Acα2-6GalNAcα1-O-Ser/Thr, respectively). In other cases, such as tumoral calcinosis associated with O-glycosylation of the fibroblast growth factor 23, O-glycans are absent or less abundant. Significant progress has been made in determining the three-dimensional structures of biomolecules that recognize GalNAc, such as antibodies, lectins, mucinases, GalNAc-transferases, and other glycosyltransferases. Analysis of the complexes between these entities and GalNAc-containing glycopeptides, in most cases derived from crystallographic or NMR analysis, provides an understanding of the key structural elements that control molecular recognition of these glycopeptides. Here, we describe and compare the binding sites of these proteins in detail, focusing on how the GalNAc moieties interact selectively with them. We also summarize the differences and similarities in GalNAc recognition. In general, the recognition of GalNAc-containing glycopeptides is determined by hydrogen bonds between hydroxyl groups and the N-acetyl group of GalNAc with proteins, as well as CH-π contacts in which the hydrophobic α-face of the sugar and the methyl group of NHAc can be involved. The latter interaction usually provides the basis for selectivity. It is worth noting that binding of these glycopeptides depends primarily on recognition of the sugar moiety, with some exceptions such as a few anti-MUC1 antibodies that primarily recognize the peptide backbone and use the sugar to facilitate shape complementarity or to establish a limited number of interactions with the protein. Focusing specifically on the GalNAc moiety, we can observe that there is some degeneracy of interactions within the same protein families, likely due to substrate flexibility. However, when all studied proteins are considered together, despite the commonalities within each protein family, no pattern can be discerned between the different families, apart from the presence of common residues such as Tyr, His, or Asp, which are responsible for hydrogen bonds. The lack of a pattern can be anticipated, given the diverse functions of mucinases, glycosyltransferases, antibodies, and lectins. Finally, it is important to point out that the conformational differences observed in solution in glycopeptides bearing GalNAc-α-1-O-Ser or GalNAc-α-1-O-Thr also can be found in the bound state. This unique characteristic is exploited, for instance, by the enzyme C1GalT1 to broadly glycosylate both acceptor substrates. The findings summarized in this review may contribute to the rational structure-guided development of therapeutic vaccines, novel diagnostic tools for early cancer detection, and new cancer treatments for cancer with tailored anti-Tn or anti-STn antibodies or new drugs to inhibit GalNAc-T isoenzymes.

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Key References

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  • de Las Rivas, M.; Paul Daniel, E. J.; Narimatsu, Y.; Compañón, I.; Kato, K.; Hermosilla, P.; Thureau, A.; Ceballos-Laita, L.; Coelho, H.; Bernadó, P.; Marcelo, F.; Hansen, L.; Maeda, R.; Lostao, A.; Corzana, F.; Clausen, H.; Gerken, T. A.; Hurtado-Guerrero, R. Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3. Nat. Chem. Biol.2020, 16, 351–360. (1) GalNAc-T3 specifically glycosylates the fibroblast growth factor 23 (FGF23), regulating its physiological function. Here we present compelling evidence that FGF23 is a poor substrate of GalNAc-T3, implying that this inefficient glycosylation of FGF23 is the key limiting step in regulating intact biological active FGF23 and phosphate homeostasis.

  • González-Ramírez, A. M.; Grosso, A. S.; Yang, Z.; Compañón, I.; Coelho, H.; Narimatsu, Y.; Clausen, H.; Marcelo, F.; Corzana, F.; Hurtado-Guerrero, R. Structural basis for the synthesis of the core 1 structure by C1GalT1. Nat. Commun.2022, 13, 2398. (2) The glycosyltransferase C1GalT1 directs a key step in protein O-glycosylation that is important for the expression of the cancer-associated Tn and T antigens. Here, we provide molecular insights into the function of C1GalT1 by solving the crystal structure of the Drosophila enzyme–substrate complex.

  • Taleb, V.; Liao, Q.; Narimatsu, Y.; García-García, A.; Compañón, I.; Borges, R. J.; González-Ramírez, A. M.; Corzana, F.; Clausen, H.; Rovira, C.; Hurtado-Guerrero, R. Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut. Nat. Commun.2022, 13, 4324. (3) AM0627 is a bis-O-glycan mucinase that might work in the last steps of degradation of the mucus, thus providing a source of carbon and nitrogen for Akkermansia muciniphila. Here, we provide molecular insights into AM0627 function from X-ray crystallography and computer simulations.

  • Bermejo, I. A.; Usabiaga, I.; Compañón, I.; Castro-López, J.; Insausti, A.; Fernández, J. A.; Avenoza, A.; Busto, J. H.; Jiménez-Barbero, J.; Asensio, J. L.; Peregrina, J. M.; Jiménez-Osés, G.; Hurtado-Guerrero, R.; Cocinero, E. J.; Corzana, F. Water Sculpts the Distinctive Shapes and Dynamics of the Tumor-Associated Carbohydrate Tn Antigens: Implications for Their Molecular Recognition. J. Am. Chem. Soc.2018, 140, 9952–9960. (4) Here we provide experimental evidence for the conformational differences between the two Tn antigens (GalNAc-α-1-O-Thr/Ser) and confirm the importance of water molecules in the 3D structures explored by each antigen.

Introduction

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N-Acetylgalactosamine (GalNAc) constitutes one of the 10 essential monosaccharides that, when activated as a sugar nucleotide (UDP-GalNAc), is used to build part of the human glycome. (5) The addition of GalNAc onto Ser/Thr residues (and possibly Tyr) of proteins defines one of the most important, abundant, and complex regulated types of protein O-glycosylation pathways present in eukaryotes, the so-called GalNAc- or mucin-type O-glycosylation. (5−7) This pathway is chiefly found in the densely clustered, heavily glycosylated domains of mucins and mucin-domain-containing glycoproteins (Figure 1). However, it is also clear that many other proteins contain isolated sites of GalNAc-O-glycosylation, where more than ∼5000 human proteins trafficking the secretory pathway have been identified to date containing one or more mucin-type O-glycans. (8)

Figure 1

Figure 1. Scheme of the mucin-type O-glycosylation pathway. All of the GTs involved in initiation and core extension are mentioned together with the stereochemistry and glycosidic linkages. The proteins under review in this Account are highlighted in red.

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The O-glycosylation pathway is initiated by a large family of GalNAc-transferases (GalNAc-Ts). These enzymes orchestrate with high fidelity the initial patterns of O-glycosylation on diverse proteins, including the high-density regions in mucins, where 30–50% of the amino acids may be glycosylated. (9) After the synthesis of the Tn antigen by the GalNAc-Ts, core extension takes place by different glycosyltransferases (GTs). Of particular relevance are C1GalT1, which needs the assistance of the Cosmc chaperone to be functional, and B3GNT6 because they synthesize core 1 and core 3, which act as substrates for core 2 and core 4 production, respectively. (10) Elongation/branching steps elaborate further the different core structures that are terminated by a capping step (Figure 1).
Mucinases, a type of O-glycoprotease that depends on neighboring O-glycans for activity, play a fundamental role in degrading mucins as an important step for infectivity (11,12) or as a nutrient source. (3) Therefore, mucin-type O-glycans can function by acting as a shield and nutrient source for pathogenic microorganisms and commensals/symbionts, respectively, (13) and have additional roles in protecting proteins from proteolytic cleavage (1,14,15) and mediating binding of protein receptors toward their protein ligands. Due to these functions, mucin-type O-glycans may conceivably interact with almost all physiological processes. (16) However, the importance of this pathway is not reflected by the single knockouts of GalNAc-T genes in mice, which lead to mild or absent phenotypes likely due to the redundant activities between these isoenzymes. (17) On the contrary, the knockouts of either C1GalT1 or Cosmc are essential at the embryonic level, emphasizing the relevance of this pathway in physiology. (18,19)
At a disease level and particularly in cancer, the controversial relocation of GalNAc-Ts from their usual location in the Golgi apparatus to the endoplasmic reticulum (20,21) and the well-accepted hypermethylation of the Cosmc promoter leading to its silencing (22) together with overexpression of ST6GalNAc-I (Figure 1), (23) have been suggested to be behind of the presence of truncated and aberrant O-glycans. (22) These are tumor-associated carbohydrate antigens (TACAs) found in clinical specimens of different types of cancers, with the most relevant ones being the Tn, T (or core 1, Galβ1-3GalNAc-α-1-O-Thr/Ser), and STn antigens. Due to the importance of these TACAs in cancer, numerous antibodies have been developed for cancer treatment and as diagnostic tools. (24) Lectins such as the human macrophage galactose lectin (MGL) also bind to these TACAs and particularly to Tn antigen, which makes this protein useful for therapeutic applications. (25)
In this Account, we focus on enzymes, antibodies, and lectins (Figure 1) that recognize the GalNAc moiety in glycopeptides. To this end, we will show examples from our laboratories of 3D structures of these proteins in complex with glycopeptides determined by X-ray crystallography or NMR experiments. We will also discuss the commonalities between these different systems in recognition of this important monosaccharide.

Key Elements in GalNAc-Ts for Substrate Recognition

GalNAc-T isoenzymes display an N-terminal GT-A fold catalytic domain followed by a C-terminal lectin domain connected by a flexible linker. For optimal binding to UDP-GalNAc, a metal, particularly manganese, is required. This is facilitated by a DxH motif and an additional His residue that coordinates the metal (Figure 2A). (26−28) Additionally, the catalytic domain contains a flexible loop that adopts two conformations, resulting in either an inactive (open flexible loop) or active form (closed flexible loop) (Figure 2B). (27) Prior to our work in this field, it was already established how GalNAc-T2 interacts with naked peptides, (27) but the catalytic mechanism and the molecular basis of how these isoenzymes recognize glycopeptides were unknown.

Figure 2

Figure 2. Five structural features define GalNAc-Ts functions. (A) Overall structure of GalNAc-T4. The catalytic and lectin domains are colored in gray, and the interdomain flexible linker is in red. The catalytic domain flexible loop, UDP, the glycopeptides, and the GalNAc moieties are depicted in yellow, brown, green, and orange, respectively. The inset shows the coordination geometry of Mn2+. (B) (Upper panel) Surface representation of the inactive form of GalNAc-T2 and the active form of GalNAc-T2. (Lower panel) Catalytic cycle of GalNAc-T2. (C) (Left panel) View of the complete sugar-nucleotide, peptide, and lectin-domain binding sites of GalNAc-T4. (Right panel) Close-up view of the GalNAc-binding sites of the lectin and catalytic domains. The residues of the lectin domain engaged in recognition of the GalNAc moieties are indicated in magenta. (D) (Upper panel) Surface representation of different GalNAc-Ts showing the location of the lectin domain GalNAc-binding sites in the overall structures. Note that GalNAc-T1 has two potential functional GalNAc-binding sites. (Lower panel) Scheme illustrating the different types of O-glycosylation and preferences found for GalNAc-T2 and T4.

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We managed to trap different structures that enabled us to determine the catalytic cycle of GalNAc-T2. (29) This, together with our group’s characterization of an inactive GalNAc-T2 (F104S) mutant, which is responsible for low levels of high-density lipoprotein cholesterol in humans, (30) prompted us to propose a UDP-GalNAc-dependent induced-fit mechanism within an overall BiBi kinetic mechanism (see Figure 2B for details of the mechanism). (31) Metadynamics calculations of trapped complexes indicated that GalNAc-T2 follows the typical front-face SNi-type reaction, where the β-phosphate acts as the catalytic base. (29) Note that the interactions of GalNAc-T2 with the UDP-GalNAc moiety are discussed in ref (29). Additionally, we hypothesized that the preference of these isoenzymes for glycosylating Thr over Ser residues was due to the Thr methyl group interacting with nearby hydrophobic/aromatic residues. (29) This was supported by kinetic experiments using different GalNAc-T isoenzymes. (32)
To figure out how these isoenzymes interact with the GalNAc moiety to direct either long-range or neighboring O-glycosylation, we trapped several crystal structures of GalNAc-Ts complexed to different glycopeptides. (1,33−35) Five features that were indispensable for the recognition of the glycopeptides were revealed. (i) The first feature is the peptide-binding site that is required to recognize the peptide sequences. For example, whereas for the GalNAc-T4-UDP-Mn2+-diglycopeptide 6 complex, most of the direct interactions with the peptide were hydrogen bonds (H-bonds) and to a lesser extent hydrophobic interactions (Figure 2C); (33) for the GalNAc-T2-UDP-Mn2+-MUC5AC-13 complex, most of the direct interactions were hydrophobic with few direct H-bond interactions. (34) Three highly conserved aromatic residues (Phe284, Phe364, and Trp286 in GalNAc-T4) were key residues in the recognition of common peptide motifs such as Pro-X-Pro (X is a small hydrophobic residue) found in acceptor substrates. These examples together with our structures of GalNAc-T3 with glycopeptides illustrated the differences and similarities between these isoenzymes at the peptide-binding site level, which are likely behind the redundancy found for some of the isoenzymes and even the specificity for GalNAc-T3 and GalNAc-T11 toward FGF23 (1) and LA repeats of low-density lipoprotein receptor (LDLR)-related proteins, respectively. (36) (ii) The flexible loop not only is involved in the catalytic cycle but also contributes to the recognition of the peptide sequences, as seen, for example, for the interactions between Arg372GalNAc-T4 with the diglycopeptide 6 (Figure 2C). Of special interest was our study of how the less stable flexible loop closed conformation and the interaction of the acceptor site of a FGF23 glycopeptide with UDP β-phosphate together with the unusual conformation of Trp385 explained why GalNAc-T3 was a poor enzyme on glycosylating FGF23. Note that Trp385, which is conserved between all isoenzymes except in those of the Y family in which the Trp residue is replaced by a Tyr residue, (37) is highly important in catalysis and also adopts different conformations through the catalytic cycle. (29) (iii) Isoenzymes such as GalNAc-T4, T7, T10, and T12 were predicted to contain an additional GalNAc-binding site in the catalytic domain to account for their neighboring O-glycosylation capacity. (38) This was demonstrated by our structure of GalNAc-T4 complexed to the diglycopeptide 6 (Figure 2C). In particular, the GalNAc moiety was mainly recognized by H-bond interactions and to a minor extent by hydrophobic contacts. (33) We explained why GalNAc-T4 is capable of glycosylating acceptor sites located at +1 with respect to a prior contiguous glycosite (Figure 2D). The molecular basis of the neighboring glycosylation of GalNAc-T12 was further provided, explaining why this isoenzyme prefers to glycosylate acceptor sites located at −1 from a prior glycosite. (39) For other isoenzymes such as GalNAc-T7 and T10, the molecular basis of how they glycosylate proximal sites from a prior glycosite is not known and merits further investigation. (iv) Our structures also provided the molecular basis of how a distant glycosite from potential acceptor sites can direct long-range O-glycosylation. It is well-accepted that these isoenzymes contain three potential GalNAc-binding sites in the lectin domain. All our structures revealed a single functional GalNAc-binding site, located on opposing sides of the lectin domain when the overall structures were superimposed (Figure 2C and D). The latter clearly explained why isoenzymes such as GalNAc-T2 had different long-range glycosylation preferences to those of GalNAc-T3 or T4 (see Figure 2D). Although it was demonstrated that the peptide sequences around the O-glycosite influenced O-glycosylation in a lectin-dependent manner, (40) in all of our structures the GalNAc moiety was mainly the only feature recognized by hydrogen bonds and CH-π interactions by mostly conserved residues of the lectin domain (Figure 2C). (v) Finally, to solve why the lectin GalNAc-binding site was located in different sides of the lectin domain depending on the isoenzyme, we conducted molecular dynamics (MD) simulations of GalNAc-T2 chimeras and kinetics experiments that revealed that the flexible linker was responsible for the location of the GalNAc-binding site in the lectin domain. This exemplified for the first time that the flexible linker was responsible for the location of the GalNAc-binding site in the lectin domain, which clearly is behind the different long-range glycosylation preferences. (35) A previous study from our group further demonstrated that the flexible linker is also responsible for the dynamics of these isoenzymes, explaining why they can adopt compact and extended forms in solution. (34)
Nevertheless, more experiments are needed to determine, for example, how GalNAc-T11 glycosylates LA repeats or GalNAc-T7/T10 to achieve multiple O-glycosylations mainly in mucins. Other functions such as the hierarchical functions of these isoenzymes on complex substrates such as MUC1 has been revealed recently by using a combination of NMR and computational experiments. (41)

Molecular Recognition of GalNAc O-Glycans by C1GalT1

We have recently reported the X-ray structure of Drosophila melanogaster C1GalT1 (DmC1GalT1), which does not appear to require a chaperone for expression, in complex with the MUC1-related glycopeptide APDT*RP (* denotes GalNAc). (2) This enzyme is responsible for adding a galactose at carbon 3 of GalNAc through a β-O-glycosidic linkage to form core 1. The GalNAc moiety of the glycopeptide forms several H-bonds with the enzyme, while the peptide sequence is recognized by hydrogen bonds and CH-π interactions (Figure 3A). Thus, in contrast to the lectin domain of GalNAc-Ts described earlier, in this enzyme the sugar does not establish CH-π interactions with the aromatic residues.

Figure 3

Figure 3. Molecular recognition of glycopeptides by C1GalT1. (A) Close-up view of the active site for the DmC1GalT1-UDP-APDT*RP complex (PDB entry 7Q4I). The carbon atoms of the residues interacting with the glycopeptide are shown in magenta. The UDP carbon atoms are shown in gray. (B) Main conformation found in solution for the Tn antigen with Thr GalNAc-α-1-O-Thr (left panel) and for the Tn antigen with Ser GalNAc-α-1-O-Thr (right panel). The CH-π interaction between the methyl group of Thr4 and Trp300 is shown by the black dashed line.

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The crystal structure shows that the glycosidic bond of α-GalNAc-Thr has a staggered conformation (torsion angle ψ ≈ 180°, Figure 3B) that does not occur in aqueous solution for the Tn-Thr-containing glycopeptides but is exhibited by the Ser variant. (4,42) MD simulations performed for DmC1GalT1 in complex with UDP and APDT*RP glycopeptide also predicted the staggered conformation for the GalNAc-α-1-O-Ser moiety and demonstrated that the eclipsed conformation fixed in α-GalNAc-Thr displayed a loss of interactions between the peptide and the enzyme compared to those found in the X-ray structure. On this basis, we hypothesize that C1GalT1 recognizes a high-energy conformation of the GalNAc-α-1-O-Thr linkage that somehow compensated for the additional CH-π interaction observed for the methyl group of Tn-Thr (see black dashed line in Figure 3A between this group and Trp300). As a result, the enzyme exhibits a comparable affinity for both Tn-Ser and Tn-Thr.

Molecular Recognition of GalNAc O-Glycans by Mucinases

The enzymatic action of mucinases depends on prior recognition of an O-glycopeptide by the enzyme, and GalNAc is a crucial moiety for this purpose. Only five mucinases (AM0627, BT4244, IMPA, ZmpB, and ZmpC) have been fully characterized to date. The different structural characteristics and recognition preferences reveal a notable capacity to adapt to the diverse patterns of the glycopeptides, suggesting an appreciable tolerance for the structure of the O-glycopeptide. On the other hand, mucinases share several similarities with conservation of various residues in the active site, but they vary for others that are responsible for the recognition preferences and specificity of the enzyme.
Common features to all mucinases regarding recognition of GalNAc comprise H-bonds of the N-acetyl group with a Trp and with the backbone of an Asn (or Gln for IMPa) as well as one or two H-bonds between the hydroxyl groups and an Arg. All mucinases have an additional aromatic residue (Phe, Trp, or Tyr) presenting different interactions with GalNAc that could be responsible, in part, for the selectivity to different O-glycopeptides (Figure 4). A residue of Tyr leads to a H-bond with a hydroxyl group of GalNAc in AM0627 and BT4244, (13,43) while in ZmpB and ZmpC a residue of phenylalanine forms a CH-π interaction with the sialic acid. These differences represent the ability of mucinases to accept different O-glycopeptides as mentioned earlier, but with restricted substrate specificities when necessary. (44)

Figure 4

Figure 4. Comparison between the different active sites of mucinases. (A) Superimposition of the active sites of IMPa (green) and BT4244 (cyan) showing main interactions with the sugar units of the O-glycopeptides (orange for IMPa and magenta for BT4244). (B) Superimposition of the active sites of ZmpB (green) and ZmpC (cyan) showing main interactions with the corresponding O-glycopeptides (orange for ZmpB and magenta for ZmpC). (C) Superimposition of the active sites of complexes formed between BT4244 (cyan) and AM0627 (green) with a Tn O-glycan (magenta) and bis-T-glycopeptide (orange), respectively. PDB entries for the structures of AM0627, IMPa, BT4244, ZmpB, and ZmpC are 7SCI, 5KDX, 5KD8, 5KDU, and 6XT1, respectively.

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Whereas ZmpB, ZmpC, and IMPa only recognize monoglycosylated peptides, mucinases like AM0627 and BT4244 are capable of recognizing bis-O-glycans through Tyr470 (AM0627) and likely Tyr723 (BT4244) by forming H-bond interactions with O6 of GalNAc and CH-π interactions with GalNAc. The importance of this interaction has been experimentally confirmed to be essential for their activity. (3)

Molecular Recognition of GalNAc by Antibodies

Anti-MUC1 and anti-Tn antibodies have long been used clinically in cancer diagnosis and therapy. (24,45) Initial efforts were focused on IgG1 antibodies that block MUC1 on the surface of cancer cells. However, anti-MUC1 antibodies have also been conjugated to radioisotopes to use in both imaging and cancer therapy, as well as to small-molecule toxins, resulting in antibody–drug conjugates (ADCs) approved for the treatment of various cancers, including trastuzumab emtansine, brentuximab vedotin, and gemtuzumab ozogamicin. The hypothesis that cancer-specific antibodies can be fused with anti-CD3 antibodies to reverse T-cell-mediated killing in cancer patients has also led to the approval of catumaxomab and blinatumomab. On the other hand, several anti-MUC1 antibodies have been tested for CAR-T cells, such as HFMG-2, SM3, and more recently 5E5. (46) These anti-MUC1 antibodies mainly recognize the peptide fragment, but glycosylation of the peptide epitope generally enhances binding (see below). In some cases, the carbohydrate either promotes proper presentation of the peptide or makes additional contact with the protein. In other cases, the antibody may accurately recognize the GalNAc (or sugar) component, representing the primary epitope binding. (47)
The first high-resolution X-ray structure that shed some light on the role of the GalNAc residue in recognition processes between antibodies and a Tn-glycopeptide was reported for a complex with the 273 antibody and shows that each hydroxyl group of GalNAc forms at least one hydrogen bond with the antibody (Figure 5A). (48) The NH and methyl groups of the acetamide interact via a hydrogen bond and a CH-π interaction with the antibody. The peptide fragment is involved in several hydrogen bonds, and the two Pro residues of the sequence are engaged in CH-π contacts. Of note, the glycosidic linkage of the GalNAc-α-1-O-Thr residue displays the eclipsed conformation found for the Tn-Thr antigen in solution. (4)

Figure 5

Figure 5. Molecular recognition of Tn-glycopeptides by antibodies. (A) Close-up view of the binding site of the 273 mAb bound to the ERGT*KPPLEEL glycopeptide, showing the interactions of the protein with the GalNAc unit (PDB entry 3IET). (B) Close-up view of the binding site of the AR20.5 antibody and the glycopeptide APDT*RP (PDB entry 5T78). (C) Close-up view of the binding site of the SM3 antibody and glycopeptide APDT*RP (PDB entry 5A2K). (D) Close-up view of the binding site of the SN-101 antibody and a MUC1-like glycopeptide (PDB entry 6KX1). Only the interactions between the protein and the GalNAc unit are shown. (E) Close-up view of the binding site of the antibody 5E5 and glycopeptide APGST*AP (PDB entry 6TNP). (F) Close-up view of the binding site of the antibody G10C and αGalNAc-PNP (PDB entry 7UT3). Note that in all panels the residues of the antibody engaged in recognition of the glycopeptides and/or the GalNAc moieties are indicated in magenta.

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The X-ray structure of a MUC1-like peptide and the corresponding Tn-glycopeptide bound to the AR20.5 antibody (Figure 5B) underwent a successful phase I clinical trial, showing that the GalNAc moiety has no specific contacts with the antibody. (49) The higher affinity for the glycopeptide compared to the parent peptide indicates that the GalNAc moiety improves the shape compatibility of the peptide with the antibody. Again, the conformation of the glycosidic linkage between GalNAc and Thr recognized by the antibody adopts the eclipsed conformation. Our group provided the first crystal structure of the Tn-glycosylated MUC1 epitope (APDT*RP) with antibody SM3 (Figure 5C). (50) The crystallographic analysis of the structure with the Tn-Thr glycopeptide showed that the GalNAc motif does not significantly affect the conformation of the peptide backbone in the SM3-bound state. The glycosidic linkage of the glycopeptide adopts the expected exoanomeric/syn conformation with an eclipsed arrangement. (4) This geometry allows the formation of an intermolecular hydrogen bond between the hydroxymethyl group of GalNAc and the antibody, and the N-acetyl group of this sugar stacks with the aromatic ring of Trp33H, providing the impetus for the observed selectivity of SM3 for GalNAc-containing antigens. In sharp contrast, the GalNAc moiety in the bound state of the Tn-Ser and Tn-Cys glycopeptides with the SM3 antibody (50) does not establish stabilizing contacts with the protein, and the glycosidic linkage displays a high-energy conformation that is not populated in solution. These results may explain the higher affinity of the SM3 antibody for the glycopeptide with Tn-Thr. (50)
The epitope mapping of a MUC1 library containing peptides and Tn-glycopeptides with several glycosylation sites and two families of cancer-related antibodies has been recently characterized. (51) The first family includes two anti-MUC1 antibodies, VU-3C6 and VU-11E2, that recognize tumor-associated MUC1 in breast cancer. The second group comprises two anti-Tn antibodies, 8D4 and 14D6. In all cases, the H2 proton and the N-acetyl moiety of GalNAc are in contact with the antibody. Interestingly, NMR data suggest that 14D6 and 8D4 antibodies prefer binding to the Tn-Ser over the Tn-Thr. The extra flexibility of the GalNAc-Ser pair may allow the glycopeptide to display the required conformation in the bound state without a significant entropy penalty.
The crystallographic analysis of the antibody SN-101 bound to a Tn-glycopeptide related to MUC1 (Figure 5D) (52) shows that the antibody interacts with the sugar through only two H-bonds. The Tn-Thr antigen displays the glycosidic linkage’s characteristic eclipsed conformation. Related to this, our group has described the X-ray structure of antibody 5E5 with a Tn-glycopeptide derived from APGSTAP, which recognizes the entire GalNAc unit as a primary epitope (Figure 5E). (53) The X-ray structure reveals that all hydroxyl groups of GalNAc, except OH6, are engaged in hydrogen bonds with the antibody. The carbonyl group of the sugar is involved in two hydrogen bonds, and the methyl group is engaged in a CH-π interaction with His50H. The lack of interactions with the hydroxymethyl group explains why this antibody can recognize STn-containing glycopeptides. (54) In addition, the serine residue is exposed to the solvent, which allows the antibody to interact with the diglycosylated peptide. The glycosidic linkage of the glycopeptide in complex with the antibody adopts the typical eclipsed conformation. (4,55) The peptide moiety forms only one hydrogen bond with the protein in the C-terminal region, allowing some degree of promiscuity. Indeed, our studies and others indicate that the antibody might interact with glycopeptides that comprise the −T*–X–P– motif, which is present in the MUC1 tandem-repeat sequence but also in many other proteins.
Antibody G10C, which recognizes GalNAc bound to the side chain of Tyr in natural peptides and proteins with high affinity and selectivity, has been recently developed (Figure 5F). (46) The X-ray structure of G10C-Fab in complex with a GalNAc-Tyr mimic shows that hydroxyl groups and the endocyclic oxygen form hydrogen bonds with the antibody. The hydrogen bonds involving the N-acetyl group explain the selectivity toward this sugar.

Molecular Recognition of GalNAc by Lectins

In the context of cancer, various lectins that bind to Tn-glycopeptides have been successfully employed as biomarkers (56) and cancer progression and anticancer agents. (57) The binding mode in the human macrophage galactose lectin (hMGL) bound to a GalNAc residue, and the Tn-Ser antigen (Figure 6A) (58) is nearly identical for both substrates, with the vicinal groups OH3 and OH4 coordinated with the protein and a structural calcium ion, confirming that this is a Ca2+-dependent (C-type) lectin. The carbonyl group of the acetamide is hydrogen-bonded with the side chain of His286, and its methyl group and the hydrophobic face of the sugar are involved in CH-π interactions. Regarding lectins as biomarkers, there are some other interesting X-ray structures. For example, complexes with the Tn-Ser antigen have been described for Helix pomatia agglutinin (HPA), (59) SNA-II, (60) winged bean lectin (Psophocarpus tetragonolobus), (61) and Vatairea macrocarpa (VML). (62) In these cases, all hydroxyl groups as well as the oxygen of the N-acetyl of the sugar are involved in hydrogen bonds with the residues of the lectin. Except for HPA, CH-π interactions occur with aromatic residues (Phe or Trp) on the α-face of the sugar. Remarkably, the underlying Ser does not interact with the lectin, except at SNA-II. In all of these complexes, the Tn-Ser antigen exhibits a staggered conformation. Recently, the complex between Bauhinia forficata lectin and Tn-containing glycopeptides was reported. (63) The binding of the GalNAc residue and the lectin is shown in Figure 6B. It is noteworthy that the carboxylate group of Glu126 forms hydrogen bonds with both the N-acetyl of GalNAc and the NH of glycosylated threonine, promoting recognition of this MUC1-derived fragment.

Figure 6

Figure 6. Molecular recognition of Tn-glycopeptides by lectins. (A) Close-up view of the binding site of the lectin hMGL bound to Tn-Ser (PDB entry 6W12). (B) Close-up view of the binding site of the Bauhinia forficata lectin bound to a Tn-containing glycopeptide derived from MUC1 (PDB entry 5T5O). (C) Close-up view of the binding site of SBA lectin bound to PDT*R glycopeptide (PDB entry 4D69). Note that in all panels the residues of lectin engaged in the recognition of the glycopeptides and/or the GalNAc moieties are indicated in magenta.

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We have reported that the peptide sequence flanking the Tn antigen can modulate Tn recognition by two plant lectins (soybean agglutinin (SBA) and Vicia villosa B-4 agglutinin (VVA)) that show higher affinity for the Tn antigen when incorporated into the most immunogenic peptide region of mucin MUC1 (sequence PDTR). (64) The charged residues Arg and Asp in this sequence provide relevant interactions with the lectin surface likely favoring the presentation of the sugar moiety to the lectin. We could determine by X-ray of SBA lectin bound to the glycopeptide PDT*R (Figure 6C). (64) Interestingly, while two different binding modes were observed for the peptide, the GalNAc unit exhibits the same interaction pattern in both modes. GalNAc-lectins can also discriminate between glycopeptides containing the Tn-Thr or the Tn-Ser, which can be explained by the different conformational behavior and dynamics of these two Tn units. (65)
hMGL and several plant lectins bind with high affinity to GalNAc-Tyr. (66) Saturation transfer difference (STD) NMR experiments between this lectin and a small glycopeptide containing this moiety show that this lectin is in close contact with H1, H4, and the acetamide group of the GalNAc residue, with weaker STD signals observed for the remaining protons. These results indicate close interactions between hMGL and the entire GalNAc residue of the glycopeptide.

Differences and Commonalities between Proteins in Recognizing O-GalNAc Glycans

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Molecular recognition of GalNAc-containing glycopeptides strongly depends on the recognition of the sugar moiety, with the exception of some anti-MUC1 antibodies. However, focusing exclusively on the GalNAc unit, a certain level of degeneracy is observed within the same families of the proteins. The flexibility of the substrate might facilitate the variability in the interactions, allowing changes in crucial residues. An example of this situation is observed in ZmpB, (67,68) which has a pocket presenting a considerable basic surface complementary with the acidic carboxylate of a sialic acid unit present in the glycopeptide. The presence of two additional subsites for the sialic acid stabilizes the primary interactions with GalNAc at the active site. A similar situation takes place with mucinase IMPa, which has an open region that is susceptible to forming π-interactions with a Trp, where AM0627 and BT4244 have a Tyr and an Asp residue that interact with 6-OH of GalNAc, blocking recognition of substrates with substituents at that position.
In GalNAc-Ts, the lectin domain presents equivalent residues, leading to similar interactions. (6,34) While most H-bonds and CH-π interactions are conserved for several GalNAc-Ts (see Figure 2C), some nonconserved interactions were observed, such as those found in GalNAc-T3. (1)
Some antibodies, such as SM3, present hydrogen bonds between a residue of Tyr and the hydroxymethyl group of GalNAc, and its methyl group interacts weakly through a CH-π contact with a Tyr residue (see, e.g., PDB entries 5A2K, (48)5OWP, (69) and 6FRJ (70)). For antibody 237-mAB, a stacking interaction between the methyl group of the N-acetyl moiety and a Trp residue was also found. (50)
When all of these proteins are considered together, despite the commonalities that are found inside each family of proteins (Figure 7), there is not an observable pattern between the different families, other than some common residues like Tyr, His, or Asp, which are responsible for hydrogen-bond interactions. This is not surprising because of the different functions exerted by mucinases, glycosyltransferases, antibodies, and lectins. In antibodies the role of GalNAc is not so crucial as in other cases, and recognition is limited to a minimum, although there are some exceptions, such as those found for the 5E5 antibody. In contrast, the affinity of lectins by their ligands is determined by their recognition to the GalNAc moiety by a series of hydrogen bonds, as well as CH-π interactions.

Figure 7

Figure 7. Main common recognition modes for the different families of proteins.

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Conclusions and Future Perspectives

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This Account demonstrates the important role that the GalNAc moiety plays in the molecular recognition of relevant glycopeptides, such as those that are hallmarks of cancer, by their corresponding biological targets, such as lectins and antibodies. Moreover, GalNAc is an active bystander involved in the synthesis and degradation of O-glycans by GTs and mucinases, respectively, by assisting these enzymes in localizing the glycopeptide in the correct position. At the interaction level, GalNAc is recognized by several hydrogen bonds involving its hydroxyl groups, especially OH3 and OH4, located in the same plane, as well as CH-π interactions, engaging the hydrophobic alpha side of the sugar or the −CH2– of the hydroxymethyl group. In general, selectivity toward this carbohydrate is achieved through hydrogen bonds with the N-acetyl group of GalNAc, which in some cases is complemented by CH-π interactions via the methyl group. The structural information on GalNAc recognition described in this work may be useful in developing drug-design strategies for GTs or mucinases to treat diseases in which they are involved. In addition, the information obtained with antibody and lectin complexes may be a promising tool for developing more effective cancer vaccines as well as tools for cancer diagnosis.

Author Information

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  • Corresponding Authors
    • Pedro Merino - Institute of Biocomputation and Physics of Complex Systems (BIFI), Glycobiology Unit, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, 50018 Zaragoza, Spain;  Department of Organic Chemistry, Faculty of Sciences, University of Zaragoza, Campus San Francisco, 50009 Zaragoza, Spain;  Orcidhttps://orcid.org/0000-0002-2202-3460;  Email: [email protected]
    • Francisco Corzana - Department of Chemistry, Centro de Investigación en Síntesis Química, University of La Rioja, Madre de Dios 53, 26006 Logroño, Spain;  Orcidhttps://orcid.org/0000-0001-5597-8127;  Email: [email protected]
    • Ramon Hurtado-Guerrero - Institute of Biocomputation and Physics of Complex Systems (BIFI), Glycobiology Unit, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, 50018 Zaragoza, Spain;  Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen DK-2200, Denmark;  Fundación ARAID, 50018 Zaragoza, Spain;  Orcidhttps://orcid.org/0000-0002-3122-9401;  Email: [email protected]
  • Authors
    • Ignacio Sanz-Martinez - Institute of Biocomputation and Physics of Complex Systems (BIFI), Glycobiology Unit, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, 50018 Zaragoza, Spain;  Department of Organic Chemistry, Faculty of Sciences, University of Zaragoza, Campus San Francisco, 50009 Zaragoza, Spain
    • Sandra Pereira - Institute of Biocomputation and Physics of Complex Systems (BIFI), Glycobiology Unit, University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, 50018 Zaragoza, Spain;  Department of Organic Chemistry, Faculty of Sciences, University of Zaragoza, Campus San Francisco, 50009 Zaragoza, Spain
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Biographies

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Ignacio Sanz-Martinez received his Bachelor’s and Master’s degrees from the University of Zaragoza (Spain). He joined Prof. Merino’s group in 2019 for his Ph.D. degree in Biological Chemistry. He is exploring the mechanisms of NAc-galactosyltransferases by QM/MM methods.

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Sandra Pereira received her Bachelor’s and Master’s degrees from the University of Alcala de Henares (Spain). She joined Prof. Merino’s group in 2017 for her Ph.D. degree in Biological Chemistry, which was focused on the structures of ligands for GalNAc-Ts.

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Pedro Merino studied Chemistry at University of Zaragoza (Spain). He obtained his Ph.D. in 1989. After postdoctoral work at University of Ferrara (Italy) with Prof. A. Dondoni, he started his independent career in 1992 at the University of Zaragoza, where he is a Full Professor since 2006. His research interests focus on Chemical Biology with particular focus on glycobiology and elucidation of enzymatic and non-enzymatic reaction mechanisms, using diverse computational methods (DFT, QM/MM, MD, metadynamics).

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Francisco Corzana studied Chemistry at the University of La Rioja (Spain) where he obtained his Ph.D. in 2001. As a postdoctoral fellow, he worked with Prof. Jiménez-Barbero and Dr. Asensio in Biological Research Center (CSIC), Madrid. In 2005 he started his research career at the University of La Rioja, where he is currently an Associate Professor. His research interests include synthetic approaches and conformational analysis of O-glycopeptides with applications in cancer therapy and diagnosis.

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Ramón Hurtado-Guerrero studied Pharmacy at the University of Granada (Spain). He obtained his Ph.D. in 2001, and subsequently undertook postdoctoral work at the University of Leeds with Professor Mike McPherson, UCL (U.K.) with Professor Vallance, and at the University of Dundee with Professor van Aalten. In 2009, he embarked on his academic career at the University of Zaragoza as a senior ARAID researcher. His research interests include structural glycobiology to elucidate the role of glycans in protein recognition and catalysis.

Acknowledgments

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The authors thank the Agencia Estatal de Investigación (AEI) (Projects PID2019-104090RB-100, BFU2016-75633-P and PID2019-105451GB-I00, PID2021-127622OB-I00) for financial support and FPI predoctoral contracts to S.P. and I.S.-M. P.M. and R.H.-G. thank Gobierno de Aragon for financial support (Grupos de Referencia, E34_R17). R.H.-G. also thanks ARAID and the Gobierno de Aragón (Project LMP58_18) with FEDER (2014–2020) funds for “Building Europe from Aragón” and the COST Action CA18103 INNOGLY: Innovation with Glycans new frontiers from synthesis to new biological targets. F.C. thanks the EU (Marie-Sklodowska Curie ITN, DIRNANO, Grant agreement no. 956544), the Mizutani Foundation for Glycoscience (Grant 220115), and Asociación Española Contra el Cáncer (AECC)─La Rioja.

References

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  7. 7
    Hurtado-Guerrero, R. Recent structural and mechanistic insights into protein O-GalNAc glycosylation. Biochem. Soc. Trans. 2016, 44, 61– 67,  DOI: 10.1042/BST20150178
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    7
    Recent structural and mechanistic insights into protein O-GalNAc glycosylation
    Hurtado-Guerrero, Ramon
    Biochemical Society Transactions (2016), 44 (1), 61-67CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
    A review. Protein O-GalNAcylation is an abundant post-translational modification and predicted to occur in >80% of the proteins passing through the Golgi app. This modification is driven by 20 polypeptide GalNAc (N-acetylgalactosamine)-transferases (GalNAc-Ts), which are unique in that they possess both catalytic and lectin domains. The peptide substrate specificities of GalNAc-Ts are still poorly defined and the understanding of the sequence and structural features that direct O-glycosylation of proteins is limited. Part of this may be attributed to the complex regulation by coordinated action of multiple GalNAc-T isoforms, and part of this may also be attributed to the 2 functional domains of GalNAc-Ts that both seem to be involved in directing the substrate specificities. Recent studies have resulted in 3-dimensional structures of GalNAc-Ts and detn. of the reaction mechanism of this family of enzymes. Key advances include the trapping of binary/ternary complexes in combination with computational simulations and AFM/small-SAXS expts., which have allowed for the dissection of the reaction coordinates and the mechanism by which the lectin domains modulate the glycosylation. These studies not only broaden the knowledge of the modes-of-action of this family of enzymes but also open up potential avenues for the rational design of effective and selective inhibitors of O-glycosylation.
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  8. 8
    Schjoldager, K. T.; Joshi, H. J.; Kong, Y.; Goth, C. K.; King, S. L.; Wandall, H. H.; Bennett, E. P.; Vakhrushev, S. Y.; Clausen, H. Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome. EMBO Rep 2015, 16, 1713– 1722,  DOI: 10.15252/embr.201540796
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    8
    Deconstruction of O-glycosylation-GalNAc-T isoforms direct distinct subsets of the O-glycoproteome
    Schjoldager, Katrine T.; Joshi, Hiren J.; Kong, Yun; Goth, Christoffer K.; King, Sarah Louise; Wandall, Hans H.; Bennett, Eric P.; Vakhrushev, Sergey Y.; Clausen, Henrik
    EMBO Reports (2015), 16 (12), 1713-1722CODEN: ERMEAX; ISSN:1469-221X. (Wiley-VCH Verlag GmbH & Co. KGaA)
    GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biol. functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and GalNAc-T2) in liver cells and explore how the GalNAc-T repertoire quant. affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and GalNAc-T2. The non-redundant O-glycoproteome subsets and specific transcriptional responses for each isoform are related to different cellular processes; for the GalNAc-T2 isoform, these support a role in lipid metab. The results demonstrate that GalNAc-Ts have different non-redundant glycosylation functions, which may affect distinct cellular processes. The data serves as a comprehensive resource for unique GalNAc-T substrates. Our study provides a new view of the differential regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.
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  9. 9
    Bennett, E. P.; Mandel, U.; Clausen, H.; Gerken, T. A.; Fritz, T. A.; Tabak, L. A. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology 2012, 22, 736– 756,  DOI: 10.1093/glycob/cwr182
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    9
    Control of mucin-type O-glycosylation: A classification of the polypeptide GalNAc-transferase gene family
    Bennett, Eric P.; Mandel, Ulla; Clausen, Henrik; Gerken, Thomas A.; Fritz, Timothy A.; Tabak, Lawrence A.
    Glycobiology (2012), 22 (6), 736-756CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
    A review. Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The GalNAc-T family is the largest glycosyltransferase enzyme family covering a single known glycosidic linkage and it is highly conserved throughout animal evolution, although absent in bacteria, yeast and plants. Emerging studies have shown that the large no. of genes (GALNTs) in the GalNAc-T family do not provide full functional redundancy and single GalNAc-T genes have been shown to be important in both animals and human. Here, we present an overview of the GalNAc-T gene family in animals and propose a classification of the genes into subfamilies, which appear to be conserved in evolution structurally as well as functionally.
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  10. 10
    Brockhausen, I.; Schachter, H.; Stanley, P. O-GalNAc Glycans. In Essentials of Glycobiology, 2nd ed.; Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E., Eds.; Cold Spring Harbor Laboratory Press: 2008.
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  11. 11
    Ho, T. D.; Davis, B. M.; Ritchie, J. M.; Waldor, M. K. Type 2 secretion promotes enterohemorrhagic Escherichia coli adherence and intestinal colonization. Infect. Immun. 2008, 76, 1858– 1865,  DOI: 10.1128/IAI.01688-07
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    11
    Type 2 secretion promotes enterohemorrhagic Escherichia coli adherence and intestinal colonization
    Ho, Theresa D.; Davis, Brigid M.; Ritchie, Jennifer M.; Waldor, Matthew K.
    Infection and Immunity (2008), 76 (5), 1858-1865CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
    Enterohemorrhagic Escherichia coli (EHEC) is a noninvasive food-borne pathogen that colonizes the distal ileum and colon. Proteins encoded in the EHEC locus of enterocyte effacement (LEE) pathogenicity island are known to contribute to this pathogen's adherence to epithelial cells and intestinal colonization. The role of non-LEE-encoded proteins in these processes is not as clear. The authors found that the Z2053 gene (designated adfO here), a gene located in a cryptic EHEC prophage, exhibits similarity to adherence and/or colonization factor genes found in several other enteric pathogens. An EHEC adfO mutant exhibited marked redns. in adherence to HeLa cells and in the secretion of several proteins into the supernatant. YodA, one of these secreted proteins, was found to be a substrate of the EHEC pO157-encoded type 2 secretion system (T2SS). Both the T2SS and YodA proved to be essential for EHEC adherence to cultured HeLa cell monolayers. Using an infant rabbit model of infection, the authors found that the adfO mutation did not affect colonization but that the colonization of an etpC (T2SS) mutant was reduced ∼5-fold. A strain deficient in YodA had a more severe colonization defect; however, this strain also exhibited a growth defect in vitro. Overall, the authors' findings indicate that the pO157-encoded T2SS contributes to EHEC adherence and intestinal colonization and thus show that EHEC pathogenicity depends on type 2 secretion as well as type 3 secretion.
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  12. 12
    Nason, R.; Bull, C.; Konstantinidi, A.; Sun, L.; Ye, Z.; Halim, A.; Du, W.; Sorensen, D. M.; Durbesson, F.; Furukawa, S.; Mandel, U.; Joshi, H. J.; Dworkin, L. A.; Hansen, L.; David, L.; Iverson, T. M.; Bensing, B. A.; Sullam, P. M.; Varki, A.; Vries, E.; de Haan, C. A. M.; Vincentelli, R.; Henrissat, B.; Vakhrushev, S. Y.; Clausen, H.; Narimatsu, Y. Nat. Commun. 2021, 12, 4070,  DOI: 10.1038/s41467-021-24366-4
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    12
    Display of the human mucinome with defined O-glycans by gene engineered cells
    Nason, Rebecca; Bull, Christian; Konstantinidi, Andriana; Sun, Lingbo; Ye, Zilu; Halim, Adnan; Du, Wenjuan; Soerensen, Daniel M.; Durbesson, Fabien; Furukawa, Sanae; Mandel, Ulla; Joshi, Hiren J.; Dworkin, Leo Alexander; Hansen, Lars; David, Leonor; Iverson, Tina M.; Bensing, Barbara A.; Sullam, Paul M.; Varki, Ajit; Vries, Erik de; de Haan, Cornelis A. M.; Vincentelli, Renaud; Henrissat, Bernard; Vakhrushev, Sergey Y.; Clausen, Henrik; Narimatsu, Yoshiki
    Nature Communications (2021), 12 (1), 4070CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromols. in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and prodn. of human TR O-glycodomains (∼200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances exptl. studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for mol. dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.
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  13. 13
    Shon, D. J.; Kuo, A.; Ferracane, M. J.; Malaker, S. A. Classification, structural biology, and applications of mucin domain-targeting proteases. Biochem.J. 2021, 478, 1585– 1603,  DOI: 10.1042/BCJ20200607
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    13
    Classification, structural biology, and applications of mucin domain-targeting proteases
    Shon, D. Judy; Kuo, Angel; Ferracane, Michael J.; Malaker, Stacy A.
    Biochemical Journal (2021), 478 (8), 1585-1603CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    A review. Epithelial surfaces throughout the body are coated by mucins, a class of proteins carrying domains characterized by a high d. of O-glycosylated serine and threonine residues. The resulting mucosal layers form crucial host-microbe interfaces that prevent the translocation of microbes while also selecting for distinct bacteria via the presented glycan repertoire. The intricate interplay between mucus prodn. and breakdown thus dets. the compn. of the microbiota maintained within these mucosal environments, which can have a large influence on the host during both homeostasis and disease. Most research to date on mucus breakdown has focused on glycosidases that trim glycan structures to release monosaccharides as a source of nutrients. More recent work has uncovered the existence of mucin-type O-glycosylation-dependent proteases that are secreted by pathogens, commensals, and mutualists to facilitate mucosal colonization and penetration. Addnl., IgA (IgA) proteases promote bacterial colonization in the presence of neutralizing secretory IgA through selective cleavage of the heavily O-glycosylated hinge region. In this review, we summarize families of O-glycoproteases and IgA proteases, discuss known structural features, and review applications of these enzymes to glycobiol.
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  14. 14
    Schjoldager, K. T.-B. G.; Clausen, H. Site-specific protein O-glycosylation modulates proprotein processing - deciphering specific functions of the large polypeptide GalNAc-transferase gene family. Biochim. Biophys. Acta. 2012, 1820, 2079– 2094,  DOI: 10.1016/j.bbagen.2012.09.014
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    14
    Site-specific protein O-glycosylation modulates proprotein processing - Deciphering specific functions of the large polypeptide GalNAc-transferase gene family
    Schjoldager, Katrine T.-B. G.; Clausen, Henrik
    Biochimica et Biophysica Acta, General Subjects (2012), 1820 (12), 2079-2094CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
    A review. Posttranslational modifications (PTMs) greatly expand the function and regulation of proteins, and glycosylation is the most abundant and diverse PTM. Of the many different types of protein glycosylation, one is quite unique; GalNAc-type (or mucin-type) O-glycosylation, where biosynthesis is initiated in the Golgi by up to twenty distinct UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). These GalNAc-Ts are differentially expressed in cells and have different (although partly overlapping) substrate specificities, which provide for both unique functions and considerable redundancy. Recently we have begun to uncover human diseases assocd. with deficiencies in GalNAc-T genes (GALNTs). Thus deficiencies in individual GALNTs produce cell and protein specific effects and subtle distinct phenotypes such as hyperphosphatemia with hyperostosis (GALNT3) and dysregulated lipid metab. (GALNT2). These phenotypes appear to be caused by deficient site-specific O-glycosylation that co-regulates proprotein convertase (PC) processing of FGF23 and ANGPTL3, resp. Here we summarize recent progress in uncovering the interplay between human O-glycosylation and protease regulated processing and describes other important functions of site-specific O-glycosylation in health and disease. Site-specific O-glycosylation modifies pro-protein processing and other proteolytic events such as ADAM processing and thus emerges as an important co-regulator of limited proteolytic processing events. Our appreciation of this function may have been hampered by our sparse knowledge of the O-glycoproteome and in particular sites of O-glycosylation. New strategies for identification of O-glycoproteins have emerged and recently the concept of SimpleCells, i.e. human cell lines made deficient in O-glycan extension by zinc finger nuclease gene targeting, was introduced for broad O-glycoproteome anal.
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  15. 15
    Goth, C. K.; Vakhrushev, S. Y.; Joshi, H. J.; Clausen, H.; Schjoldager, K. T. Fine-Tuning Limited Proteolysis: A Major Role for Regulated Site-Specific O-Glycosylation. Trends Biochem. Sci. 2018, 43, 269– 284,  DOI: 10.1016/j.tibs.2018.02.005
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    15
    Fine-Tuning Limited Proteolysis: A Major Role for Regulated Site-Specific O-Glycosylation
    Goth, Christoffer K.; Vakhrushev, Sergey Y.; Joshi, Hiren J.; Clausen, Henrik; Schjoldager, Katrine T.
    Trends in Biochemical Sciences (2018), 43 (4), 269-284CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
    A review. Limited proteolytic processing is an essential and ubiquitous post-translational modification (PTM) affecting secreted proteins; failure to regulate the process is often assocd. with disease. Glycosylation is also a ubiquitous protein PTM and site-specific O-glycosylation in close proximity to sites of proteolysis can regulate and direct the activity of proprotein convertases, a disintegrin and metalloproteinases (ADAMs), and metalloproteinases affecting the activation or inactivation of many classes of proteins, including G-protein-coupled receptors (GPCRs). Here, we summarize the emerging data that suggest O-glycosylation to be a key regulator of limited proteolysis, and highlight the potential for crosstalk between multiple PTMs.
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  16. 16
    Wandall, H. H.; Nielsen, M. A. I.; King-Smith, S.; de Haan, N.; Bagdonaite, I. Global functions of O-glycosylation: promises and challenges in O-glycobiology. FEBS J. 2021, 288, 7183– 7212,  DOI: 10.1111/febs.16148
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    16
    Global functions of O-glycosylation: promises and challenges in O-glycobiology
    Wandall, Hans H.; Nielsen, Mathias A. I.; King-Smith, Sarah; de Haan, Noortje; Bagdonaite, Ieva
    FEBS Journal (2021), 288 (24), 7183-7212CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
    A review. Mucin type O-glycosylation is one of the most diverse types of glycosylation, playing essential roles in tissue development and homeostasis. In complex organisms, O-GalNAc glycans comprise a substantial proportion of the glycocalyx, with defined functions in hemostatic, gastrointestinal, and respiratory systems. Furthermore, O-GalNAc glycans are important players in host-microbe interactions, and changes in O-glycan compn. are assocd. with certain diseases and metabolic conditions, which in some instances can be used for diagnosis or therapeutic intervention. Breakthroughs in O-glycobiol. have gone hand in hand with the development of new technologies, such as advancements in mass spectrometry, as well as facilitation of genetic engineering in mammalian cell lines. High-throughput O-glycoproteomics have enabled us to draw a comprehensive map of O-glycosylation, and mining this information has supported the definition and confirmation of functions related to site-specific O-glycans. This includes protection from proteolytic cleavage, as well as modulation of binding affinity or receptor function. Yet, there is still much to discover, and among the important next challenges will be to define the context-dependent functions of O-glycans in different stages of cellular differentiation, cellular metab., host-microbiome interactions, and in disease. In this review, we present the achievements and the promises in O-GalNAc glycobiol. driven by technol. advances in anal. methods, genetic engineering, and systems biol.
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  17. 17
    Brockhausen, I.; Wandall, H. H.; Hagen, K. G. T.; Stanley, P. O-GalNAc Glycans. In Essentials of Glycobiology, 4th ed.; Varki, A., Cummings, R. D., Esko, J. D., Stanley, P., Hart, G. W., Aebi, M., Mohnen, D., Kinoshita, T., Packer, N. H., Prestegard, J. H., Schnaar, R. L., Seeberger, P. H., Eds.; Cold Spring Harbor Laboratory Press: 2022; pp 117– 128.
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  18. 18
    Xia, L.; Ju, T.; Westmuckett, A.; An, G.; Ivanciu, L.; McDaniel, J. M.; Lupu, F.; Cummings, R. D.; McEver, R. P. Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans. J. Cell Biol. 2004, 164, 451– 459,  DOI: 10.1083/jcb.200311112
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    18
    Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans
    Xia, Lijun; Ju, Tongzhong; Westmuckett, Andrew; An, Guangyu; Ivanciu, Lacramioara; McDaniel, J. Michael; Lupu, Florea; Cummings, Richard D.; McEver, Rodger P.
    Journal of Cell Biology (2004), 164 (3), 451-459CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    The core 1 β1-3-galactosyltransferase (T-synthase) transfers Gal from UDP-Gal to GalNAcα1-Ser/Thr (Tn antigen) to form the core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr (T antigen). The T antigen is a precursor for extended and branched O-glycans of largely unknown function. We found that wild-type mice expressed the NeuAcα2-3Galβ1-3GalNAcα1-Ser/Thr primarily in endothelial, hematopoietic, and epithelial cells during development. Gene-targeted mice lacking T-synthase instead expressed the nonsialylated Tn antigen in these cells and developed brain hemorrhage that was uniformly fatal by embryonic day 14. T-synthase-deficient brains formed a chaotic microvascular network with distorted capillary lumens and defective assocn. of endothelial cells with pericytes and extracellular matrix. These data reveal an unexpected requirement for core 1-derived O-glycans during angiogenesis.
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  19. 19
    Wang, Y.; Ju, T.; Ding, X.; Xia, B.; Wang, W.; Xia, L.; He, M.; Cummings, R. D. Cosmc is an essential chaperone for correct protein O-glycosylation. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 9228– 9233,  DOI: 10.1073/pnas.0914004107
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    19
    Cosmc is an essential chaperone for correct protein O-glycosylation
    Wang, Yingchun; Ju, Tongzhong; Ding, Xiaokun; Xia, Baoyun; Wang, Wenyi; Xia, Lijun; He, Miao; Cummings, Richard D.
    Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (20), 9228-9233, S9228/1-S9228/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Cosmc is a mol. chaperone thought to be required for expression of active T-synthase, the only enzyme that galactosylates the Tn antigen (GalNAcα1-Ser/Thr-R) to form core 1 Galβ-3GalNAcα1-Ser/Thr (T antigen) during mucin type O-glycan biosynthesis. Here the authors show that ablation of the X-linked Cosmc gene in mice causes embryonic lethality and Tn antigen expression. Loss of Cosmc is assocd. with loss of T-synthase but not other enzymes required for glycoprotein biosynthesis, demonstrating that Cosmc is specific in vivo for the T-synthase. The authors generated genetically mosaic mice with a targeted Cosmc deletion and survivors exhibited abnormalities correlated with Tn antigen expression that are related to several human diseases.
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  20. 20
    Gill, D. J.; Tham, K. M.; Chia, J.; Wang, S. C.; Steentoft, C.; Clausen, H.; Bard-Chapeau, E. A.; Bard, F. A. Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, E3152– 3161,  DOI: 10.1073/pnas.1305269110
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    20
    Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness
    Gill, David J.; Tham, Keit Min; Chia, Joanne; Wang, Shyi Chyi; Steentoft, Catharina; Clausen, Henrik; Bard-Chapeau, Emilie A.; Bard, Frederic A.
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (34), E3152-E3161,SE3152/1-SE3152/32CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Invasiveness underlies cancer aggressiveness and is a hallmark of malignancy. Most malignant tumors have elevated levels of Tn, an O-GalNAc glycan. Mechanisms underlying Tn up-regulation and its effects remain unclear. Here we show that Golgi-to-endoplasmic reticulum relocation of polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) drives high Tn levels in cancer cell lines and in 70% of malignant breast tumors. This process stimulates cell adhesion to the extracellular matrix, as well as migration and invasiveness. The GalNAc-Ts lectin domain, mediating high-d. glycosylation, is crit. for these effects. Interfering with the lectin domain function inhibited carcinoma cell migration in vitro and metastatic potential in mice. We also show that stimulation of cell migration is dependent on Tn-bearing proteins present in lamellipodia of migrating cells. Our findings suggest that relocation of GalNAc-Ts to the endoplasmic reticulum frequently occurs upon cancerous transformation to enhance tumor cell migration and invasiveness through modification of cell surface proteins.
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  21. 21
    Herbomel, G. G.; Rojas, R. E.; Tran, D. T.; Ajinkya, M.; Beck, L.; Tabak, L. A. The GalNAc-T Activation Pathway (GALA) is not a general mechanism for regulating mucin-type O-glycosylation. PLoS One 2017, 12, e0179241  DOI: 10.1371/journal.pone.0179241
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    21
    The GalNAc-T activation pathway (GALA) is not a general mechanism for regulating mucintype O-glycosylation
    Herbomel, Gaetan G.; Rojas, Raul E.; Tran, Duy T.; Ajinkya, Monica; Beck, Lauren; Tabak, Lawrence A.
    PLoS One (2017), 12 (7), e0179241/1-e0179241/14CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Mucin-type O-glycosylation is initiated by the UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferase (GalNAc-T) family of enzymes. Their activity results in the GalNAc α1-OThr/ Ser structure, termed the Tn antigen, which is further decorated with addnl. sugars. In neoplastic cells, the Tn antigen is often overexpressed. Because O-glycosylation is controlled by the activity of GalNAc-Ts, their regulation is of great interest. Previous reports suggest that growth factors, EGF or PDGF, induce Golgi complex-to-endoplasmic reticulum (ER) relocation of both GalNAc-Ts and Tn antigen in HeLa cells, offering a mechanism for Tn antigen overexpression termed "GALA". However, we were unable to reproduce these findings. Upon treatment of HeLa cells with either EGF or PDGF we obsd. no change in the co-localization of endogenous GalNAc-T1, GalNAc-T2 or Tn antigen with the Golgi complex marker TGN46. There was also no enhancement of localization with the ER marker calnexin. We conclude that growth factors do not cause redistribution of GalNAc-Ts from the Golgi complex to the ER in HeLa cells.
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  22. 22
    Radhakrishnan, P.; Dabelsteen, S.; Madsen, F. B.; Francavilla, C.; Kopp, K. L.; Steentoft, C.; Vakhrushev, S. Y.; Olsen, J. V.; Hansen, L.; Bennett, E. P.; Woetmann, A.; Yin, G.; Chen, L.; Song, H.; Bak, M.; Hlady, R. A.; Peters, S. L.; Opavsky, R.; Thode, C.; Qvortrup, K.; Schjoldager, K. T.; Clausen, H.; Hollingsworth, M. A.; Wandall, H. H. Immature truncated O-glycophenotype of cancer directly induces oncogenic features. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, E4066– 4075,  DOI: 10.1073/pnas.1406619111
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    Immature truncated O-glycophenotype of cancer directly induces oncogenic features
    Radhakrishnan, Prakash; Dabelsteen, Sally; Madsen, Frey Brus; Francavilla, Chiara; Kopp, Katharina L.; Steentoft, Catharina; Vakhrushev, Sergey Y.; Olsen, Jesper V.; Hansen, Lars; Bennett, Eric P.; Woetmann, Anders; Yin, Guangliang; Chen, Longyun; Song, Haiyan; Bak, Mads; Hlady, Ryan A.; Peters, Staci L.; Opavsky, Rene; Thode, Christenze; Qvortrup, Klaus; Schjoldager, Katrine T.-B. G.; Clausen, Henrik; Hollingsworth, Michael A.; Wandall, Hans H.
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (39), E4066-E4075CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Aberrant expression of immature truncated O-glycans is a characteristic feature obsd. on virtually all epithelial cancer cells, and a very high frequency is obsd. in early epithelial premalignant lesions that precede the development of adenocarcinomas. Expression of the truncated O-glycan structures Tn and sialyl-Tn is strongly assocd. with poor prognosis and overall low survival. The genetic and biosynthetic mechanisms leading to accumulation of truncated O-glycans are not fully understood and include mutation or dysregulation of glycosyltransferases involved in elongation of O-glycans, as well as relocation of glycosyltransferases controlling initiation of O-glycosylation from Golgi to endoplasmic reticulum. Truncated O-glycans have been proposed to play functional roles for cancer-cell invasiveness, but our understanding of the biol. functions of aberrant glycosylation in cancer is still highly limited. Here, we used exome sequencing of most glycosyltransferases in a large series of primary and metastatic pancreatic cancers to rule out somatic mutations as a cause of expression of truncated O-glycans. Instead, we found hypermethylation of core 1 β3-Gal-T-specific mol. chaperone, a key chaperone for O-glycan elongation, as the most prevalent cause. We next used gene editing to produce isogenic cell systems with and without homogenous truncated O-glycans that enabled, to our knowledge, the first polyomic and side-by-side evaluation of the cancer O-glycophenotype in an organotypic tissue model and in xenografts. The results strongly suggest that truncation of O-glycans directly induces oncogenic features of cell growth and invasion. The study provides support for targeting cancer-specific truncated O-glycans with immunotherapeutic measures.
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  23. 23
    Marcos, N. T.; Pinho, S.; Grandela, C.; Cruz, A.; Samyn-Petit, B.; Harduin-Lepers, A.; Almeida, R.; Silva, F.; Morais, V.; Costa, J.; Kihlberg, J.; Clausen, H.; Reis, C. A. Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated sialyl-Tn antigen. Cancer Res. 2004, 64, 7050– 7057,  DOI: 10.1158/0008-5472.CAN-04-1921
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    23
    Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated Sialyl-Tn antigen
    Marcos, Nuno T.; Pinho, Sandra; Grandela, Catarina; Cruz, Andrea; Samyn-Petit, Benedicte; Harduin-Lepers, Anne; Almeida, Raquel; Silva, Filipe; Morais, Vanessa; Costa, Julia; Kihlberg, Jan; Clausen, Henrik; Reis, Celso A.
    Cancer Research (2004), 64 (19), 7050-7057CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    The Sialyl-Tn antigen (Neu5Acα2-6GalNAc-O-Ser/Thr) is highly expressed in several human carcinomas and is assocd. with carcinoma aggressiveness and poor prognosis. The authors characterized two human sialyltransferases, CMP-Neu5Ac:GalNAc-R α2,6-sialyltransferase (ST6GalNAc)-I and ST6GalNAc-II, that are candidate enzymes for Sialyl-Tn synthases. The authors expressed sol. recombinant hST6GalNAc-I and hST6GalNAc-II and characterized the substrate specificity of both enzymes toward a panel of glycopeptides, glycoproteins, and other synthetic glycoconjugates. The recombinant ST6GalNAc-I and ST6GalNAc-II showed similar substrate specificity toward glycoproteins and GalNAcα-O-Ser/Thr glycopeptides, such as glycopeptides derived from the MUC2 mucin and the HIVgp120. The authors also obsd. that the amino acid sequence of the acceptor glycopeptide contributes to the in vitro substrate specificity of both enzymes. The authors addnl. established a gastric cell line, MKN45, stably transfected with the full length of either ST6GalNAc-I or ST6GalNAc-II and evaluated the carbohydrate antigens expression profile induced by each enzyme. MKN45 transfected with ST6GalNAc-I showed high expression of Sialyl-Tn, whereas MKN45 transfected with ST6GalNAc-II showed the biosynthesis of the Sialyl-6T structure [Galβ1-3 (Neu5Acα2-6)GalNAc-O-Ser/Thr]. In conclusion, although both enzymes show similar in vitro activities when Tn antigen alone is available, whenever both Tn and T antigens are present, ST6GalNAc-I acts preferentially on Tn antigen, whereas the ST6GalNAc-II acts preferentially on T antigen. These results show that ST6GalNAc-I is the major Sialyl-Tn synthase and strongly support the hypothesis that the expression of the Sialyl-Tn antigen in cancer cells is due to ST6GalNAc-I activity.
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  24. 24
    Martínez-Sáez, N.; Peregrina, J. M.; Corzana, F. Principles of mucin structure: implications for the rational design of cancer vaccines derived from MUC1-glycopeptides. Chem. Soc. Rev. 2017, 46, 7154– 7175,  DOI: 10.1039/C6CS00858E
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    24
    Principles of mucin structure: implications for the rational design of cancer vaccines derived from MUC1-glycopeptides
    Martinez-Saez, Nuria; Peregrina, Jesus M.; Corzana, Francisco
    Chemical Society Reviews (2017), 46 (23), 7154-7175CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    Cancer is currently one of the world's most serious public health problems. Significant efforts are being made to develop new strategies that can eradicate tumors selectively without detrimental effects to healthy cells. One promising approach is focused on the design of vaccines that contain partially glycosylated mucins in their formulation. Although some of these vaccines are in clin. trials, a lack of knowledge about the mol. basis that governs the antigen presentation, and the interactions between antigens and the elicited antibodies has limited their success thus far. This review focuses on the most significant milestones achieved to date in the conformational anal. of tumor-assocd. MUC1 derivs. both in soln. and bound to antibodies. The effect that the carbohydrate scaffold has on the peptide backbone structure and the role of the sugar in mol. recognition by antibodies are emphasized. The outcomes summarised in this review may be a useful guide to develop new antigens for the design of cancer vaccines in the near future.
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  25. 25
    Bulteau, F.; Thépaut, M.; Henry, M.; Hurbin, A.; Vanwonterghem, L.; Vivès, C.; Le Roy, A.; Ebel, C.; Renaudet, O.; Fieschi, F.; Coll, J.-L. Targeting Tn-Antigen-Positive Human Tumors with a Re combinant Human Macrophage Galactose C-Type Lectin. Mol. Pharmaceutics 2022, 19, 235– 245,  DOI: 10.1021/acs.molpharmaceut.1c00744
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    25
    Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin
    Bulteau, Francois; Thepaut, Michel; Henry, Maxime; Hurbin, Amandine; Vanwonterghem, Laetitia; Vives, Corinne; Le Roy, Aline; Ebel, Christine; Renaudet, Olivier; Fieschi, Franck; Coll, Jean-Luc
    Molecular Pharmaceutics (2022), 19 (1), 235-245CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    Alterations in glycosylation cause the emergence of tumor-assocd. carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-pos. human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.
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  26. 26
    Fritz, T. A.; Hurley, J. H.; Trinh, L. B.; Shiloach, J.; Tabak, L. A. The beginnings of mucin biosynthesis: the crystal structure of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-T1. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15307– 15312,  DOI: 10.1073/pnas.0405657101
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    26
    The beginnings of mucin biosynthesis: The crystal structure of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase-T1
    Fritz, Timothy A.; Hurley, James H.; Trinh, Loc-Ba; Shiloach, Joseph; Tabak, Lawrence A.
    Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (43), 15307-15312CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    UDP-N-acetylgalactosamine-polypeptide N-acetylgalactosaminyltransferases (UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases; ppGaNTases) initiate the formation of mucin-type, O-linked glycans by catalyzing the transfer of α-N-acetylgalactosamine from UDP-GalNAc to Ser or Thr residues of core proteins to form the Tn antigen (GalNAc-α-1-O-Ser/Thr). The ppGaNTases are unique among glycosyltransferases in contg. a C-terminal lectin domain. Here, the authors present the x-ray crystal structure of a ppGaNTase, murine ppGaNTase-T1, and show that it folds to form distinct catalytic and lectin domains. The assocn. of the 2 domains formed a large cleft in the surface of the enzyme that contained a Mn2+ ion complexed by invariant Asp-209 and His-211 of the "DXH" motif and by invariant His-344. Each of the 3 potential lectin domain carbohydrate-binding sites (α, β, and γ) was located on the active site face of the enzyme, suggesting a mechanism by which the transferase may accommodate multiple conformations of glycosylated acceptor substrates. A model of a mucin 1 glycopeptide (MUC1) substrate bound to the enzyme showed that the spatial sepn. between the lectin α site and a modeled active site UDP-GalNAc was consistent with the in vitro pattern of glycosylation obsd. for this peptide catalyzed by ppGaNTase-T1. The structure also provided a template for the larger ppGaNTase family, and homol. models of several ppGaNTase isoforms predict dramatically different surface chemistries consistent with isoform-selective acceptor substrate recognition.
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    Fritz, T. A.; Raman, J.; Tabak, L. A. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-2. J. Biol. Chem. 2006, 281, 8613– 8619,  DOI: 10.1074/jbc.M513590200
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    27
    Dynamic Association between the Catalytic and Lectin Domains of Human UDP-GalNAc:Polypeptide α-N-Acetylgalactosaminyltransferase-2
    Fritz, Timothy A.; Raman, Jayalakshmi; Tabak, Lawrence A.
    Journal of Biological Chemistry (2006), 281 (13), 8613-8619CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The family of (ppGalNAcTs) is unique among glycosyltransferases, contg. both catalytic and lectin domains that we have previously shown to be closely assocd. Here we describe the x-ray crystal structures of human ppGalNAcT-2 (hT2) bound to the product UDP at 2.75 Å resoln. and to UDP and an acceptor peptide substrate EA2 (PTTDSTTPAPTTK) at 1.64 Å resoln. The conformations of both UDP and residues Arg362-Ser372 vary greatly between the two structures. In the hT2-UDP-EA2 complex, residues Arg362-Ser373 comprise a loop that forms a lid over UDP, sealing it in the active site, whereas in the hT2-UDP complex this loop is folded back, exposing UDP to bulk solvent. EA2 binds in a shallow groove with threonine 7 positioned consistent with in vitro data showing it to be the preferred site of glycosylation. The relative orientations of the hT2 catalytic and lectin domains differ dramatically from that of murine ppGalNAcT-1 and also vary considerably between the two hT2 complexes. Indeed, in the hT2-UDP-EA2 complex essentially no contact is made between the catalytic and lectin domains except for the peptide bridge between them. Thus, the hT2 structures reveal an unexpected flexibility between the catalytic and lectin domains and suggest a new mechanism used by hT2 to capture glycosylated substrates. Kinetic anal. of hT2 lacking the lectin domain confirmed the importance of this domain in acting on glycopeptide but not peptide substrates. The structure of the hT2-UDP-EA2 complex also resolves longstanding questions regarding ppGalNAcT acceptor substrate specificity.
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  28. 28
    Kubota, T.; Shiba, T.; Sugioka, S.; Furukawa, S.; Sawaki, H.; Kato, R.; Wakatsuki, S.; Narimatsu, H. Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10). J. Mol. Biol. 2006, 359, 708– 727,  DOI: 10.1016/j.jmb.2006.03.061
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    28
    Structural Basis of Carbohydrate Transfer Activity by Human UDP-GalNAc: Polypeptide α-N-Acetylgalactosaminyltransferase (pp-GalNAc-T10)
    Kubota, Tomomi; Shiba, Tomoo; Sugioka, Shigemi; Furukawa, Sanae; Sawaki, Hiromichi; Kato, Ryuich; Wakatsuki, Soichi; Narimatsu, Hisashi
    Journal of Molecular Biology (2006), 359 (3), 708-727CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
    Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isoenzyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin β sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isoenzymes to produce a wide range of O-glycans.
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    Lira-Navarrete, E.; Iglesias-Fernandez, J.; Zandberg, W. F.; Companon, I.; Kong, Y.; Corzana, F.; Pinto, B. M.; Clausen, H.; Peregrina, J. M.; Vocadlo, D. J.; Rovira, C.; Hurtado-Guerrero, R. Substrate-Guided Front-Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N-Acetylgalactosaminyltransferase 2. Angew. Chem., Int. Ed. 2014, 53, 8206– 8210,  DOI: 10.1002/anie.201402781
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    29
    Substrate-Guided Front-Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N-Acetylgalactosaminyltransferase 2
    Lira-Navarrete, Erandi; Iglesias-Fernandez, Javier; Zandberg, Wesley F.; Companon, Ismael; Kong, Yun; Corzana, Francisco; Pinto, B. Mario; Clausen, Henrik; Peregrina, Jesus M.; Vocadlo, David J.; Rovira, Carme; Hurtado-Guerrero, Ramon
    Angewandte Chemie, International Edition (2014), 53 (31), 8206-8210CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/mol.-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the at.-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals crit. aspects of substrate recognition, which dictate the specificity for acceptor Thr vs. Ser residues and enforce a front-face SNi-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.
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  30. 30
    Khetarpal, S. A.; Schjoldager, K. T.; Christoffersen, C.; Raghavan, A.; Edmondson, A. C.; Reutter, H. M.; Ahmed, B.; Ouazzani, R.; Peloso, G. M.; Vitali, C.; Zhao, W.; Somasundara, A. V.; Millar, J. S.; Park, Y.; Fernando, G.; Livanov, V.; Choi, S.; Noé, E.; Patel, P.; Ho, S. P.; Kirchgessner, T. G.; Wandall, H. H.; Hansen, L.; Bennett, E. P.; Vakhrushev, S. Y.; Saleheen, D.; Kathiresan, S.; Brown, C. D.; Abou Jamra, R.; LeGuern, E.; Clausen, H.; Rader, D. J. Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents. Cell Metab 2016, 24, 234– 245,  DOI: 10.1016/j.cmet.2016.07.012
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    30
    Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents
    Khetarpal, Sumeet A.; Schjoldager, Katrine T.; Christoffersen, Christina; Raghavan, Avanthi; Edmondson, Andrew C.; Reutter, Heiko M.; Ahmed, Bouhouche; Ouazzani, Reda; Peloso, Gina M.; Vitali, Cecilia; Zhao, Wei; Somasundara, Amritha Varshini Hanasoge; Millar, John S.; Park, YoSon; Fernando, Gayani; Livanov, Valentin; Choi, Seungbum; Noe, Eric; Patel, Pritesh; Ho, Siew Peng; Kirchgessner, Todd G.; Wandall, Hans H.; Hansen, Lars; Bennett, Eric P.; Vakhrushev, Sergey Y.; Saleheen, Danish; Kathiresan, Sekar; Brown, Christopher D.; Abou Jamra, Rami; LeGuern, Eric; Clausen, Henrik; Rader, Daniel J.
    Cell Metabolism (2016), 24 (2), 234-245CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)
    Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a regulator of high-d. lipoprotein cholesterol (HDL-C) metab., but the mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss of function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2-deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Addnl. glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs assocd. with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metab. across mammals.
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    de las Rivas, M.; Coelho, H.; Diniz, A.; Lira-Navarrete, E.; Companon, I.; Jimenez-Barbero, J.; Schjoldager, K. T.; Bennett, E. P.; Vakhrushev, S. Y.; Clausen, H.; Corzana, F.; Marcelo, F.; Hurtado-Guerrero, R. Structural Analysis of a GalNAc-T2Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts. Chem.─Eur. J. 2018, 24, 8382– 8392,  DOI: 10.1002/chem.201800701
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    Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts
    de las Rivas, Matilde; Coelho, Helena; Diniz, Ana; Lira-Navarrete, Erandi; Companon, Ismael; Jimenez-Barbero, Jesus; Schjoldager, Katrine T.; Bennett, Eric P.; Vakhrushev, Sergey Y.; Clausen, Henrik; Corzana, Francisco; Marcelo, Filipa; Hurtado-Guerrero, Ramon
    Chemistry - A European Journal (2018), 24 (33), 8382-8392CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-d. lipoprotein cholesterol (HDL-C) in humans. Herein, the mol. basis for F104S mutant inactivation has been deciphered. Satn. transfer difference NMR spectroscopy expts. demonstrate that the mutation induces loss of binding to peptide substrates. Anal. of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with mol. dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19F NMR spectroscopy expts. reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation.
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    Daniel, E. J. P.; Las Rivas, M.; Lira-Navarrete, E.; Garcia-Garcia, A.; Hurtado-Guerrero, R.; Clausen, H.; Gerken, T. A. Ser and Thr acceptor preferences of the GalNAc-Ts vary among isoenzymes to modulate mucin-type O-glycosylation. Glycobiology 2020, 30, 910– 922,  DOI: 10.1093/glycob/cwaa036
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    Ser and Thr acceptor preferences of the GalNAc-Ts vary among isoenzymes to modulate mucin-type O-glycosylation
    Daniel, Earnest James Paul; De Las Rivas, Matilde; Lira-Navarrete, Erandi; Garcia-Garcia, Ana; Hurtado-Guerrero, Ramon; Clausen, Henrik; Gerken, Thomas A.
    Glycobiology (2020), 30 (11), 910-922CODEN: GLYCE3; ISSN:1460-2423. (Oxford University Press)
    A family of polypeptide GalNAc-transferases (GalNAc-Ts) initiates mucin-type O-glycosylation, transferring GalNAc onto hydroxyl groups of Ser and Thr residues of target substrates. The 20 GalNAc-T isoenzymes in humans are classified into nine subfamilies according to sequence similarity. GalNAc-Ts select their sites of glycosylation based on weak and overlapping peptide sequence motifs, as well prior substrate O-GalNAc glycosylation at sites both remote (long-range) and neighboring (short-range) the acceptor. Together, these preferences vary among GalNAc-Ts imparting each isoenzyme with its own unique specificity. Studies on the first identified GalNAc-Ts showed Thr acceptors were preferred over Ser acceptors; however studies comparing Thr vs.Ser glycosylation across the GalNAc-T family are lacking. Using a series of identical random peptide substrates, with single Thr or Ser acceptor sites, we detd. the rate differences (Thr/Ser rate ratio) between Thr and Ser substrate glycosylation for 12 isoenzymes (representing 7 GalNAcT subfamilies). These Thr/Ser rate ratios varied across subfamilies, ranging from ~ 2 to ~ 18 (for GalNAc-T4/GalNAc-T12 and GalNAc-T3/GalNAc-T6, resp.), while nearly identical Thr/Ser rate ratios were obsd. for isoenzymes within subfamilies. Furthermore, the Thr/Ser rate ratios did not appreciably vary over a series of fixed sequence substrates of different relative activities, suggesting the ratio is a const. for each isoenzyme against single acceptor substrates. Finally, based on GalNAc-T structures, the different Thr/Ser rate ratios likely reflect differences in the strengths of the Thr acceptor Me group binding to the active site pocket. With this work, another activity that further differentiates substrate specificity among the GalNAc-Ts has been identified.
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    de las Rivas, M.; Paul Daniel, E. J.; Coelho, H.; Lira-Navarrete, E.; Raich, L.; Companon, I.; Diniz, A.; Lagartera, L.; Jimenez-Barbero, J.; Clausen, H.; Rovira, C.; Marcelo, F.; Corzana, F.; Gerken, T. A.; Hurtado-Guerrero, R. Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of GalNAc-T4. ACS Cent. Sci. 2018, 4, 1274– 1290,  DOI: 10.1021/acscentsci.8b00488
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    Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of GalNAc-T4
    de las Rivas, Matilde; Paul Daniel, Earnest James; Coelho, Helena; Lira-Navarrete, Erandi; Raich, Lluis; Companon, Ismael; Diniz, Ana; Lagartera, Laura; Jimenez-Barbero, Jesus; Clausen, Henrik; Rovira, Carme; Marcelo, Filipa; Corzana, Francisco; Gerken, Thomas A.; Hurtado-Guerrero, Ramon
    ACS Central Science (2018), 4 (9), 1274-1290CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, resp. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the mol. basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (contg. two α-GalNAc glycosylated Thr (T*), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
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    Lira-Navarrete, E.; de las Rivas, M.; Companon, I.; Pallares, M. C.; Kong, Y.; Iglesias-Fernandez, J.; Bernardes, G. J. L.; Peregrina, J. M.; Rovira, C.; Bernado, P.; Bruscolini, P.; Clausen, H.; Lostao, A.; Corzana, F.; Hurtado-Guerrero, R. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Nat. Commun. 2015, 6, 6937,  DOI: 10.1038/ncomms7937
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    Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation
    Lira-Navarrete, Erandi; de las Rivas, Matilde; Companon, Ismael; Pallares, Maria Carmen; Kong, Yun; Iglesias-Fernandez, Javier; Bernardes, Goncalo J. L.; Peregrina, Jesus M.; Rovira, Carme; Bernado, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon
    Nature Communications (2015), 6 (), 6937CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying mol. basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling mol. dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering expts. further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.
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  35. 35
    de Las Rivas, M.; Lira-Navarrete, E.; Daniel, E. J. P.; Companon, I.; Coelho, H.; Diniz, A.; Jimenez-Barbero, J.; Peregrina, J. M.; Clausen, H.; Corzana, F.; Marcelo, F.; Jimenez-Oses, G.; Gerken, T. A.; Hurtado-Guerrero, R. The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences. Nat. Commun. 2017, 8, 1959,  DOI: 10.1038/s41467-017-02006-0
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    35
    The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences
    de Las Rivas Matilde; Lira-Navarrete Erandi; Hurtado-Guerrero Ramon; Lira-Navarrete Erandi; Clausen Henrik; Daniel Earnest James Paul; Gerken Thomas A; Companon Ismael; Peregrina Jesus M; Corzana Francisco; Jimenez-Oses Gonzalo; Coelho Helena; Diniz Ana; Marcelo Filipa; Coelho Helena; Jimenez-Barbero Jesus; Coelho Helena; Jimenez-Barbero Jesus; Jimenez-Barbero Jesus; Gerken Thomas A; Gerken Thomas A; Hurtado-Guerrero Ramon
    Nature communications (2017), 8 (1), 1959 ISSN:.
    The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- and/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2. The GalNAc-T4 structure bound to a monoglycopeptide shows that the GalNAc-binding site of its lectin domain is rotated relative to the homologous GalNAc-T2 structure, explaining their different long-range preferences. Kinetics and molecular dynamics simulations on several GalNAc-T2 flexible linker constructs show altered remote prior glycosylation preferences, confirming that the flexible linker dictates the rotation of the lectin domain, thus modulating the GalNAc-Ts' long-range preferences. This work for the first time provides the structural basis for the different remote prior glycosylation preferences of the GalNAc-Ts.
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  36. 36
    Wang, S.; Mao, Y.; Narimatsu, Y.; Ye, Z.; Tian, W.; Goth, C. K.; Lira-Navarrete, E.; Pedersen, N. B.; Benito-Vicente, A.; Martin, C.; Uribe, K. B.; Hurtado-Guerrero, R.; Christoffersen, C.; Seidah, N. G.; Nielsen, R.; Christensen, E. I.; Hansen, L.; Bennett, E. P.; Vakhrushev, S. Y.; Schjoldager, K. T.; Clausen, H. Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions. J. Biol. Chem. 2018, 293, 7408– 7422,  DOI: 10.1074/jbc.M117.817981
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    Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions
    Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K.; Lira-Navarrete, Erandi; Pedersen, Nis B.; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B.; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G.; Nielsen, Rikke; Christensen, Erik I.; Hansen, Lars; Bennett, Eric P.; Vakhrushev, Sergey Y.; Schjoldager, Katrine T.; Clausen, Henrik
    Journal of Biological Chemistry (2018), 293 (19), 7408-7422CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The low-d. lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomols. across cell membranes and barriers. Their functions are esp. relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomols. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites were selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrated that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation was not required for transport and cell-surface expression and stability of these receptors but markedly enhanced LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased the affinity for LDL by ∼5-fold. The mol. basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.
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  37. 37
    Raman, J.; Guan, Y.; Perrine, C. L.; Gerken, T. A.; Tabak, L. A. UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases: completion of the family tree. Glycobiology 2012, 22, 768– 777,  DOI: 10.1093/glycob/cwr183
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    UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases: Completion of the family tree
    Raman, Jayalakshmi; Guan, Yu; Perrine, Cynthia L.; Gerken, Thomas A.; Tabak, Lawrence A.
    Glycobiology (2012), 22 (6), 768-777CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
    The formation of mucin-type O-glycans is initiated by an evolutionarily conserved family of enzymes, the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). The human genome encodes 20 transferases; 17 of which have been characterized functionally. The complexity of the GalNAc-T family reflects the differential patterns of expression among the individual enzyme isoforms and the unique substrate specificities which are required to form the dense arrays of glycans that are essential for mucin function. We report the expression patterns and enzymic activity of the remaining three members of the family and the further characterization of a recently reported isoform, GalNAc-T17. One isoform, GalNAcT-16 that is most homologous to GalNAc-T14, is widely expressed (abundantly in the heart) and has robust polypeptide transferase activity. The second isoform GalNAc-T18, most similar to GalNAc-T8, -T9 and -T19, completes a discrete subfamily of GalNAc-Ts. It is widely expressed and has low, albeit detectable, activity. The final isoform, GalNAc-T20, is most homologous to GalNAc-T11 but lacks a lectin domain and has no detectable transferase activity with the panel of substrates tested. We have also identified and characterized enzymically active splice variants of GalNAc-T13 that differ in the sequence of their lectin domain. The variants differ in their affinities for glycopeptide substrates. Our findings provide a comprehensive view of the complexities of mucin-type O-glycan formation and provide insight into the underlying mechanisms employed to heavily decorate mucins and mucin-like domains with carbohydrate.
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  38. 38
    Revoredo, L.; Wang, S.; Bennett, E. P.; Clausen, H.; Moremen, K. W.; Jarvis, D. L.; Ten Hagen, K. G.; Tabak, L. A.; Gerken, T. A. Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family. Glycobiology 2016, 26, 360– 376,  DOI: 10.1093/glycob/cwv108
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    Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family
    Revoredo, Leslie; Wang, Shengjun; Bennett, Eric Paul; Clausen, Henrik; Moremen, Kelley W.; Jarvis, Donald L.; Ten Hagen, Kelly G.; Tabak, Lawrence A.; Gerken, Thomas A.
    Glycobiology (2016), 26 (4), 360-376CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
    A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities against glycopeptide substrates. All but one isoform contains a C-terminal carbohydrate-binding lectin domain whose roles in modulating glycopeptide specificity is just being understood. Study have previously shown for several peptide-preferring isoforms that the presence of a remote Thr-O-Gal-NAc, 6-17 residues from a Ser/Thr acceptor site, may enhance overall catalytic activity in an N- or C-terminal direction. This enhancement varies with isoform and is attributed to Thr-O-GalNAc interactions at the lectin domain. This work now report on the glycopeptide substrate utilization of a series of glycopeptide (human-ppGalNAc-T4, T7, T10, T12 and fly PGANT7) and peptide-preferring transferases (T2, T3 and T5) by exploiting a series of random glycopeptide substrates designed to probe the functions of their catalytic and lectin domains. Glycosylation was obsd. at the -3, -1 and +1 residues relative to a neighboring Thr-O-GalNAc, depending on isoform, which this study attribute to specific Thr-O-GalNAc binding at the catalytic domain. Addnl., these glycopeptide-preferring isoforms show remote lectin domain-assisted Thr-O-GalNAc enhancements that vary from modest to none. This paper conclude that the glycopeptide specificity of the glycopeptide-preferring isoforms predominantly resides in their catalytic domain but may be further modulated by remote lectin domain interactions. These studies further demonstrate that both domains of the ppGalNAc-Ts have specialized and unique functions that work in concert to control and order mucin-type O-glycosylation.
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    Fernandez, A. J.; Daniel, E. J. P.; Mahajan, S. P.; Gray, J. J.; Gerken, T. A.; Tabak, L. A.; Samara, N. L. The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 20404– 20410,  DOI: 10.1073/pnas.1902211116
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    The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function
    Fernandez, Amy J.; Paul Daniel, Earnest James; Mahajan, Sai Pooja; Gray, Jeffrey J.; Gerken, Thomas A.; Tabak, Lawrence A.; Samara, Nadine L.
    Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (41), 20404-20410CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochem. studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the mol. basis for this distinct substrate selectivity remains elusive. Here we examine the mol. basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addn., the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.
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  40. 40
    Pedersen, J. W.; Bennett, E. P.; Schjoldager, K. T.; Meldal, M.; Holmer, A. P.; Blixt, O.; Clo, E.; Levery, S. B.; Clausen, H.; Wandall, H. H. Lectin domains of polypeptide GalNAc transferases exhibit glycopeptide binding specificity. J. Biol. Chem. 2011, 286, 32684– 32696,  DOI: 10.1074/jbc.M111.273722
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    Lectin Domains of Polypeptide GalNAc Transferases Exhibit Glycopeptide Binding Specificity
    Pedersen, Johannes W.; Bennett, Eric P.; Schjoldager, Katrine T.-B. G.; Meldal, Morten; Holmer, Andreas P.; Blixt, Ola; Clo, Emiliano; Levery, Steven B.; Clausen, Henrik; Wandall, Hans H.
    Journal of Biological Chemistry (2011), 286 (37), 32684-32696, S32684/1-S32684/2CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, resp. Thus far, only the catalytic domain has been shown to have peptide sequence specificity, whereas the primary function of the lectin domain is to increase affinity to previously glycosylated substrates. Whether the lectin domain also has peptide sequence selectivity has remained unclear. Using a glycopeptide array with a library of synthetic and recombinant glycopeptides based on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examd. the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate an addnl. level of complexity in the initiation step of O-glycosylation by GalNAc-Ts.
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  41. 41
    Coelho, H.; Rivas, M. d. l.; Grosso, A. S.; Diniz, A.; Soares, C. O.; Francisco, R. A.; Dias, J. S.; Companon, I.; Sun, L.; Narimatsu, Y.; Vakhrushev, S. Y.; Clausen, H.; Cabrita, E. J.; Jimenez-Barbero, J.; Corzana, F.; Hurtado-Guerrero, R.; Marcelo, F. Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling. JACS Au 2022, 2, 631– 645,  DOI: 10.1021/jacsau.1c00529
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    41
    Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling
    Coelho, Helena; Rivas, Matilde de las; Grosso, Ana S.; Diniz, Ana; Soares, Catia O.; Francisco, Rodrigo A.; Dias, Jorge S.; Companon, Ismael; Sun, Lingbo; Narimatsu, Yoshiki; Vakhrushev, Sergey Y.; Clausen, Henrik; Cabrita, Eurico J.; Jimenez-Barbero, Jesus; Corzana, Francisco; Hurtado-Guerrero, Ramon; Marcelo, Filipa
    JACS Au (2022), 2 (3), 631-645CODEN: JAAUCR; ISSN:2691-3704. (American Chemical Society)
    The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by mol. modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addn. of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our expts. suggest that during this process, neighboring tandem repeats are crit. for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addn., in-cell anal. shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodol. established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to det. the specificity toward complex mucin acceptor substrates.
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    Corzana, F.; Busto, J. H.; Jiménez-Osés, G.; Asensio, J. L.; Jiménez-Barbero, J.; Peregrina, J. M.; Avenoza, A. New Insights into α-GalNAc–Ser Motif: Influence of Hydrogen Bonding versus Solvent Interactions on the Preferred Conformation. J. Am. Chem. Soc. 2006, 128, 14640– 14648,  DOI: 10.1021/ja064539u
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    New Insights into α-GalNAc-Ser Motif: Influence of Hydrogen Bonding versus Solvent Interactions on the Preferred Conformation
    Corzana, Francisco; Busto, Jesus H.; Jimenez-Oses, Gonzalo; Asensio, Juan L.; Jimenez-Barbero, Jesus; Peregrina, Jesus M.; Avenoza, Alberto
    Journal of the American Chemical Society (2006), 128 (45), 14640-14648CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The structural features of the mucin-type simplest model, namely, the glycopeptide α-O-GalNAc-L-Ser diamide, have been investigated by combining NMR spectroscopy, mol. dynamics simulations, and DFT calcns. In contrast to previous reports, the study reveals that intramol. hydrogen bonds between sugar and peptide residues are very weak and, as a consequence, not strong enough to maintain the well-defined conformation of this type of mol. In fact, the obsd. conformation of this model glycopeptide can be satisfactorily explained by the presence of water pockets/bridges between the sugar and the peptide moieties. Addnl., DFT calcns. reveal that not only the bridging water mols. but also the surrounding water mols. in the first hydration shell are essential to keep the existing conformation.
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  43. 43
    Shon, D. J.; Fernandez, D.; Riley, N. M.; Ferracane, M. J.; Bertozzi, C. R. Structure-guided mutagenesis of a mucin-selective metalloprotease from Akkermansia muciniphila alters substrate preferences. J. Biol. Chem. 2022, 298, 101917,  DOI: 10.1016/j.jbc.2022.101917
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    Structure-guided mutagenesis of a mucin-selective metalloprotease from Akkermansia muciniphila alters substrate preferences
    Shon, D. Judy; Fernandez, Daniel; Riley, Nicholas M.; Ferracane, Michael J.; Bertozzi, Carolyn R.
    Journal of Biological Chemistry (2022), 298 (5), 101917CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
    Akkermansia muciniphila, a mucin-degrading microbe found in the human gut, is often assocd. with pos. health outcomes. The abundance of A. muciniphila is modulated by the presence and accessibility of nutrients, which can be derived from diet or host glycoproteins. In particular, the ability to degrade host mucins, a class of proteins carrying densely O-glycosylated domains, provides a competitive advantage in the sustained colonization of niche mucosal environments. Although A. muciniphila is known to rely on mucins as a carbon and nitrogen source, the enzymic machinery used by this microbe to process mucins in the gut is not yet fully characterized. Here, we focus on the mucin-selective metalloprotease, Amuc_0627 (AM0627), which is known to cleave between adjacent residues carrying truncated core 1 O-glycans. We showed that this enzyme is capable of degrading purified mucin 2 (MUC2), the major protein component of mucus in the gut. An X-ray crystal structure of AM0627 (1.9 Å resoln.) revealed O-glycan-binding residues that are conserved between structurally characterized enzymes from the same family. We further rationalized the substrate cleavage motif using mol. modeling to identify nonconserved glycan-interacting residues. We conclude that mutagenesis of these residues resulted in altered substrate preferences down to the glycan level, providing insight into the structural determinants of O-glycan recognition.
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  44. 44
    Konstantinidi, A.; Nason, R.; Caval, T.; Sun, L.; Soerensen, D. M.; Furukawa, S.; Ye, Z.; Vincentelli, R.; Narimatsu, Y.; Vakhrushev, S. Y.; Clausen, H. Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells. J. Biol. Chem. 2022, 298, 101784,  DOI: 10.1016/j.jbc.2022.101784
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    44
    Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells
    Konstantinidi, Andriana; Nason, Rebecca; Caval, Tomislav; Sun, Lingbo; Soerensen, Daniel M.; Furukawa, Sanae; Ye, Zilu; Vincentelli, Renaud; Narimatsu, Yoshiki; Vakhrushev, Sergey Y.; Clausen, Henrik
    Journal of Biological Chemistry (2022), 298 (4), 101784CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
    Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and prodn. of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific anal. for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom-up anal. of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider anal. of native mucins.
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  45. 45
    Zhou, D.; Xu, L.; Huang, W.; Tonn, T. Epitopes of MUC1 Tandem Repeats in Cancer as Revealed by Antibody Crystallography: Toward Glycopeptide Signature-Guided Therapy. Molecules 2018, 23, 1326,  DOI: 10.3390/molecules23061326
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    45
    Epitopes of MUC1 tandem repeats in cancer as revealed by antibody crystallography: toward glycopeptide signature-guided therapy
    Zhou, Dapeng; Xu, Lan; Huang, Wei; Tonn, Torsten
    Molecules (2018), 23 (6), 1326/1-1326/27CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    Abnormally O-glycosylated MUC1 tandem repeat glycopeptide epitopes expressed by multiple types of cancer have long been attractive targets for therapy in the race against genetic mutations of tumor cells. Glycopeptide signature-guided therapy might be a more promising avenue than mutation signature-guided therapy. Three O-glycosylated peptide motifs, PDTR, GSTA, and GVTS, exist in a tandem repeat HGVTSAPDTRPAPGSTAPPA, contg. five O-glycosylation sites. The exact peptide and sugar residues involved in antibody binding are poorly defined. Co-crystal structures of glycopeptides and resp. monoclonal antibodies are very few. Here we review 3 groups of monoclonal antibodies: antibodies which only bind to peptide portion, antibodies which only bind to sugar portion, and antibodieswhich bind to both peptide and sugar portions. The antigenicity of peptide and sugar portions of glyco-MUC1 tandemrepeat were analyzed according to available biochem. and structural data, esp. the GSTA and GVTS motifs independent from the most studied PDTR. Tn is focused as a peptide-modifying residue in vaccine design, to induce glycopeptide-binding antibodies with cross reactivity to Tn-related tumor glycans, but not glycans of healthy cells. The unique requirement for the designs of antibody in antibody-drug conjugate, bi-specific antibodies, and chimeric antigen receptors are also discussed.
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  46. 46
    Posey, A. D.; Schwab, R. D.; Boesteanu, A. C.; Steentoft, C.; Mandel, U.; Engels, B.; Stone, J. D.; Madsen, T. D.; Schreiber, K.; Haines, K. M.; Cogdill, A. P.; Chen, T. J.; Song, D.; Scholler, J.; Kranz, D. M.; Feldman, M. D.; Young, R.; Keith, B.; Schreiber, H.; Clausen, H.; Johnson, L. A.; June, C. H. Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma. Immunity 2016, 44, 1444– 1454,  DOI: 10.1016/j.immuni.2016.05.014
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    46
    Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma
    Posey, Avery D. Jr.; Schwab, Robert D.; Boesteanu, Alina C.; Steentoft, Catharina; Mandel, Ulla; Engels, Boris; Stone, Jennifer D.; Madsen, Thomas D.; Schreiber, Karin; Haines, Kathleen M.; Cogdill, Alexandria P.; Chen, Taylor J.; Song, Decheng; Scholler, John; Kranz, David M.; Feldman, Michael D.; Young, Regina; Keith, Brian; Schreiber, Hans; Clausen, Henrik; Johnson, Laura A.; June, Carl H.
    Immunity (2016), 44 (6), 1444-1454CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)
    Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematol. cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-assocd. Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.
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  47. 47
    Karsten, U. Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1). Glycobiology 2004, 14, 681– 692,  DOI: 10.1093/glycob/cwh090
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    47
    Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1)
    Karsten, Uwe; Serttas, Nida; Paulsen, Hans; Danielczyk, Antje; Goletz, Steffen
    Glycobiology (2004), 14 (8), 681-692CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
    Glycosylation dets. essential biol. functions of epithelial mucins in health and disease. We report on the influence of glycosylation of the immunodominant DTR motif of MUC1 on its antigenicity. Sets of novel glycopeptides were synthesized that enabled us to examine sole and combined effects of peptide length (no. of repeats) and O-glycosylation with GalNAc at the DTR motif on the binding patterns of 22 monoclonal antibodies recognizing this motif. In case of unglycosylated peptides almost all antibodies bound better to multiple MUC1 tandem repeats. Glycosylation at the DTR led to enhanced binding in 11 cases, whereas 10 antibodies were not influenced in binding, and one was inhibited. In nine of the former cases both length and DTR glycosylation were additive in their influence on antibody binding, suggesting that both effects are different. Improved binding to the glycosylated DTR motif was exclusively found with antibodies generated against tumor-derived MUC1. Based on these data a tumor-specific MUC1 epitope is defined comprising the ...PDTRP... sequence in a particular conformation essentially detd. by O-glycosylation at its threonine with either GalNAcα1 or a related short glycan. The results can find application in the field of MUC1-based immunotherapy.
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  48. 48
    Brooks, C. L.; Schietinger, A.; Borisova, S. N.; Kufer, P.; Okon, M.; Hirama, T.; Mackenzie, C. R.; Wang, L. X.; Schreiber, H.; Evans, S. V. Antibody recognition of a unique tumor-specific glycopeptide antigen. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10056– 10061,  DOI: 10.1073/pnas.0915176107
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    48
    Antibody recognition of a unique tumor-specific glycopeptide antigen
    Brooks, Cory L.; Schietinger, Andrea; Borisova, Svetlana N.; Kufer, Peter; Okon, Mark; Hirama, Tomoko; MacKenzie, C. Roger; Wang, Lai-Xi; Schreiber, Hans; Evans, Stephen V.
    Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (22), 10056-10061, S10056/1-S10056/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Aberrant glycosylation and the over expression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-assocd. oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the "Tn antigen.". Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been obsd. on embryonic and non-malignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide contg. the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no obsd. physiol. cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of antigen.
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    Movahedin, M.; Brooks, T. M.; Supekar, N. T.; Gokanapudi, N.; Boons, G.-J.; Brooks, C. L. Glycosylation of MUC1 influences the binding of a therapeutic antibody by altering the conformational equilibrium of the antigen. Glycobiology 2016, 27, 677– 687,  DOI: 10.1093/glycob/cww131
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    Martínez-Sáez, N.; Castro-Lopez, J.; Valero-Gonzalez, J.; Madariaga, D.; Companon, I.; Somovilla, V. J.; Salvado, M.; Asensio, J. L.; Jimenez-Barbero, J.; Avenoza, A.; Busto, J. H.; Bernardes, G. J. L.; Peregrina, J. M.; Hurtado-Guerrero, R.; Corzana, F. Deciphering the non-equivalence of serine and threonine O-glycosylation points: Implications for molecular recognition of the Tn antigen by an anti-MUC1 antibody. Angew. Chem., Int. Ed. 2015, 54, 9830– 9834,  DOI: 10.1002/anie.201502813
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    50
    Deciphering the non-equivalence of serine and threonine O-glycosylation points: Implications for molecular recognition of the Tn antigen by an anti-MUC1 antibody
    Martinez-Saez, Nuria; Castro-Lopez, Jorge; Valero-Gonzalez, Jessika; Madariaga, David; Companon, Ismael; Somovilla, Victor J.; Salvado, Miriam; Asensio, Juan L.; Jimenez-Barbero, Jesus; Avenoza, Alberto; Busto, Jesus H.; Bernardes, Goncalo J. L.; Peregrina, Jesus M.; Hurtado-Guerrero, Ramon; Corzana, Francisco
    Angewandte Chemie, International Edition (2015), 54 (34), 9830-9834CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at at. resoln. For the α-O-GalNAc-Ser deriv., the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also obsd. in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the Me group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in mol. recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.
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  51. 51
    Coelho, H.; Matsushita, T.; Artigas, G.; Hinou, H.; Cañada, F. J.; Lo-Man, R.; Leclerc, C.; Cabrita, E. J.; Jiménez-Barbero, J.; Nishimura, S.-I.; Garcia-Martín, F.; Marcelo, F. The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR. J. Am. Chem. Soc. 2015, 137, 12438– 12441,  DOI: 10.1021/jacs.5b06787
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    51
    The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR
    Coelho, Helena; Matsushita, Takahiko; Artigas, Gerard; Hinou, Hiroshi; Canada, F. Javier; Lo-Man, Richard; Leclerc, Claude; Cabrita, Eurico J.; Jimenez-Barbero, Jesus; Nishimura, Shin-Ichiro; Garcia-Martin, Fayna; Marcelo, Filipa
    Journal of the American Chemical Society (2015), 137 (39), 12438-12441CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The identification of MUC1 tumor-assocd. Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and satn. transfer difference NMR, the authors have characterized the fine-epitope mapping of a MUC1 chem. library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides contg. the Tn-Ser antigen rather than the Tn-Thr analog, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.
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  52. 52
    Wakui, H.; Tanaka, Y.; Ose, T.; Matsumoto, I.; Kato, K.; Min, Y.; Tachibana, T.; Sato, M.; Naruchi, K.; Martin, F. G.; Hinou, H.; Nishimura, S.-I. A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes. Chem. Sci. 2020, 11, 4999– 5006,  DOI: 10.1039/D0SC00317D
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    A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes
    Wakui, Hajime; Tanaka, Yoshikazu; Ose, Toyoyuki; Matsumoto, Isamu; Kato, Koji; Min, Yao; Tachibana, Taro; Sato, Masaharu; Naruchi, Kentaro; Martin, Fayna Garcia; Hinou, Hiroshi; Nishimura, Shin-Ichiro
    Chemical Science (2020), 11 (19), 4999-5006CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Aberrantly truncated immature O-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly assocd. with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immature O-glycans, no anti-MUC1 antibody recognizes carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure anal., epitope mapping and biochem. anal. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognizes specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its soln. NMR structure, an extended conformation induced by site-specific glycosylation.
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  53. 53
    Macias-Leon, J.; Bermejo, I. A.; Asin, A.; Garcia-Garcia, A.; Companon, I.; Jimenez-Moreno, E.; Coelho, H.; Mangini, V.; Albuquerque, I. S.; Marcelo, F.; Asensio, J. L.; Bernardes, G. J. L.; Joshi, H. J.; Fiammengo, R.; Blixt, O.; Hurtado-Guerrero, R.; Corzana, F. Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody. Chem. Commun. 2020, 56, 15137– 15140,  DOI: 10.1039/D0CC06349E
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    53
    Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody
    Macias-Leon, Javier; Bermejo, Iris A.; Asin, Alicia; Garcia-Garcia, Ana; Companon, Ismael; Jimenez-Moreno, Ester; Coelho, Helena; Mangini, Vincenzo; Albuquerque, Ines S.; Marcelo, Filipa; Asensio, Juan L.; Bernardes, Goncalo J. L.; Joshi, Hiren J.; Fiammengo, Roberto; Blixt, Ola; Hurtado-Guerrero, Ramon; Corzana, Francisco
    Chemical Communications (Cambridge, United Kingdom) (2020), 56 (96), 15137-15140CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    The mol. basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chem. design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.
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  54. 54
    Yoshimura, Y.; Denda-Nagai, K.; Takahashi, Y.; Nagashima, I.; Shimizu, H.; Kishimoto, T.; Noji, M.; Shichino, S.; Chiba, Y.; Irimura, T. Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies. Sci. Rep. 2019, 9, 16641,  DOI: 10.1038/s41598-019-53052-1
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    54
    Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies
    Yoshimura Yayoi; Kishimoto Toshimitsu; Yoshimura Yayoi; Takahashi Yoshie; Chiba Yasunori; Denda-Nagai Kaori; Noji Miki; Irimura Tatsuro; Nagashima Izuru; Shimizu Hiroki; Shichino Shigeyuki
    Scientific reports (2019), 9 (1), 16641 ISSN:.
    Anti-mucin1 (MUC1) antibodies have long been used clinically in cancer diagnosis and therapy and specific bindings of some of them are known to be dependent on the differential glycosylation of MUC1. However, a systematic comparison of the binding specificities of anti-MUC1 antibodies was not previously conducted. Here, a total of 20 glycopeptides including the tandem repeat unit of MUC1, APPAHGVTSAPDTRPAPGSTAPPAHGV with GalNAc (Tn-antigen), Galβ1-3GalNAc (T-antigen), NeuAcα2-3Galβ1-3GalNAc (sialyl-T-antigen), or NeuAcα2-6GalNAc (sialyl-Tn-antigen) at each threonine or serine residue were prepared by a combination of chemical glycopeptide synthesis and enzymatic extension of carbohydrate chains. These glycopeptides were tested by the enzyme-linked immunosorbent assay (ELISA) for their capacity to bind 13 monoclonal antibodies (mAbs) known to be specific for MUC1. The results indicated that anti-MUC1 mAbs have diverse specificities but can be classified into a few characteristic groups based on their binding pattern toward glycopeptides in some cases having a specific glycan at unique glycosylation sites. Because the clinical significance of some of these antibodies was already established, the structural features identified by these antibodies as revealed in the present study should provide useful information relevant to their further clinical use and the biological understanding of MUC1.
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  55. 55
    Corzana, F.; Busto, J. H.; Jiménez-Osés, G.; García De Luis, M.; Asensio, J. L.; Jiménez-Barbero, J.; Peregrina, J. M.; Avenoza, A. Serine versus Threonine Glycosylation: The Methyl Group Causes a Drastic Alteration on the Carbohydrate Orientation and on the Surrounding Water Shell. J. Am. Chem. Soc. 2007, 129, 9458– 9467,  DOI: 10.1021/ja072181b
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    Serine versus Threonine Glycosylation: The Methyl Group Causes a Drastic Alteration on the Carbohydrate Orientation and on the Surrounding Water Shell
    Corzana, Francisco; Busto, Jesus H.; Jimenez-Oses, Gonzalo; Garcia de Luis, Marisa; Asensio, Juan L.; Jimenez-Barbero, Jesus; Peregrina, Jesus M.; Avenoza, Alberto
    Journal of the American Chemical Society (2007), 129 (30), 9458-9467CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Different behavior has been obsd. for the ψ torsion angle of the glycosidic linkages of D-GalNAc-Ser and D-GalNAc-Thr motifs, allowing the carbohydrate moiety to adopt a completely different orientation. In addn., the fact that the water pockets found in α-D-GalNAc-Thr differ from those obtained for its serine analog could be related to the different capability that the two model glycopeptides have to structure the surrounding water. This fact could have important biol. inferences (i.e., antifreeze activity).
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    (a) Clark, D.; Mao, L. Cancer biomarker discovery: lectin-based strategies targeting glycoproteins. Dis. Markers 2012, 33, 1– 10,  DOI: 10.1155/2012/308738
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    Cancer biomarker discovery: Lectin-based strategies targeting glycoproteins
    Clark, David; Mao, Li
    Disease Markers (2012), 33 (1), 1-10CODEN: DMARD3; ISSN:0278-0240. (IOS Press)
    A review. Biomarker discovery can identify mol. markers in various cancers that can be used for detection, screening, diagnosis, and monitoring of disease progression. Lectin-affinity is a technique that can be used for the enrichment of glycoproteins from a complex sample, facilitating the discovery of novel cancer biomarkers assocd. with a disease state.
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    (b) Lastovickova, M.; Strouhalova, D.; Bobalova, J. Use of Lectin-based Affinity Techniques in Breast Cancer Glycoproteomics: A Review. J. Proteome Res. 2020, 19, 1885– 1899,  DOI: 10.1021/acs.jproteome.9b00818
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    Use of Lectin-based Affinity Techniques in Breast Cancer Glycoproteomics: A Review
    Lastovickova, Marketa; Strouhalova, Dana; Bobalova, Janette
    Journal of Proteome Research (2020), 19 (5), 1885-1899CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    A review. Changes in glycoprotein content, altered glycosylations, and aberrant glycan structures are increasingly recognized as cancer hallmarks. Because breast cancer is one of the most common causes of cancer deaths in the world, it is highly urgent to find other reliable biomarkers for its initial diagnosis and to learn as much as possible about this disease. In this Review, the applications of lectins to a screening of potential breast cancer biomarkers published during recent years are overviewed. These data provide a deeper insight into the use of modern strategies, technologies, and scientific knowledge in glycoproteomic breast cancer research. Particular attention is concd. on the use of lectin-based affinity techniques, applied independently or most frequently in combination with mass spectrometry, as an effective tool for the targeting, sepn., and reliable identification of glycoprotein mols. Individual procedures and lectins used in published glycoproteomic studies of breast-cancer-related glycoproteins are discussed. The summarized approaches have the potential for use in diagnostic and predictive applications. Finally, the use of lectins is briefly discussed from the view of their future applications in the anal. of glycoproteins in cancer.
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    de Oliveira Figueirôa, E.; Albuquerque da Cunha, C. R.; Albuquerque, P. B. S.; de Paula, R. A.; Aranda-Souza, M. A.; Alves, M. S.; Zagmignan, A.; Carneiro-da-Cunha, M. G.; Nascimento da Silva, L. C.; dos Santos Correia, M. T. Lectin-carbohydrate interactions: Implications for the development of new anticancer agents. Curr. Med. Chem. 2017, 24, 3667– 3680,  DOI: 10.2174/0929867324666170523110400
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    57
    Lectin-Carbohydrate Interactions: Implications for the Development of New Anticancer Agents
    de Oliveira Figueiroa, Evellyne; Albuquerque da Cunha, Cassia Regina; Albuquerque, Priscilla B. S.; de Paula, Raiana Apolinario; Aranda-Souza, Mary Angela; Alves, Matheus Silva; Zagmignan, Adrielle; Carneiro-da-Cunha, Maria G.; Nascimento da Silva, Luis Claudio; dos Santos Correia, Maria Tereza
    Current Medicinal Chemistry (2017), 24 (34), 3667-3680CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
    Lectins are a large group of proteins found in animals, plants, fungi, and bacteria that recognize specific carbohydrate targets and play an important role in cell recognition and communication, host-pathogen interactions, embryogenesis, and tissue development. Recently, lectins have emerged as important biomedical tools that have been used in the development of immunomodulatory, antipathogenic, and anticancer agents. Several lectins have been shown to have the ability to discriminate between normal cells and tumor cells as a result of their different glycosylation patterns. Furthermore, the specific binding of lectins to cancer cells has been shown to trigger mechanisms that can promote the death of these abnormal cells. Here, we review the importance of lectins-carbohydrates interactions in cancer therapy and diagnosis. We examine the use of lectins in the modification of nanoparticles (liposomes, solid lipid nanoparticles and other polymers) for anticancer drug delivery. The development of drug delivery systems (liposomes, alginate/chitosan microcapsules, alginate beads) carrying some antitumor lectins is also discussed. In these cases, the processes of cell death induced by these antitumor lectins were also showed (if available). In both cases (lectin-conjugated polymers or encapsulated lectins), these new pharmaceutical prepns. showed improved intracellular delivery, bioavailability and targetability leading to enhanced therapeutic index and significantly less side effects.
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    Gabba, A.; Bogucka, A.; Luz, J. G.; Diniz, A.; Coelho, H.; Corzana, F.; Cañada, F. J.; Marcelo, F.; Murphy, P. V.; Birrane, G. Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen. Biochemistry 2021, 60, 1327– 1336,  DOI: 10.1021/acs.biochem.1c00009
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    Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen
    Gabba, Adele; Bogucka, Agnieszka; Luz, John G.; Diniz, Ana; Coelho, Helena; Corzana, Francisco; Canada, Francisco Javier; Marcelo, Filipa; Murphy, Paul V.; Birrane, Gabriel
    Biochemistry (2021), 60 (17), 1327-1336CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The human macrophage galactose lectin (MGL) is an endocytic type ii transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N-acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins were identified, the mol. basis for substrate recognition has remained elusive due to the lack of high-resoln. structures. Here the authors present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-assocd. Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, addnl. calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the mol. basis for preferential binding of N-acetylgalactosamine over galactose and prompted the reevaluation of the binding modes previously proposed in soln. Satn. transfer difference NMR data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in soln. Models of MGL1 and MGL2, the mouse homologs of MGL, explain how these proteins might recognize LewisX and GalNAc, resp.
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    Lescar, J.; Sanchez, J.-F.; Audfray, A.; Coll, J.-L.; Breton, C.; Mitchell, E. P.; Imberty, A. Structural basis for recognition of breast and colon cancer epitopes Tn antigen and Forssman disaccharide by Helix pomatia lectin. Glycobiology 2007, 17, 1077– 1083,  DOI: 10.1093/glycob/cwm077
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    Structural basis for recognition of breast and colon cancer epitopes Tn antigen and Forssman disaccharide by Helix pomatia lectin
    Lescar, Julien; Sanchez, Jean-Frederic; Audfray, Aymeric; Coll, Jean-Luc; Breton, Christelle; Mitchell, Edward P.; Imberty, Anne
    Glycobiology (2007), 17 (10), 1077-1083CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
    Helix pomatia agglutinin (HPA) is a lectin that has been used extensively in histopathol., since its binding to tissue sections from breast and colon cancers is correlated with the worst prognosis for the patients. The lectin recognizes α-D-N-acetylgalactosamine (αGalNAc) contg. epitopes which are only present in cancer cell lines having a high likelihood to undergo metastasis, such as the HT29 cancer colon cell line. Several breast cancer cell lines have also been shown to be labeled, although IGROV1, an ovarian cancer cell line, is not. Inhibition studies, using GalNAc monosaccharides, are reported here, showing that the labeling is dependent upon the presence of carbohydrate epitopes. The crystal structures of the lectin complexed with two GalNAc contg. epitopes assocd. with cancer, the Tn (αGalNAc-Ser) and Forssman (αGalNAc1-3GalNAc) antigens, show the lectin's specificity for GalNAc is due to a particular network of hydrogen bonds. A histidine residue makes hydrophobic contact with the aglycon, rationalizing the preference for GalNAc bearing an addnl. sugar or amino acid in the α position. These structures provide the mol. basis for the use of HPA in metastasis research.
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    Maveyraud, L.; Niwa, H.; Guillet, V.; Svergun, D. I.; Konarev, P. V.; Palmer, R. A.; Peumans, W. J.; Rougé, P.; Van Damme, E. J.; Reynolds, C. D.; Mourey, L. Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra. Proteins 2009, 75, 89– 103,  DOI: 10.1002/prot.22222
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    Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra
    Maveyraud, Laurent; Niwa, Hideaki; Guillet, Valerie; Svergun, Dmitri I.; Konarev, Peter V.; Palmer, Rex A.; Peumans, Willy J.; Rouge, Pierre; Van Damme, Els J. M.; Reynolds, Colin D.; Mourey, Lionel
    Proteins: Structure, Function, and Bioinformatics (2009), 75 (1), 89-103CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
    Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional x-ray structure of SNA-II has been detd. in two distinct crystal forms, hexagonal and tetragonal, at 1.90 Å and 1.35 Å, resp. In both crystal forms, the SNA-II mol. folds into two linked β-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is obsd. at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resoln. structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, α1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were detd. at 1.55 Å resoln. or better. Binding is obsd. in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, addnl. interactions with the protein involve the O6 atom. Anal. gel filtration, small angle x-ray scattering studies and crystal packing anal. indicate that, although some oligomeric species are present, the monomeric species predominate in soln.
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  61. 61
    Kulkarni, K. A.; Sinha, S.; Katiyar, S.; Surolia, A.; Vijayan, M.; Suguna, K. Structural basis for the specificity of basic winged bean lectin for the Tn-antigen: A crystallographic, thermodynamic and modelling study. FEBS Lett. 2005, 579, 6775– 6780,  DOI: 10.1016/j.febslet.2005.11.011
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    Structural basis for the specificity of basic winged bean lectin for the Tn-antigen: A crystallographic, thermodynamic and modelling study
    Kulkarni, Kiran A.; Sinha, Sharmistha; Katiyar, Samiksha; Surolia, Avadhesha; Vijayan, Mamannamana; Suguna, Kaza
    FEBS Letters (2005), 579 (30), 6775-6780CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
    The crystal structure of winged bean basic agglutinin in complex with GalNAc-α-O-Ser (Tn-antigen) has been elucidated at 2.35 Å resoln. in order to characterize the mode of binding of Tn-antigen with the lectin. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its Me group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qual. explanation for the thermodn. parameters of the complexation of the lectin with Tn-antigen. Modeling studies indicate the possibility of an addnl. hydrogen bond with the lectin when the antigen is part of a glycoprotein.
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  62. 62
    Sousa, B. L.; Silva Filho, J. C.; Kumar, P.; Pereira, R. I.; Łyskowski, A.; Rocha, B. A.; Delatorre, P.; Bezerra, G. A.; Nagano, C. S.; Gruber, K.; Cavada, B. S. High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins. Int. J. Biochem. Cell Biol. 2015, 59, 103– 110,  DOI: 10.1016/j.biocel.2014.12.002
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    High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins
    Sousa, Bruno Lopes; Silva Filho, Jose Caetano; Kumar, Prashant; Pereira, Ronniery Ilario; Lyskowski, Andrzej; Rocha, Bruno Anderson Matias; Delatorre, Plinio; Bezerra, Gustavo Arruda; Nagano, Celso Shiniti; Gruber, Karl; Cavada, Benildo Sousa
    International Journal of Biochemistry & Cell Biology (2015), 59 (), 103-110CODEN: IJBBFU; ISSN:1357-2725. (Elsevier Ltd.)
    Plant lectins have been studied as histol. markers and promising antineoplastic mols. for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been detd. at the resoln. of 1.4 Å and 1.7 Å, resp. Mol. docking anal. of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addn. to the specific biol. effects of VML, the structural and binding similarities between it and other lectins commonly used as histol. markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.
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  63. 63
    Lubkowski, J.; Durbin, S. V.; Silva, M. C. C.; Farnsworth, D.; Gildersleeve, J. C.; Oliva, M. L. V.; Wlodawer, A. Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth. FEBS J. 2017, 284, 429– 450,  DOI: 10.1111/febs.13989
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    63
    Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth
    Lubkowski, Jacek; Durbin, Sarah V.; Silva, Mariana C. C.; Farnsworth, David; Gildersleeve, Jeffrey C.; Oliva, Maria Luiza V.; Wlodawer, Alexander
    FEBS Journal (2017), 284 (3), 429-450CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
    Lectins have been used at length for basic research and clin. applications. New insights into the mol. recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallog. to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in soln. and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have esp. strong binding (apparent Kd = 0.5-1.0 nM) to the tumor-assocd. Tn antigen. High-resoln. crystal structures were detd. for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive anal. of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the mol. recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. Database : Structural data are available in the PDB under the accession nos. , , , , , , , and .
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  64. 64
    Madariaga, D.; Martínez-Sáez, N.; Somovilla, V. J.; Coelho, H.; Valero-González, J.; Castro-López, J.; Asensio, J. L.; Jiménez-Barbero, J.; Busto, J. H.; Avenoza, A.; Marcelo, F.; Hurtado-Guerrero, R.; Corzana, F.; Peregrina, J. M. Detection of tumor-associated glycopeptides by lectins: the peptide context modulates carbohydrate recognition. ACS Chem. Biol. 2015, 10, 747– 756,  DOI: 10.1021/cb500855x
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    64
    Detection of Tumor-Associated Glycopeptides by Lectins: The Peptide Context Modulates Carbohydrate Recognition
    Madariaga, David; Martinez-Saez, Nuria; Somovilla, Victor J.; Coelho, Helena; Valero-Gonzalez, Jessika; Castro-Lopez, Jorge; Asensio, Juan L.; Jimenez-Barbero, Jesus; Busto, Jesus H.; Avenoza, Alberto; Marcelo, Filipa; Hurtado-Guerrero, Ramon; Corzana, Francisco; Peregrina, Jesus M.
    ACS Chemical Biology (2015), 10 (3), 747-756CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is obsd. when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide-lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.
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  65. 65
    Madariaga, D.; Martínez-Sáez, N.; Somovilla, V. J.; García-García, L.; Berbis, M. Á.; Valero-Gónzalez, J.; Martín-Santamaría, S.; Hurtado-Guerrero, R.; Asensio, J. L.; Jiménez-Barbero, J.; Avenoza, A.; Busto, J. H.; Corzana, F.; Peregrina, J. M. Serine versus Threonine Glycosylation with α-O-GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins. Chem. Eur. J. 2014, 20, 12616– 12627,  DOI: 10.1002/chem.201403700
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    65
    Serine versus Threonine Glycosylation with α-O-GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins
    Madariaga, David; Martinez-Saez, Nuria; Somovilla, Victor J.; Garcia-Garcia, Laura; Berbis, M. Alvaro; Valero-Gonzalez, Jessika; Martin-Santamaria, Sonsoles; Hurtado-Guerrero, Ramon; Asensio, Juan L.; Jimenez-Barbero, Jesus; Avenoza, Alberto; Busto, Jesus H.; Corzana, Francisco; Peregrina, Jesus M.
    Chemistry - A European Journal (2014), 20 (39), 12616-12627CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The mol. recognition of several glycopeptides bearing Tn antigen (α-O-GalNAc-Ser or α-O-GalNAc-Thr) in their structure by three lectins with affinity for this determinant has been analyzed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the mol. recognition. In fact, while Soybean agglutinin (SBA) and Vicia villosa agglutinin (VVA) lectins prefer Tn-threonine, Helix pomatia agglutinin (HPA) shows a higher affinity for the glycopeptides carrying Tn-serine. The different conformational behavior of the two Tn biol. entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topol. of the binding site of the lectins are at the origin of these differences.
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  66. 66
    Gibadullin, R.; Farnsworth, D. W.; Barchi, J. J.; Gildersleeve, J. C. GalNAc-Tyrosine Is a Ligand of Plant Lectins, Antibodies, and Human and Murine Macrophage Galactose-Type Lectins. ACS Chem. Biol. 2017, 12, 2172– 2182,  DOI: 10.1021/acschembio.7b00471
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    66
    GalNAc-Tyrosine Is a Ligand of Plant Lectins, Antibodies, and Human and Murine Macrophage Galactose-Type Lectins
    Gibadullin, Ruslan; Farnsworth, David Wayne; Barchi, Joseph J.; Gildersleeve, Jeffrey C.
    ACS Chemical Biology (2017), 12 (8), 2172-2182CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    In 2011, a new type of protein O-glycosylation was discovered in which N-acetylgalactosamine is attached to the side chain of tyrosine (GalNAc-Tyr). While present on dozens of proteins, the biol. roles of GalNAc-Tyr are unknown. To gain insight into this new type of modification, we synthesized a group of GalNAc-Tyr glycopeptides, constructed microarrays, and evaluated potential recognition of GalNAc-Tyr by a series of glycan-binding proteins. Through a series of >150 microarray expts., we assessed binding properties of a variety of plant lectins, monoclonal antibodies, and endogenous lectins. VVL, HPA, and SBA were all found to bind tightly to GalNAc-Tyr, and several Tn binding antibodies and blood group A antibodies were found to cross-react with GalNAc-Tyr. Thus, detection of GalNAc-Tyr modified proteins is an important consideration when analyzing results from these reagents. Addnl., we evaluated potential recognition by two mammalian lectins, human (hMGL) and murine (mMGL-2) macrophage galactose type C-type lectins. Both hMGL and mMGL-2 bound tightly to GalNAc-Tyr determinants. The apparent Kd values (∼1-40 nM) were on par with some of the best known ligands for MGL, such as the Tn antigen. hMGL also bound the natural beta-amyloid peptide contg. a GalNAc-Tyr epitope. STD NMR expts. provided structural insights into the mol. basis of recognition. Finally, GalNAc-Tyr was selectively captured by mMGL-2 pos. dendritic cells. These results provide the first evidence that GalNAc-Tyr modified proteins and/or peptides may be ligands for hMGL and mMGL-2 and offer unique structures for the design of MGL targeting agents.
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  67. 67
    Pluvinage, B.; Ficko-Blean, E.; Noach, I.; Stuart, C.; Thompson, N.; McClure, H.; Buenbrazo, N.; Wakarchuk, W.; Boraston, A. B. Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis. Proc. Natl. Acad. Sci. U. S. A. 2021, 118, e2019220118  DOI: 10.1073/pnas.2019220118
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    67
    Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis
    Pluvinage, Benjamin; Ficko-Blean, Elizabeth; Noach, Ilit; Stuart, Christopher; Thompson, Nicole; McClure, Hayden; Buenbrazo, Nakita; Wakarchuk, Warren; Boraston, Alisdair B.
    Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (10), e2019220118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    A challenge faced by peptidases is the recognition of highly diverse substrates. A feature of some peptidase families is the capacity to specifically use post-translationally added glycans present on their protein substrates as a recognition determinant. This is ultimately crit. to enabling peptide bond hydrolysis. This class of enzyme is also frequently large and architecturally sophisticated. However, the mol. details underpinning glycan recognition by these O-glycopeptidases, the importance of these interactions, and the functional roles of their ancillary domains remain unclear. Here, using the Clostridium perfringens ZmpA, ZmpB, and ZmpC M60 peptidases as model proteins, we provide structural and functional insight into how these intricate proteins recognize glycans as part of catalytic and noncatalytic substrate recognition. Structural, kinetic, and mutagenic analyses support the key role of glycan recognition within the M60 domain catalytic site, though they point to ZmpA as an apparently inactive enzyme. Wider examn. of the Zmp domain content reveals noncatalytic carbohydrate binding as a feature of these proteins. The complete three-dimensional structure of ZmpB provides rare insight into the overall mol. organization of a highly multimodular enzyme and reveals how the interplay of individual domain function may influence biol. activity. O-glycopeptidases frequently occur in host-adapted microbes that inhabit or attack mucus layers. Therefore, we anticipate that these results will be fundamental to informing more detailed models of how the glycoproteins that are abundant in mucus are destroyed as part of pathogenic processes or liberated as energy sources during normal commensal lifestyles.
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  68. 68
    Noach, I.; Ficko-Blean, E.; Pluvinage, B.; Stuart, C.; Jenkins, M. L.; Brochu, D.; Buenbrazo, N.; Wakarchuk, W.; Burke, J. E.; Gilbert, M.; Boraston, A. B. Recognition of protein-linked glycans as a determinant of peptidase activity. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E679  DOI: 10.1073/pnas.1615141114
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    68
    Recognition of protein-linked glycans as a determinant of peptidase activity
    Noach, Ilit; Ficko-Blean, Elizabeth; Pluvinage, Benjamin; Stuart, Christopher; Jenkins, Meredith L.; Brochu, Denis; Buenbrazo, Nakita; Wakarchuk, Warren; Burke, John E.; Gilbert, Michel; Boraston, Alisdair B.
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (5), E679-E688CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The vast majority of proteins are post-translationally altered, with the addn. of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate post-translational modifications, including glycosylation, has not been addressed. Here, through biochem. analyses and x-ray crystallog. structures, the authors show that to hydrolyze their substrates, 3 structurally related metallopeptidases (BT4244, IMPa, and ZmpB) require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The 3 peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight built upon the paradigm of how peptidases recognize substrates and provides a mol. understanding of glycoprotein degrdn.
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  69. 69
    Somovilla, V. J.; Bermejo, I. A.; Albuquerque, I. S.; Martinez-Saez, N.; Castro-Lopez, J.; Garcia-Martin, F.; Companon, I.; Hinou, H.; Nishimura, S.-I.; Jimenez-Barbero, J.; Asensio, J. L.; Avenoza, A.; Busto, J. H.; Hurtado-Guerrero, R.; Peregrina, J. M.; Bernardes, G. J. L.; Corzana, F. The use of fluoroproline in MUC1 antigen enables efficient detection of antibodies in patients with prostate cancer. J. Am. Chem. Soc. 2017, 139, 18255– 18261,  DOI: 10.1021/jacs.7b09447
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    A structure-based design of a new generation of tumor-assocd. glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-L-proline or 4,4-difluoro-L-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallog. and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-assocd. anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivs. can be considered better diagnostic tools than the natural antigen for prostate cancer.
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    Companon, I.; Guerreiro, A.; Mangini, V.; Castro-Lopez, J.; Escudero-Casao, M.; Avenoza, A.; Busto, J. H.; Castillon, S.; Jimenez-Barbero, J.; Asensio, J. L.; Jimenez-Oses, G.; Boutureira, O.; Peregrina, J. M.; Hurtado-Guerrero, R.; Fiammengo, R.; Bernardes, G. J. L.; Corzana, F. Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage. J. Am. Chem. Soc. 2019, 141, 4063– 4072,  DOI: 10.1021/jacs.8b13503
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    GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-assocd. antigens. α-O-glycosylation forces the peptide to adopt an extended conformation in soln., which is far from the structure obsd. in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chem. modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-assocd. MUC1 antigen mimics, the deriv. bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiol. context. Clin., the exploitation of tumor-assocd. antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodol. presented here is of general interest for applications because it may be extended to modulate the affinity of biol. relevant glycopeptides toward their receptors.
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  1. Yiran Li, Haiqi Wang, Yunlong Chen, Lin Ding, Huangxian Ju. In Situ Glycan Analysis and Editing in Living Systems. JACS Au 2024, 4 (2) , 384-401. https://doi.org/10.1021/jacsau.3c00717
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  • Abstract

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    Figure 1

    Figure 1. Scheme of the mucin-type O-glycosylation pathway. All of the GTs involved in initiation and core extension are mentioned together with the stereochemistry and glycosidic linkages. The proteins under review in this Account are highlighted in red.

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    Figure 2

    Figure 2. Five structural features define GalNAc-Ts functions. (A) Overall structure of GalNAc-T4. The catalytic and lectin domains are colored in gray, and the interdomain flexible linker is in red. The catalytic domain flexible loop, UDP, the glycopeptides, and the GalNAc moieties are depicted in yellow, brown, green, and orange, respectively. The inset shows the coordination geometry of Mn2+. (B) (Upper panel) Surface representation of the inactive form of GalNAc-T2 and the active form of GalNAc-T2. (Lower panel) Catalytic cycle of GalNAc-T2. (C) (Left panel) View of the complete sugar-nucleotide, peptide, and lectin-domain binding sites of GalNAc-T4. (Right panel) Close-up view of the GalNAc-binding sites of the lectin and catalytic domains. The residues of the lectin domain engaged in recognition of the GalNAc moieties are indicated in magenta. (D) (Upper panel) Surface representation of different GalNAc-Ts showing the location of the lectin domain GalNAc-binding sites in the overall structures. Note that GalNAc-T1 has two potential functional GalNAc-binding sites. (Lower panel) Scheme illustrating the different types of O-glycosylation and preferences found for GalNAc-T2 and T4.

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    Figure 3

    Figure 3. Molecular recognition of glycopeptides by C1GalT1. (A) Close-up view of the active site for the DmC1GalT1-UDP-APDT*RP complex (PDB entry 7Q4I). The carbon atoms of the residues interacting with the glycopeptide are shown in magenta. The UDP carbon atoms are shown in gray. (B) Main conformation found in solution for the Tn antigen with Thr GalNAc-α-1-O-Thr (left panel) and for the Tn antigen with Ser GalNAc-α-1-O-Thr (right panel). The CH-π interaction between the methyl group of Thr4 and Trp300 is shown by the black dashed line.

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    Figure 4

    Figure 4. Comparison between the different active sites of mucinases. (A) Superimposition of the active sites of IMPa (green) and BT4244 (cyan) showing main interactions with the sugar units of the O-glycopeptides (orange for IMPa and magenta for BT4244). (B) Superimposition of the active sites of ZmpB (green) and ZmpC (cyan) showing main interactions with the corresponding O-glycopeptides (orange for ZmpB and magenta for ZmpC). (C) Superimposition of the active sites of complexes formed between BT4244 (cyan) and AM0627 (green) with a Tn O-glycan (magenta) and bis-T-glycopeptide (orange), respectively. PDB entries for the structures of AM0627, IMPa, BT4244, ZmpB, and ZmpC are 7SCI, 5KDX, 5KD8, 5KDU, and 6XT1, respectively.

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    Figure 5

    Figure 5. Molecular recognition of Tn-glycopeptides by antibodies. (A) Close-up view of the binding site of the 273 mAb bound to the ERGT*KPPLEEL glycopeptide, showing the interactions of the protein with the GalNAc unit (PDB entry 3IET). (B) Close-up view of the binding site of the AR20.5 antibody and the glycopeptide APDT*RP (PDB entry 5T78). (C) Close-up view of the binding site of the SM3 antibody and glycopeptide APDT*RP (PDB entry 5A2K). (D) Close-up view of the binding site of the SN-101 antibody and a MUC1-like glycopeptide (PDB entry 6KX1). Only the interactions between the protein and the GalNAc unit are shown. (E) Close-up view of the binding site of the antibody 5E5 and glycopeptide APGST*AP (PDB entry 6TNP). (F) Close-up view of the binding site of the antibody G10C and αGalNAc-PNP (PDB entry 7UT3). Note that in all panels the residues of the antibody engaged in recognition of the glycopeptides and/or the GalNAc moieties are indicated in magenta.

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    Figure 6

    Figure 6. Molecular recognition of Tn-glycopeptides by lectins. (A) Close-up view of the binding site of the lectin hMGL bound to Tn-Ser (PDB entry 6W12). (B) Close-up view of the binding site of the Bauhinia forficata lectin bound to a Tn-containing glycopeptide derived from MUC1 (PDB entry 5T5O). (C) Close-up view of the binding site of SBA lectin bound to PDT*R glycopeptide (PDB entry 4D69). Note that in all panels the residues of lectin engaged in the recognition of the glycopeptides and/or the GalNAc moieties are indicated in magenta.

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    Figure 7

    Figure 7. Main common recognition modes for the different families of proteins.

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  • References

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      A review. Protein O-GalNAcylation is an abundant post-translational modification and predicted to occur in >80% of the proteins passing through the Golgi app. This modification is driven by 20 polypeptide GalNAc (N-acetylgalactosamine)-transferases (GalNAc-Ts), which are unique in that they possess both catalytic and lectin domains. The peptide substrate specificities of GalNAc-Ts are still poorly defined and the understanding of the sequence and structural features that direct O-glycosylation of proteins is limited. Part of this may be attributed to the complex regulation by coordinated action of multiple GalNAc-T isoforms, and part of this may also be attributed to the 2 functional domains of GalNAc-Ts that both seem to be involved in directing the substrate specificities. Recent studies have resulted in 3-dimensional structures of GalNAc-Ts and detn. of the reaction mechanism of this family of enzymes. Key advances include the trapping of binary/ternary complexes in combination with computational simulations and AFM/small-SAXS expts., which have allowed for the dissection of the reaction coordinates and the mechanism by which the lectin domains modulate the glycosylation. These studies not only broaden the knowledge of the modes-of-action of this family of enzymes but also open up potential avenues for the rational design of effective and selective inhibitors of O-glycosylation.
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      Schjoldager, Katrine T.; Joshi, Hiren J.; Kong, Yun; Goth, Christoffer K.; King, Sarah Louise; Wandall, Hans H.; Bennett, Eric P.; Vakhrushev, Sergey Y.; Clausen, Henrik
      EMBO Reports (2015), 16 (12), 1713-1722CODEN: ERMEAX; ISSN:1469-221X. (Wiley-VCH Verlag GmbH & Co. KGaA)
      GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biol. functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and GalNAc-T2) in liver cells and explore how the GalNAc-T repertoire quant. affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and GalNAc-T2. The non-redundant O-glycoproteome subsets and specific transcriptional responses for each isoform are related to different cellular processes; for the GalNAc-T2 isoform, these support a role in lipid metab. The results demonstrate that GalNAc-Ts have different non-redundant glycosylation functions, which may affect distinct cellular processes. The data serves as a comprehensive resource for unique GalNAc-T substrates. Our study provides a new view of the differential regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.
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    9. 9
      Bennett, E. P.; Mandel, U.; Clausen, H.; Gerken, T. A.; Fritz, T. A.; Tabak, L. A. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology 2012, 22, 736– 756,  DOI: 10.1093/glycob/cwr182
      9
      Control of mucin-type O-glycosylation: A classification of the polypeptide GalNAc-transferase gene family
      Bennett, Eric P.; Mandel, Ulla; Clausen, Henrik; Gerken, Thomas A.; Fritz, Timothy A.; Tabak, Lawrence A.
      Glycobiology (2012), 22 (6), 736-756CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
      A review. Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The GalNAc-T family is the largest glycosyltransferase enzyme family covering a single known glycosidic linkage and it is highly conserved throughout animal evolution, although absent in bacteria, yeast and plants. Emerging studies have shown that the large no. of genes (GALNTs) in the GalNAc-T family do not provide full functional redundancy and single GalNAc-T genes have been shown to be important in both animals and human. Here, we present an overview of the GalNAc-T gene family in animals and propose a classification of the genes into subfamilies, which appear to be conserved in evolution structurally as well as functionally.
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    10. 10
      Brockhausen, I.; Schachter, H.; Stanley, P. O-GalNAc Glycans. In Essentials of Glycobiology, 2nd ed.; Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E., Eds.; Cold Spring Harbor Laboratory Press: 2008.
      There is no corresponding record for this reference.
    11. 11
      Ho, T. D.; Davis, B. M.; Ritchie, J. M.; Waldor, M. K. Type 2 secretion promotes enterohemorrhagic Escherichia coli adherence and intestinal colonization. Infect. Immun. 2008, 76, 1858– 1865,  DOI: 10.1128/IAI.01688-07
      11
      Type 2 secretion promotes enterohemorrhagic Escherichia coli adherence and intestinal colonization
      Ho, Theresa D.; Davis, Brigid M.; Ritchie, Jennifer M.; Waldor, Matthew K.
      Infection and Immunity (2008), 76 (5), 1858-1865CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
      Enterohemorrhagic Escherichia coli (EHEC) is a noninvasive food-borne pathogen that colonizes the distal ileum and colon. Proteins encoded in the EHEC locus of enterocyte effacement (LEE) pathogenicity island are known to contribute to this pathogen's adherence to epithelial cells and intestinal colonization. The role of non-LEE-encoded proteins in these processes is not as clear. The authors found that the Z2053 gene (designated adfO here), a gene located in a cryptic EHEC prophage, exhibits similarity to adherence and/or colonization factor genes found in several other enteric pathogens. An EHEC adfO mutant exhibited marked redns. in adherence to HeLa cells and in the secretion of several proteins into the supernatant. YodA, one of these secreted proteins, was found to be a substrate of the EHEC pO157-encoded type 2 secretion system (T2SS). Both the T2SS and YodA proved to be essential for EHEC adherence to cultured HeLa cell monolayers. Using an infant rabbit model of infection, the authors found that the adfO mutation did not affect colonization but that the colonization of an etpC (T2SS) mutant was reduced ∼5-fold. A strain deficient in YodA had a more severe colonization defect; however, this strain also exhibited a growth defect in vitro. Overall, the authors' findings indicate that the pO157-encoded T2SS contributes to EHEC adherence and intestinal colonization and thus show that EHEC pathogenicity depends on type 2 secretion as well as type 3 secretion.
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    12. 12
      Nason, R.; Bull, C.; Konstantinidi, A.; Sun, L.; Ye, Z.; Halim, A.; Du, W.; Sorensen, D. M.; Durbesson, F.; Furukawa, S.; Mandel, U.; Joshi, H. J.; Dworkin, L. A.; Hansen, L.; David, L.; Iverson, T. M.; Bensing, B. A.; Sullam, P. M.; Varki, A.; Vries, E.; de Haan, C. A. M.; Vincentelli, R.; Henrissat, B.; Vakhrushev, S. Y.; Clausen, H.; Narimatsu, Y. Nat. Commun. 2021, 12, 4070,  DOI: 10.1038/s41467-021-24366-4
      12
      Display of the human mucinome with defined O-glycans by gene engineered cells
      Nason, Rebecca; Bull, Christian; Konstantinidi, Andriana; Sun, Lingbo; Ye, Zilu; Halim, Adnan; Du, Wenjuan; Soerensen, Daniel M.; Durbesson, Fabien; Furukawa, Sanae; Mandel, Ulla; Joshi, Hiren J.; Dworkin, Leo Alexander; Hansen, Lars; David, Leonor; Iverson, Tina M.; Bensing, Barbara A.; Sullam, Paul M.; Varki, Ajit; Vries, Erik de; de Haan, Cornelis A. M.; Vincentelli, Renaud; Henrissat, Bernard; Vakhrushev, Sergey Y.; Clausen, Henrik; Narimatsu, Yoshiki
      Nature Communications (2021), 12 (1), 4070CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromols. in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and prodn. of human TR O-glycodomains (∼200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances exptl. studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for mol. dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.
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    13. 13
      Shon, D. J.; Kuo, A.; Ferracane, M. J.; Malaker, S. A. Classification, structural biology, and applications of mucin domain-targeting proteases. Biochem.J. 2021, 478, 1585– 1603,  DOI: 10.1042/BCJ20200607
      13
      Classification, structural biology, and applications of mucin domain-targeting proteases
      Shon, D. Judy; Kuo, Angel; Ferracane, Michael J.; Malaker, Stacy A.
      Biochemical Journal (2021), 478 (8), 1585-1603CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      A review. Epithelial surfaces throughout the body are coated by mucins, a class of proteins carrying domains characterized by a high d. of O-glycosylated serine and threonine residues. The resulting mucosal layers form crucial host-microbe interfaces that prevent the translocation of microbes while also selecting for distinct bacteria via the presented glycan repertoire. The intricate interplay between mucus prodn. and breakdown thus dets. the compn. of the microbiota maintained within these mucosal environments, which can have a large influence on the host during both homeostasis and disease. Most research to date on mucus breakdown has focused on glycosidases that trim glycan structures to release monosaccharides as a source of nutrients. More recent work has uncovered the existence of mucin-type O-glycosylation-dependent proteases that are secreted by pathogens, commensals, and mutualists to facilitate mucosal colonization and penetration. Addnl., IgA (IgA) proteases promote bacterial colonization in the presence of neutralizing secretory IgA through selective cleavage of the heavily O-glycosylated hinge region. In this review, we summarize families of O-glycoproteases and IgA proteases, discuss known structural features, and review applications of these enzymes to glycobiol.
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    14. 14
      Schjoldager, K. T.-B. G.; Clausen, H. Site-specific protein O-glycosylation modulates proprotein processing - deciphering specific functions of the large polypeptide GalNAc-transferase gene family. Biochim. Biophys. Acta. 2012, 1820, 2079– 2094,  DOI: 10.1016/j.bbagen.2012.09.014
      14
      Site-specific protein O-glycosylation modulates proprotein processing - Deciphering specific functions of the large polypeptide GalNAc-transferase gene family
      Schjoldager, Katrine T.-B. G.; Clausen, Henrik
      Biochimica et Biophysica Acta, General Subjects (2012), 1820 (12), 2079-2094CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
      A review. Posttranslational modifications (PTMs) greatly expand the function and regulation of proteins, and glycosylation is the most abundant and diverse PTM. Of the many different types of protein glycosylation, one is quite unique; GalNAc-type (or mucin-type) O-glycosylation, where biosynthesis is initiated in the Golgi by up to twenty distinct UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). These GalNAc-Ts are differentially expressed in cells and have different (although partly overlapping) substrate specificities, which provide for both unique functions and considerable redundancy. Recently we have begun to uncover human diseases assocd. with deficiencies in GalNAc-T genes (GALNTs). Thus deficiencies in individual GALNTs produce cell and protein specific effects and subtle distinct phenotypes such as hyperphosphatemia with hyperostosis (GALNT3) and dysregulated lipid metab. (GALNT2). These phenotypes appear to be caused by deficient site-specific O-glycosylation that co-regulates proprotein convertase (PC) processing of FGF23 and ANGPTL3, resp. Here we summarize recent progress in uncovering the interplay between human O-glycosylation and protease regulated processing and describes other important functions of site-specific O-glycosylation in health and disease. Site-specific O-glycosylation modifies pro-protein processing and other proteolytic events such as ADAM processing and thus emerges as an important co-regulator of limited proteolytic processing events. Our appreciation of this function may have been hampered by our sparse knowledge of the O-glycoproteome and in particular sites of O-glycosylation. New strategies for identification of O-glycoproteins have emerged and recently the concept of SimpleCells, i.e. human cell lines made deficient in O-glycan extension by zinc finger nuclease gene targeting, was introduced for broad O-glycoproteome anal.
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    15. 15
      Goth, C. K.; Vakhrushev, S. Y.; Joshi, H. J.; Clausen, H.; Schjoldager, K. T. Fine-Tuning Limited Proteolysis: A Major Role for Regulated Site-Specific O-Glycosylation. Trends Biochem. Sci. 2018, 43, 269– 284,  DOI: 10.1016/j.tibs.2018.02.005
      15
      Fine-Tuning Limited Proteolysis: A Major Role for Regulated Site-Specific O-Glycosylation
      Goth, Christoffer K.; Vakhrushev, Sergey Y.; Joshi, Hiren J.; Clausen, Henrik; Schjoldager, Katrine T.
      Trends in Biochemical Sciences (2018), 43 (4), 269-284CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
      A review. Limited proteolytic processing is an essential and ubiquitous post-translational modification (PTM) affecting secreted proteins; failure to regulate the process is often assocd. with disease. Glycosylation is also a ubiquitous protein PTM and site-specific O-glycosylation in close proximity to sites of proteolysis can regulate and direct the activity of proprotein convertases, a disintegrin and metalloproteinases (ADAMs), and metalloproteinases affecting the activation or inactivation of many classes of proteins, including G-protein-coupled receptors (GPCRs). Here, we summarize the emerging data that suggest O-glycosylation to be a key regulator of limited proteolysis, and highlight the potential for crosstalk between multiple PTMs.
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    16. 16
      Wandall, H. H.; Nielsen, M. A. I.; King-Smith, S.; de Haan, N.; Bagdonaite, I. Global functions of O-glycosylation: promises and challenges in O-glycobiology. FEBS J. 2021, 288, 7183– 7212,  DOI: 10.1111/febs.16148
      16
      Global functions of O-glycosylation: promises and challenges in O-glycobiology
      Wandall, Hans H.; Nielsen, Mathias A. I.; King-Smith, Sarah; de Haan, Noortje; Bagdonaite, Ieva
      FEBS Journal (2021), 288 (24), 7183-7212CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      A review. Mucin type O-glycosylation is one of the most diverse types of glycosylation, playing essential roles in tissue development and homeostasis. In complex organisms, O-GalNAc glycans comprise a substantial proportion of the glycocalyx, with defined functions in hemostatic, gastrointestinal, and respiratory systems. Furthermore, O-GalNAc glycans are important players in host-microbe interactions, and changes in O-glycan compn. are assocd. with certain diseases and metabolic conditions, which in some instances can be used for diagnosis or therapeutic intervention. Breakthroughs in O-glycobiol. have gone hand in hand with the development of new technologies, such as advancements in mass spectrometry, as well as facilitation of genetic engineering in mammalian cell lines. High-throughput O-glycoproteomics have enabled us to draw a comprehensive map of O-glycosylation, and mining this information has supported the definition and confirmation of functions related to site-specific O-glycans. This includes protection from proteolytic cleavage, as well as modulation of binding affinity or receptor function. Yet, there is still much to discover, and among the important next challenges will be to define the context-dependent functions of O-glycans in different stages of cellular differentiation, cellular metab., host-microbiome interactions, and in disease. In this review, we present the achievements and the promises in O-GalNAc glycobiol. driven by technol. advances in anal. methods, genetic engineering, and systems biol.
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    17. 17
      Brockhausen, I.; Wandall, H. H.; Hagen, K. G. T.; Stanley, P. O-GalNAc Glycans. In Essentials of Glycobiology, 4th ed.; Varki, A., Cummings, R. D., Esko, J. D., Stanley, P., Hart, G. W., Aebi, M., Mohnen, D., Kinoshita, T., Packer, N. H., Prestegard, J. H., Schnaar, R. L., Seeberger, P. H., Eds.; Cold Spring Harbor Laboratory Press: 2022; pp 117– 128.
      There is no corresponding record for this reference.
    18. 18
      Xia, L.; Ju, T.; Westmuckett, A.; An, G.; Ivanciu, L.; McDaniel, J. M.; Lupu, F.; Cummings, R. D.; McEver, R. P. Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans. J. Cell Biol. 2004, 164, 451– 459,  DOI: 10.1083/jcb.200311112
      18
      Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans
      Xia, Lijun; Ju, Tongzhong; Westmuckett, Andrew; An, Guangyu; Ivanciu, Lacramioara; McDaniel, J. Michael; Lupu, Florea; Cummings, Richard D.; McEver, Rodger P.
      Journal of Cell Biology (2004), 164 (3), 451-459CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      The core 1 β1-3-galactosyltransferase (T-synthase) transfers Gal from UDP-Gal to GalNAcα1-Ser/Thr (Tn antigen) to form the core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr (T antigen). The T antigen is a precursor for extended and branched O-glycans of largely unknown function. We found that wild-type mice expressed the NeuAcα2-3Galβ1-3GalNAcα1-Ser/Thr primarily in endothelial, hematopoietic, and epithelial cells during development. Gene-targeted mice lacking T-synthase instead expressed the nonsialylated Tn antigen in these cells and developed brain hemorrhage that was uniformly fatal by embryonic day 14. T-synthase-deficient brains formed a chaotic microvascular network with distorted capillary lumens and defective assocn. of endothelial cells with pericytes and extracellular matrix. These data reveal an unexpected requirement for core 1-derived O-glycans during angiogenesis.
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    19. 19
      Wang, Y.; Ju, T.; Ding, X.; Xia, B.; Wang, W.; Xia, L.; He, M.; Cummings, R. D. Cosmc is an essential chaperone for correct protein O-glycosylation. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 9228– 9233,  DOI: 10.1073/pnas.0914004107
      19
      Cosmc is an essential chaperone for correct protein O-glycosylation
      Wang, Yingchun; Ju, Tongzhong; Ding, Xiaokun; Xia, Baoyun; Wang, Wenyi; Xia, Lijun; He, Miao; Cummings, Richard D.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (20), 9228-9233, S9228/1-S9228/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Cosmc is a mol. chaperone thought to be required for expression of active T-synthase, the only enzyme that galactosylates the Tn antigen (GalNAcα1-Ser/Thr-R) to form core 1 Galβ-3GalNAcα1-Ser/Thr (T antigen) during mucin type O-glycan biosynthesis. Here the authors show that ablation of the X-linked Cosmc gene in mice causes embryonic lethality and Tn antigen expression. Loss of Cosmc is assocd. with loss of T-synthase but not other enzymes required for glycoprotein biosynthesis, demonstrating that Cosmc is specific in vivo for the T-synthase. The authors generated genetically mosaic mice with a targeted Cosmc deletion and survivors exhibited abnormalities correlated with Tn antigen expression that are related to several human diseases.
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    20. 20
      Gill, D. J.; Tham, K. M.; Chia, J.; Wang, S. C.; Steentoft, C.; Clausen, H.; Bard-Chapeau, E. A.; Bard, F. A. Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, E3152– 3161,  DOI: 10.1073/pnas.1305269110
      20
      Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness
      Gill, David J.; Tham, Keit Min; Chia, Joanne; Wang, Shyi Chyi; Steentoft, Catharina; Clausen, Henrik; Bard-Chapeau, Emilie A.; Bard, Frederic A.
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (34), E3152-E3161,SE3152/1-SE3152/32CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Invasiveness underlies cancer aggressiveness and is a hallmark of malignancy. Most malignant tumors have elevated levels of Tn, an O-GalNAc glycan. Mechanisms underlying Tn up-regulation and its effects remain unclear. Here we show that Golgi-to-endoplasmic reticulum relocation of polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) drives high Tn levels in cancer cell lines and in 70% of malignant breast tumors. This process stimulates cell adhesion to the extracellular matrix, as well as migration and invasiveness. The GalNAc-Ts lectin domain, mediating high-d. glycosylation, is crit. for these effects. Interfering with the lectin domain function inhibited carcinoma cell migration in vitro and metastatic potential in mice. We also show that stimulation of cell migration is dependent on Tn-bearing proteins present in lamellipodia of migrating cells. Our findings suggest that relocation of GalNAc-Ts to the endoplasmic reticulum frequently occurs upon cancerous transformation to enhance tumor cell migration and invasiveness through modification of cell surface proteins.
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    21. 21
      Herbomel, G. G.; Rojas, R. E.; Tran, D. T.; Ajinkya, M.; Beck, L.; Tabak, L. A. The GalNAc-T Activation Pathway (GALA) is not a general mechanism for regulating mucin-type O-glycosylation. PLoS One 2017, 12, e0179241  DOI: 10.1371/journal.pone.0179241
      21
      The GalNAc-T activation pathway (GALA) is not a general mechanism for regulating mucintype O-glycosylation
      Herbomel, Gaetan G.; Rojas, Raul E.; Tran, Duy T.; Ajinkya, Monica; Beck, Lauren; Tabak, Lawrence A.
      PLoS One (2017), 12 (7), e0179241/1-e0179241/14CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Mucin-type O-glycosylation is initiated by the UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferase (GalNAc-T) family of enzymes. Their activity results in the GalNAc α1-OThr/ Ser structure, termed the Tn antigen, which is further decorated with addnl. sugars. In neoplastic cells, the Tn antigen is often overexpressed. Because O-glycosylation is controlled by the activity of GalNAc-Ts, their regulation is of great interest. Previous reports suggest that growth factors, EGF or PDGF, induce Golgi complex-to-endoplasmic reticulum (ER) relocation of both GalNAc-Ts and Tn antigen in HeLa cells, offering a mechanism for Tn antigen overexpression termed "GALA". However, we were unable to reproduce these findings. Upon treatment of HeLa cells with either EGF or PDGF we obsd. no change in the co-localization of endogenous GalNAc-T1, GalNAc-T2 or Tn antigen with the Golgi complex marker TGN46. There was also no enhancement of localization with the ER marker calnexin. We conclude that growth factors do not cause redistribution of GalNAc-Ts from the Golgi complex to the ER in HeLa cells.
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    22. 22
      Radhakrishnan, P.; Dabelsteen, S.; Madsen, F. B.; Francavilla, C.; Kopp, K. L.; Steentoft, C.; Vakhrushev, S. Y.; Olsen, J. V.; Hansen, L.; Bennett, E. P.; Woetmann, A.; Yin, G.; Chen, L.; Song, H.; Bak, M.; Hlady, R. A.; Peters, S. L.; Opavsky, R.; Thode, C.; Qvortrup, K.; Schjoldager, K. T.; Clausen, H.; Hollingsworth, M. A.; Wandall, H. H. Immature truncated O-glycophenotype of cancer directly induces oncogenic features. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, E4066– 4075,  DOI: 10.1073/pnas.1406619111
      22
      Immature truncated O-glycophenotype of cancer directly induces oncogenic features
      Radhakrishnan, Prakash; Dabelsteen, Sally; Madsen, Frey Brus; Francavilla, Chiara; Kopp, Katharina L.; Steentoft, Catharina; Vakhrushev, Sergey Y.; Olsen, Jesper V.; Hansen, Lars; Bennett, Eric P.; Woetmann, Anders; Yin, Guangliang; Chen, Longyun; Song, Haiyan; Bak, Mads; Hlady, Ryan A.; Peters, Staci L.; Opavsky, Rene; Thode, Christenze; Qvortrup, Klaus; Schjoldager, Katrine T.-B. G.; Clausen, Henrik; Hollingsworth, Michael A.; Wandall, Hans H.
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (39), E4066-E4075CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Aberrant expression of immature truncated O-glycans is a characteristic feature obsd. on virtually all epithelial cancer cells, and a very high frequency is obsd. in early epithelial premalignant lesions that precede the development of adenocarcinomas. Expression of the truncated O-glycan structures Tn and sialyl-Tn is strongly assocd. with poor prognosis and overall low survival. The genetic and biosynthetic mechanisms leading to accumulation of truncated O-glycans are not fully understood and include mutation or dysregulation of glycosyltransferases involved in elongation of O-glycans, as well as relocation of glycosyltransferases controlling initiation of O-glycosylation from Golgi to endoplasmic reticulum. Truncated O-glycans have been proposed to play functional roles for cancer-cell invasiveness, but our understanding of the biol. functions of aberrant glycosylation in cancer is still highly limited. Here, we used exome sequencing of most glycosyltransferases in a large series of primary and metastatic pancreatic cancers to rule out somatic mutations as a cause of expression of truncated O-glycans. Instead, we found hypermethylation of core 1 β3-Gal-T-specific mol. chaperone, a key chaperone for O-glycan elongation, as the most prevalent cause. We next used gene editing to produce isogenic cell systems with and without homogenous truncated O-glycans that enabled, to our knowledge, the first polyomic and side-by-side evaluation of the cancer O-glycophenotype in an organotypic tissue model and in xenografts. The results strongly suggest that truncation of O-glycans directly induces oncogenic features of cell growth and invasion. The study provides support for targeting cancer-specific truncated O-glycans with immunotherapeutic measures.
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    23. 23
      Marcos, N. T.; Pinho, S.; Grandela, C.; Cruz, A.; Samyn-Petit, B.; Harduin-Lepers, A.; Almeida, R.; Silva, F.; Morais, V.; Costa, J.; Kihlberg, J.; Clausen, H.; Reis, C. A. Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated sialyl-Tn antigen. Cancer Res. 2004, 64, 7050– 7057,  DOI: 10.1158/0008-5472.CAN-04-1921
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      Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated Sialyl-Tn antigen
      Marcos, Nuno T.; Pinho, Sandra; Grandela, Catarina; Cruz, Andrea; Samyn-Petit, Benedicte; Harduin-Lepers, Anne; Almeida, Raquel; Silva, Filipe; Morais, Vanessa; Costa, Julia; Kihlberg, Jan; Clausen, Henrik; Reis, Celso A.
      Cancer Research (2004), 64 (19), 7050-7057CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      The Sialyl-Tn antigen (Neu5Acα2-6GalNAc-O-Ser/Thr) is highly expressed in several human carcinomas and is assocd. with carcinoma aggressiveness and poor prognosis. The authors characterized two human sialyltransferases, CMP-Neu5Ac:GalNAc-R α2,6-sialyltransferase (ST6GalNAc)-I and ST6GalNAc-II, that are candidate enzymes for Sialyl-Tn synthases. The authors expressed sol. recombinant hST6GalNAc-I and hST6GalNAc-II and characterized the substrate specificity of both enzymes toward a panel of glycopeptides, glycoproteins, and other synthetic glycoconjugates. The recombinant ST6GalNAc-I and ST6GalNAc-II showed similar substrate specificity toward glycoproteins and GalNAcα-O-Ser/Thr glycopeptides, such as glycopeptides derived from the MUC2 mucin and the HIVgp120. The authors also obsd. that the amino acid sequence of the acceptor glycopeptide contributes to the in vitro substrate specificity of both enzymes. The authors addnl. established a gastric cell line, MKN45, stably transfected with the full length of either ST6GalNAc-I or ST6GalNAc-II and evaluated the carbohydrate antigens expression profile induced by each enzyme. MKN45 transfected with ST6GalNAc-I showed high expression of Sialyl-Tn, whereas MKN45 transfected with ST6GalNAc-II showed the biosynthesis of the Sialyl-6T structure [Galβ1-3 (Neu5Acα2-6)GalNAc-O-Ser/Thr]. In conclusion, although both enzymes show similar in vitro activities when Tn antigen alone is available, whenever both Tn and T antigens are present, ST6GalNAc-I acts preferentially on Tn antigen, whereas the ST6GalNAc-II acts preferentially on T antigen. These results show that ST6GalNAc-I is the major Sialyl-Tn synthase and strongly support the hypothesis that the expression of the Sialyl-Tn antigen in cancer cells is due to ST6GalNAc-I activity.
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    24. 24
      Martínez-Sáez, N.; Peregrina, J. M.; Corzana, F. Principles of mucin structure: implications for the rational design of cancer vaccines derived from MUC1-glycopeptides. Chem. Soc. Rev. 2017, 46, 7154– 7175,  DOI: 10.1039/C6CS00858E
      24
      Principles of mucin structure: implications for the rational design of cancer vaccines derived from MUC1-glycopeptides
      Martinez-Saez, Nuria; Peregrina, Jesus M.; Corzana, Francisco
      Chemical Society Reviews (2017), 46 (23), 7154-7175CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Cancer is currently one of the world's most serious public health problems. Significant efforts are being made to develop new strategies that can eradicate tumors selectively without detrimental effects to healthy cells. One promising approach is focused on the design of vaccines that contain partially glycosylated mucins in their formulation. Although some of these vaccines are in clin. trials, a lack of knowledge about the mol. basis that governs the antigen presentation, and the interactions between antigens and the elicited antibodies has limited their success thus far. This review focuses on the most significant milestones achieved to date in the conformational anal. of tumor-assocd. MUC1 derivs. both in soln. and bound to antibodies. The effect that the carbohydrate scaffold has on the peptide backbone structure and the role of the sugar in mol. recognition by antibodies are emphasized. The outcomes summarised in this review may be a useful guide to develop new antigens for the design of cancer vaccines in the near future.
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    25. 25
      Bulteau, F.; Thépaut, M.; Henry, M.; Hurbin, A.; Vanwonterghem, L.; Vivès, C.; Le Roy, A.; Ebel, C.; Renaudet, O.; Fieschi, F.; Coll, J.-L. Targeting Tn-Antigen-Positive Human Tumors with a Re combinant Human Macrophage Galactose C-Type Lectin. Mol. Pharmaceutics 2022, 19, 235– 245,  DOI: 10.1021/acs.molpharmaceut.1c00744
      25
      Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin
      Bulteau, Francois; Thepaut, Michel; Henry, Maxime; Hurbin, Amandine; Vanwonterghem, Laetitia; Vives, Corinne; Le Roy, Aline; Ebel, Christine; Renaudet, Olivier; Fieschi, Franck; Coll, Jean-Luc
      Molecular Pharmaceutics (2022), 19 (1), 235-245CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      Alterations in glycosylation cause the emergence of tumor-assocd. carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-pos. human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.
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    26. 26
      Fritz, T. A.; Hurley, J. H.; Trinh, L. B.; Shiloach, J.; Tabak, L. A. The beginnings of mucin biosynthesis: the crystal structure of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-T1. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15307– 15312,  DOI: 10.1073/pnas.0405657101
      26
      The beginnings of mucin biosynthesis: The crystal structure of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferase-T1
      Fritz, Timothy A.; Hurley, James H.; Trinh, Loc-Ba; Shiloach, Joseph; Tabak, Lawrence A.
      Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (43), 15307-15312CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      UDP-N-acetylgalactosamine-polypeptide N-acetylgalactosaminyltransferases (UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases; ppGaNTases) initiate the formation of mucin-type, O-linked glycans by catalyzing the transfer of α-N-acetylgalactosamine from UDP-GalNAc to Ser or Thr residues of core proteins to form the Tn antigen (GalNAc-α-1-O-Ser/Thr). The ppGaNTases are unique among glycosyltransferases in contg. a C-terminal lectin domain. Here, the authors present the x-ray crystal structure of a ppGaNTase, murine ppGaNTase-T1, and show that it folds to form distinct catalytic and lectin domains. The assocn. of the 2 domains formed a large cleft in the surface of the enzyme that contained a Mn2+ ion complexed by invariant Asp-209 and His-211 of the "DXH" motif and by invariant His-344. Each of the 3 potential lectin domain carbohydrate-binding sites (α, β, and γ) was located on the active site face of the enzyme, suggesting a mechanism by which the transferase may accommodate multiple conformations of glycosylated acceptor substrates. A model of a mucin 1 glycopeptide (MUC1) substrate bound to the enzyme showed that the spatial sepn. between the lectin α site and a modeled active site UDP-GalNAc was consistent with the in vitro pattern of glycosylation obsd. for this peptide catalyzed by ppGaNTase-T1. The structure also provided a template for the larger ppGaNTase family, and homol. models of several ppGaNTase isoforms predict dramatically different surface chemistries consistent with isoform-selective acceptor substrate recognition.
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    27. 27
      Fritz, T. A.; Raman, J.; Tabak, L. A. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-2. J. Biol. Chem. 2006, 281, 8613– 8619,  DOI: 10.1074/jbc.M513590200
      27
      Dynamic Association between the Catalytic and Lectin Domains of Human UDP-GalNAc:Polypeptide α-N-Acetylgalactosaminyltransferase-2
      Fritz, Timothy A.; Raman, Jayalakshmi; Tabak, Lawrence A.
      Journal of Biological Chemistry (2006), 281 (13), 8613-8619CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The family of (ppGalNAcTs) is unique among glycosyltransferases, contg. both catalytic and lectin domains that we have previously shown to be closely assocd. Here we describe the x-ray crystal structures of human ppGalNAcT-2 (hT2) bound to the product UDP at 2.75 Å resoln. and to UDP and an acceptor peptide substrate EA2 (PTTDSTTPAPTTK) at 1.64 Å resoln. The conformations of both UDP and residues Arg362-Ser372 vary greatly between the two structures. In the hT2-UDP-EA2 complex, residues Arg362-Ser373 comprise a loop that forms a lid over UDP, sealing it in the active site, whereas in the hT2-UDP complex this loop is folded back, exposing UDP to bulk solvent. EA2 binds in a shallow groove with threonine 7 positioned consistent with in vitro data showing it to be the preferred site of glycosylation. The relative orientations of the hT2 catalytic and lectin domains differ dramatically from that of murine ppGalNAcT-1 and also vary considerably between the two hT2 complexes. Indeed, in the hT2-UDP-EA2 complex essentially no contact is made between the catalytic and lectin domains except for the peptide bridge between them. Thus, the hT2 structures reveal an unexpected flexibility between the catalytic and lectin domains and suggest a new mechanism used by hT2 to capture glycosylated substrates. Kinetic anal. of hT2 lacking the lectin domain confirmed the importance of this domain in acting on glycopeptide but not peptide substrates. The structure of the hT2-UDP-EA2 complex also resolves longstanding questions regarding ppGalNAcT acceptor substrate specificity.
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    28. 28
      Kubota, T.; Shiba, T.; Sugioka, S.; Furukawa, S.; Sawaki, H.; Kato, R.; Wakatsuki, S.; Narimatsu, H. Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10). J. Mol. Biol. 2006, 359, 708– 727,  DOI: 10.1016/j.jmb.2006.03.061
      28
      Structural Basis of Carbohydrate Transfer Activity by Human UDP-GalNAc: Polypeptide α-N-Acetylgalactosaminyltransferase (pp-GalNAc-T10)
      Kubota, Tomomi; Shiba, Tomoo; Sugioka, Shigemi; Furukawa, Sanae; Sawaki, Hiromichi; Kato, Ryuich; Wakatsuki, Soichi; Narimatsu, Hisashi
      Journal of Molecular Biology (2006), 359 (3), 708-727CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
      Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isoenzyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin β sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isoenzymes to produce a wide range of O-glycans.
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    29. 29
      Lira-Navarrete, E.; Iglesias-Fernandez, J.; Zandberg, W. F.; Companon, I.; Kong, Y.; Corzana, F.; Pinto, B. M.; Clausen, H.; Peregrina, J. M.; Vocadlo, D. J.; Rovira, C.; Hurtado-Guerrero, R. Substrate-Guided Front-Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N-Acetylgalactosaminyltransferase 2. Angew. Chem., Int. Ed. 2014, 53, 8206– 8210,  DOI: 10.1002/anie.201402781
      29
      Substrate-Guided Front-Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N-Acetylgalactosaminyltransferase 2
      Lira-Navarrete, Erandi; Iglesias-Fernandez, Javier; Zandberg, Wesley F.; Companon, Ismael; Kong, Yun; Corzana, Francisco; Pinto, B. Mario; Clausen, Henrik; Peregrina, Jesus M.; Vocadlo, David J.; Rovira, Carme; Hurtado-Guerrero, Ramon
      Angewandte Chemie, International Edition (2014), 53 (31), 8206-8210CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/mol.-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the at.-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals crit. aspects of substrate recognition, which dictate the specificity for acceptor Thr vs. Ser residues and enforce a front-face SNi-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.
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    30. 30
      Khetarpal, S. A.; Schjoldager, K. T.; Christoffersen, C.; Raghavan, A.; Edmondson, A. C.; Reutter, H. M.; Ahmed, B.; Ouazzani, R.; Peloso, G. M.; Vitali, C.; Zhao, W.; Somasundara, A. V.; Millar, J. S.; Park, Y.; Fernando, G.; Livanov, V.; Choi, S.; Noé, E.; Patel, P.; Ho, S. P.; Kirchgessner, T. G.; Wandall, H. H.; Hansen, L.; Bennett, E. P.; Vakhrushev, S. Y.; Saleheen, D.; Kathiresan, S.; Brown, C. D.; Abou Jamra, R.; LeGuern, E.; Clausen, H.; Rader, D. J. Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents. Cell Metab 2016, 24, 234– 245,  DOI: 10.1016/j.cmet.2016.07.012
      30
      Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents
      Khetarpal, Sumeet A.; Schjoldager, Katrine T.; Christoffersen, Christina; Raghavan, Avanthi; Edmondson, Andrew C.; Reutter, Heiko M.; Ahmed, Bouhouche; Ouazzani, Reda; Peloso, Gina M.; Vitali, Cecilia; Zhao, Wei; Somasundara, Amritha Varshini Hanasoge; Millar, John S.; Park, YoSon; Fernando, Gayani; Livanov, Valentin; Choi, Seungbum; Noe, Eric; Patel, Pritesh; Ho, Siew Peng; Kirchgessner, Todd G.; Wandall, Hans H.; Hansen, Lars; Bennett, Eric P.; Vakhrushev, Sergey Y.; Saleheen, Danish; Kathiresan, Sekar; Brown, Christopher D.; Abou Jamra, Rami; LeGuern, Eric; Clausen, Henrik; Rader, Daniel J.
      Cell Metabolism (2016), 24 (2), 234-245CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)
      Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a regulator of high-d. lipoprotein cholesterol (HDL-C) metab., but the mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss of function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2-deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Addnl. glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs assocd. with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metab. across mammals.
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    31. 31
      de las Rivas, M.; Coelho, H.; Diniz, A.; Lira-Navarrete, E.; Companon, I.; Jimenez-Barbero, J.; Schjoldager, K. T.; Bennett, E. P.; Vakhrushev, S. Y.; Clausen, H.; Corzana, F.; Marcelo, F.; Hurtado-Guerrero, R. Structural Analysis of a GalNAc-T2Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts. Chem.─Eur. J. 2018, 24, 8382– 8392,  DOI: 10.1002/chem.201800701
      31
      Structural Analysis of a GalNAc-T2 Mutant Reveals an Induced-Fit Catalytic Mechanism for GalNAc-Ts
      de las Rivas, Matilde; Coelho, Helena; Diniz, Ana; Lira-Navarrete, Erandi; Companon, Ismael; Jimenez-Barbero, Jesus; Schjoldager, Katrine T.; Bennett, Eric P.; Vakhrushev, Sergey Y.; Clausen, Henrik; Corzana, Francisco; Marcelo, Filipa; Hurtado-Guerrero, Ramon
      Chemistry - A European Journal (2018), 24 (33), 8382-8392CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-d. lipoprotein cholesterol (HDL-C) in humans. Herein, the mol. basis for F104S mutant inactivation has been deciphered. Satn. transfer difference NMR spectroscopy expts. demonstrate that the mutation induces loss of binding to peptide substrates. Anal. of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with mol. dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19F NMR spectroscopy expts. reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation.
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    32. 32
      Daniel, E. J. P.; Las Rivas, M.; Lira-Navarrete, E.; Garcia-Garcia, A.; Hurtado-Guerrero, R.; Clausen, H.; Gerken, T. A. Ser and Thr acceptor preferences of the GalNAc-Ts vary among isoenzymes to modulate mucin-type O-glycosylation. Glycobiology 2020, 30, 910– 922,  DOI: 10.1093/glycob/cwaa036
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      Ser and Thr acceptor preferences of the GalNAc-Ts vary among isoenzymes to modulate mucin-type O-glycosylation
      Daniel, Earnest James Paul; De Las Rivas, Matilde; Lira-Navarrete, Erandi; Garcia-Garcia, Ana; Hurtado-Guerrero, Ramon; Clausen, Henrik; Gerken, Thomas A.
      Glycobiology (2020), 30 (11), 910-922CODEN: GLYCE3; ISSN:1460-2423. (Oxford University Press)
      A family of polypeptide GalNAc-transferases (GalNAc-Ts) initiates mucin-type O-glycosylation, transferring GalNAc onto hydroxyl groups of Ser and Thr residues of target substrates. The 20 GalNAc-T isoenzymes in humans are classified into nine subfamilies according to sequence similarity. GalNAc-Ts select their sites of glycosylation based on weak and overlapping peptide sequence motifs, as well prior substrate O-GalNAc glycosylation at sites both remote (long-range) and neighboring (short-range) the acceptor. Together, these preferences vary among GalNAc-Ts imparting each isoenzyme with its own unique specificity. Studies on the first identified GalNAc-Ts showed Thr acceptors were preferred over Ser acceptors; however studies comparing Thr vs.Ser glycosylation across the GalNAc-T family are lacking. Using a series of identical random peptide substrates, with single Thr or Ser acceptor sites, we detd. the rate differences (Thr/Ser rate ratio) between Thr and Ser substrate glycosylation for 12 isoenzymes (representing 7 GalNAcT subfamilies). These Thr/Ser rate ratios varied across subfamilies, ranging from ~ 2 to ~ 18 (for GalNAc-T4/GalNAc-T12 and GalNAc-T3/GalNAc-T6, resp.), while nearly identical Thr/Ser rate ratios were obsd. for isoenzymes within subfamilies. Furthermore, the Thr/Ser rate ratios did not appreciably vary over a series of fixed sequence substrates of different relative activities, suggesting the ratio is a const. for each isoenzyme against single acceptor substrates. Finally, based on GalNAc-T structures, the different Thr/Ser rate ratios likely reflect differences in the strengths of the Thr acceptor Me group binding to the active site pocket. With this work, another activity that further differentiates substrate specificity among the GalNAc-Ts has been identified.
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    33. 33
      de las Rivas, M.; Paul Daniel, E. J.; Coelho, H.; Lira-Navarrete, E.; Raich, L.; Companon, I.; Diniz, A.; Lagartera, L.; Jimenez-Barbero, J.; Clausen, H.; Rovira, C.; Marcelo, F.; Corzana, F.; Gerken, T. A.; Hurtado-Guerrero, R. Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of GalNAc-T4. ACS Cent. Sci. 2018, 4, 1274– 1290,  DOI: 10.1021/acscentsci.8b00488
      33
      Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of GalNAc-T4
      de las Rivas, Matilde; Paul Daniel, Earnest James; Coelho, Helena; Lira-Navarrete, Erandi; Raich, Lluis; Companon, Ismael; Diniz, Ana; Lagartera, Laura; Jimenez-Barbero, Jesus; Clausen, Henrik; Rovira, Carme; Marcelo, Filipa; Corzana, Francisco; Gerken, Thomas A.; Hurtado-Guerrero, Ramon
      ACS Central Science (2018), 4 (9), 1274-1290CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, resp. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the mol. basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (contg. two α-GalNAc glycosylated Thr (T*), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
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    34. 34
      Lira-Navarrete, E.; de las Rivas, M.; Companon, I.; Pallares, M. C.; Kong, Y.; Iglesias-Fernandez, J.; Bernardes, G. J. L.; Peregrina, J. M.; Rovira, C.; Bernado, P.; Bruscolini, P.; Clausen, H.; Lostao, A.; Corzana, F.; Hurtado-Guerrero, R. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Nat. Commun. 2015, 6, 6937,  DOI: 10.1038/ncomms7937
      34
      Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation
      Lira-Navarrete, Erandi; de las Rivas, Matilde; Companon, Ismael; Pallares, Maria Carmen; Kong, Yun; Iglesias-Fernandez, Javier; Bernardes, Goncalo J. L.; Peregrina, Jesus M.; Rovira, Carme; Bernado, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon
      Nature Communications (2015), 6 (), 6937CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying mol. basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling mol. dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering expts. further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtF2lu7%252FL&md5=882286292a933b2c4c54f9ef700fe087
    35. 35
      de Las Rivas, M.; Lira-Navarrete, E.; Daniel, E. J. P.; Companon, I.; Coelho, H.; Diniz, A.; Jimenez-Barbero, J.; Peregrina, J. M.; Clausen, H.; Corzana, F.; Marcelo, F.; Jimenez-Oses, G.; Gerken, T. A.; Hurtado-Guerrero, R. The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences. Nat. Commun. 2017, 8, 1959,  DOI: 10.1038/s41467-017-02006-0
      35
      The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences
      de Las Rivas Matilde; Lira-Navarrete Erandi; Hurtado-Guerrero Ramon; Lira-Navarrete Erandi; Clausen Henrik; Daniel Earnest James Paul; Gerken Thomas A; Companon Ismael; Peregrina Jesus M; Corzana Francisco; Jimenez-Oses Gonzalo; Coelho Helena; Diniz Ana; Marcelo Filipa; Coelho Helena; Jimenez-Barbero Jesus; Coelho Helena; Jimenez-Barbero Jesus; Jimenez-Barbero Jesus; Gerken Thomas A; Gerken Thomas A; Hurtado-Guerrero Ramon
      Nature communications (2017), 8 (1), 1959 ISSN:.
      The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- and/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2. The GalNAc-T4 structure bound to a monoglycopeptide shows that the GalNAc-binding site of its lectin domain is rotated relative to the homologous GalNAc-T2 structure, explaining their different long-range preferences. Kinetics and molecular dynamics simulations on several GalNAc-T2 flexible linker constructs show altered remote prior glycosylation preferences, confirming that the flexible linker dictates the rotation of the lectin domain, thus modulating the GalNAc-Ts' long-range preferences. This work for the first time provides the structural basis for the different remote prior glycosylation preferences of the GalNAc-Ts.
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    36. 36
      Wang, S.; Mao, Y.; Narimatsu, Y.; Ye, Z.; Tian, W.; Goth, C. K.; Lira-Navarrete, E.; Pedersen, N. B.; Benito-Vicente, A.; Martin, C.; Uribe, K. B.; Hurtado-Guerrero, R.; Christoffersen, C.; Seidah, N. G.; Nielsen, R.; Christensen, E. I.; Hansen, L.; Bennett, E. P.; Vakhrushev, S. Y.; Schjoldager, K. T.; Clausen, H. Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions. J. Biol. Chem. 2018, 293, 7408– 7422,  DOI: 10.1074/jbc.M117.817981
      36
      Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions
      Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K.; Lira-Navarrete, Erandi; Pedersen, Nis B.; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B.; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G.; Nielsen, Rikke; Christensen, Erik I.; Hansen, Lars; Bennett, Eric P.; Vakhrushev, Sergey Y.; Schjoldager, Katrine T.; Clausen, Henrik
      Journal of Biological Chemistry (2018), 293 (19), 7408-7422CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The low-d. lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomols. across cell membranes and barriers. Their functions are esp. relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomols. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites were selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrated that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation was not required for transport and cell-surface expression and stability of these receptors but markedly enhanced LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased the affinity for LDL by ∼5-fold. The mol. basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.
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    37. 37
      Raman, J.; Guan, Y.; Perrine, C. L.; Gerken, T. A.; Tabak, L. A. UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases: completion of the family tree. Glycobiology 2012, 22, 768– 777,  DOI: 10.1093/glycob/cwr183
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      UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases: Completion of the family tree
      Raman, Jayalakshmi; Guan, Yu; Perrine, Cynthia L.; Gerken, Thomas A.; Tabak, Lawrence A.
      Glycobiology (2012), 22 (6), 768-777CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
      The formation of mucin-type O-glycans is initiated by an evolutionarily conserved family of enzymes, the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). The human genome encodes 20 transferases; 17 of which have been characterized functionally. The complexity of the GalNAc-T family reflects the differential patterns of expression among the individual enzyme isoforms and the unique substrate specificities which are required to form the dense arrays of glycans that are essential for mucin function. We report the expression patterns and enzymic activity of the remaining three members of the family and the further characterization of a recently reported isoform, GalNAc-T17. One isoform, GalNAcT-16 that is most homologous to GalNAc-T14, is widely expressed (abundantly in the heart) and has robust polypeptide transferase activity. The second isoform GalNAc-T18, most similar to GalNAc-T8, -T9 and -T19, completes a discrete subfamily of GalNAc-Ts. It is widely expressed and has low, albeit detectable, activity. The final isoform, GalNAc-T20, is most homologous to GalNAc-T11 but lacks a lectin domain and has no detectable transferase activity with the panel of substrates tested. We have also identified and characterized enzymically active splice variants of GalNAc-T13 that differ in the sequence of their lectin domain. The variants differ in their affinities for glycopeptide substrates. Our findings provide a comprehensive view of the complexities of mucin-type O-glycan formation and provide insight into the underlying mechanisms employed to heavily decorate mucins and mucin-like domains with carbohydrate.
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    38. 38
      Revoredo, L.; Wang, S.; Bennett, E. P.; Clausen, H.; Moremen, K. W.; Jarvis, D. L.; Ten Hagen, K. G.; Tabak, L. A.; Gerken, T. A. Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family. Glycobiology 2016, 26, 360– 376,  DOI: 10.1093/glycob/cwv108
      38
      Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family
      Revoredo, Leslie; Wang, Shengjun; Bennett, Eric Paul; Clausen, Henrik; Moremen, Kelley W.; Jarvis, Donald L.; Ten Hagen, Kelly G.; Tabak, Lawrence A.; Gerken, Thomas A.
      Glycobiology (2016), 26 (4), 360-376CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
      A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities against glycopeptide substrates. All but one isoform contains a C-terminal carbohydrate-binding lectin domain whose roles in modulating glycopeptide specificity is just being understood. Study have previously shown for several peptide-preferring isoforms that the presence of a remote Thr-O-Gal-NAc, 6-17 residues from a Ser/Thr acceptor site, may enhance overall catalytic activity in an N- or C-terminal direction. This enhancement varies with isoform and is attributed to Thr-O-GalNAc interactions at the lectin domain. This work now report on the glycopeptide substrate utilization of a series of glycopeptide (human-ppGalNAc-T4, T7, T10, T12 and fly PGANT7) and peptide-preferring transferases (T2, T3 and T5) by exploiting a series of random glycopeptide substrates designed to probe the functions of their catalytic and lectin domains. Glycosylation was obsd. at the -3, -1 and +1 residues relative to a neighboring Thr-O-GalNAc, depending on isoform, which this study attribute to specific Thr-O-GalNAc binding at the catalytic domain. Addnl., these glycopeptide-preferring isoforms show remote lectin domain-assisted Thr-O-GalNAc enhancements that vary from modest to none. This paper conclude that the glycopeptide specificity of the glycopeptide-preferring isoforms predominantly resides in their catalytic domain but may be further modulated by remote lectin domain interactions. These studies further demonstrate that both domains of the ppGalNAc-Ts have specialized and unique functions that work in concert to control and order mucin-type O-glycosylation.
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    39. 39
      Fernandez, A. J.; Daniel, E. J. P.; Mahajan, S. P.; Gray, J. J.; Gerken, T. A.; Tabak, L. A.; Samara, N. L. The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 20404– 20410,  DOI: 10.1073/pnas.1902211116
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      The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function
      Fernandez, Amy J.; Paul Daniel, Earnest James; Mahajan, Sai Pooja; Gray, Jeffrey J.; Gerken, Thomas A.; Tabak, Lawrence A.; Samara, Nadine L.
      Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (41), 20404-20410CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochem. studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the mol. basis for this distinct substrate selectivity remains elusive. Here we examine the mol. basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addn., the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFGhtbzF&md5=08f79b942a6f904a58e5f42f1bfe250a
    40. 40
      Pedersen, J. W.; Bennett, E. P.; Schjoldager, K. T.; Meldal, M.; Holmer, A. P.; Blixt, O.; Clo, E.; Levery, S. B.; Clausen, H.; Wandall, H. H. Lectin domains of polypeptide GalNAc transferases exhibit glycopeptide binding specificity. J. Biol. Chem. 2011, 286, 32684– 32696,  DOI: 10.1074/jbc.M111.273722
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      Lectin Domains of Polypeptide GalNAc Transferases Exhibit Glycopeptide Binding Specificity
      Pedersen, Johannes W.; Bennett, Eric P.; Schjoldager, Katrine T.-B. G.; Meldal, Morten; Holmer, Andreas P.; Blixt, Ola; Clo, Emiliano; Levery, Steven B.; Clausen, Henrik; Wandall, Hans H.
      Journal of Biological Chemistry (2011), 286 (37), 32684-32696, S32684/1-S32684/2CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, resp. Thus far, only the catalytic domain has been shown to have peptide sequence specificity, whereas the primary function of the lectin domain is to increase affinity to previously glycosylated substrates. Whether the lectin domain also has peptide sequence selectivity has remained unclear. Using a glycopeptide array with a library of synthetic and recombinant glycopeptides based on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examd. the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate an addnl. level of complexity in the initiation step of O-glycosylation by GalNAc-Ts.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFCju7bK&md5=f92d759d50c5fc44f419feac493eaa99
    41. 41
      Coelho, H.; Rivas, M. d. l.; Grosso, A. S.; Diniz, A.; Soares, C. O.; Francisco, R. A.; Dias, J. S.; Companon, I.; Sun, L.; Narimatsu, Y.; Vakhrushev, S. Y.; Clausen, H.; Cabrita, E. J.; Jimenez-Barbero, J.; Corzana, F.; Hurtado-Guerrero, R.; Marcelo, F. Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling. JACS Au 2022, 2, 631– 645,  DOI: 10.1021/jacsau.1c00529
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      Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling
      Coelho, Helena; Rivas, Matilde de las; Grosso, Ana S.; Diniz, Ana; Soares, Catia O.; Francisco, Rodrigo A.; Dias, Jorge S.; Companon, Ismael; Sun, Lingbo; Narimatsu, Yoshiki; Vakhrushev, Sergey Y.; Clausen, Henrik; Cabrita, Eurico J.; Jimenez-Barbero, Jesus; Corzana, Francisco; Hurtado-Guerrero, Ramon; Marcelo, Filipa
      JACS Au (2022), 2 (3), 631-645CODEN: JAAUCR; ISSN:2691-3704. (American Chemical Society)
      The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by mol. modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addn. of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our expts. suggest that during this process, neighboring tandem repeats are crit. for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addn., in-cell anal. shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodol. established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to det. the specificity toward complex mucin acceptor substrates.
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    42. 42
      Corzana, F.; Busto, J. H.; Jiménez-Osés, G.; Asensio, J. L.; Jiménez-Barbero, J.; Peregrina, J. M.; Avenoza, A. New Insights into α-GalNAc–Ser Motif: Influence of Hydrogen Bonding versus Solvent Interactions on the Preferred Conformation. J. Am. Chem. Soc. 2006, 128, 14640– 14648,  DOI: 10.1021/ja064539u
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      New Insights into α-GalNAc-Ser Motif: Influence of Hydrogen Bonding versus Solvent Interactions on the Preferred Conformation
      Corzana, Francisco; Busto, Jesus H.; Jimenez-Oses, Gonzalo; Asensio, Juan L.; Jimenez-Barbero, Jesus; Peregrina, Jesus M.; Avenoza, Alberto
      Journal of the American Chemical Society (2006), 128 (45), 14640-14648CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The structural features of the mucin-type simplest model, namely, the glycopeptide α-O-GalNAc-L-Ser diamide, have been investigated by combining NMR spectroscopy, mol. dynamics simulations, and DFT calcns. In contrast to previous reports, the study reveals that intramol. hydrogen bonds between sugar and peptide residues are very weak and, as a consequence, not strong enough to maintain the well-defined conformation of this type of mol. In fact, the obsd. conformation of this model glycopeptide can be satisfactorily explained by the presence of water pockets/bridges between the sugar and the peptide moieties. Addnl., DFT calcns. reveal that not only the bridging water mols. but also the surrounding water mols. in the first hydration shell are essential to keep the existing conformation.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFWju7rK&md5=dc2408fe1e0db83d44d34a426de93fdd
    43. 43
      Shon, D. J.; Fernandez, D.; Riley, N. M.; Ferracane, M. J.; Bertozzi, C. R. Structure-guided mutagenesis of a mucin-selective metalloprotease from Akkermansia muciniphila alters substrate preferences. J. Biol. Chem. 2022, 298, 101917,  DOI: 10.1016/j.jbc.2022.101917
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      Structure-guided mutagenesis of a mucin-selective metalloprotease from Akkermansia muciniphila alters substrate preferences
      Shon, D. Judy; Fernandez, Daniel; Riley, Nicholas M.; Ferracane, Michael J.; Bertozzi, Carolyn R.
      Journal of Biological Chemistry (2022), 298 (5), 101917CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
      Akkermansia muciniphila, a mucin-degrading microbe found in the human gut, is often assocd. with pos. health outcomes. The abundance of A. muciniphila is modulated by the presence and accessibility of nutrients, which can be derived from diet or host glycoproteins. In particular, the ability to degrade host mucins, a class of proteins carrying densely O-glycosylated domains, provides a competitive advantage in the sustained colonization of niche mucosal environments. Although A. muciniphila is known to rely on mucins as a carbon and nitrogen source, the enzymic machinery used by this microbe to process mucins in the gut is not yet fully characterized. Here, we focus on the mucin-selective metalloprotease, Amuc_0627 (AM0627), which is known to cleave between adjacent residues carrying truncated core 1 O-glycans. We showed that this enzyme is capable of degrading purified mucin 2 (MUC2), the major protein component of mucus in the gut. An X-ray crystal structure of AM0627 (1.9 Å resoln.) revealed O-glycan-binding residues that are conserved between structurally characterized enzymes from the same family. We further rationalized the substrate cleavage motif using mol. modeling to identify nonconserved glycan-interacting residues. We conclude that mutagenesis of these residues resulted in altered substrate preferences down to the glycan level, providing insight into the structural determinants of O-glycan recognition.
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    44. 44
      Konstantinidi, A.; Nason, R.; Caval, T.; Sun, L.; Soerensen, D. M.; Furukawa, S.; Ye, Z.; Vincentelli, R.; Narimatsu, Y.; Vakhrushev, S. Y.; Clausen, H. Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells. J. Biol. Chem. 2022, 298, 101784,  DOI: 10.1016/j.jbc.2022.101784
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      Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells
      Konstantinidi, Andriana; Nason, Rebecca; Caval, Tomislav; Sun, Lingbo; Soerensen, Daniel M.; Furukawa, Sanae; Ye, Zilu; Vincentelli, Renaud; Narimatsu, Yoshiki; Vakhrushev, Sergey Y.; Clausen, Henrik
      Journal of Biological Chemistry (2022), 298 (4), 101784CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
      Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and prodn. of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific anal. for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom-up anal. of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider anal. of native mucins.
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    45. 45
      Zhou, D.; Xu, L.; Huang, W.; Tonn, T. Epitopes of MUC1 Tandem Repeats in Cancer as Revealed by Antibody Crystallography: Toward Glycopeptide Signature-Guided Therapy. Molecules 2018, 23, 1326,  DOI: 10.3390/molecules23061326
      45
      Epitopes of MUC1 tandem repeats in cancer as revealed by antibody crystallography: toward glycopeptide signature-guided therapy
      Zhou, Dapeng; Xu, Lan; Huang, Wei; Tonn, Torsten
      Molecules (2018), 23 (6), 1326/1-1326/27CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      Abnormally O-glycosylated MUC1 tandem repeat glycopeptide epitopes expressed by multiple types of cancer have long been attractive targets for therapy in the race against genetic mutations of tumor cells. Glycopeptide signature-guided therapy might be a more promising avenue than mutation signature-guided therapy. Three O-glycosylated peptide motifs, PDTR, GSTA, and GVTS, exist in a tandem repeat HGVTSAPDTRPAPGSTAPPA, contg. five O-glycosylation sites. The exact peptide and sugar residues involved in antibody binding are poorly defined. Co-crystal structures of glycopeptides and resp. monoclonal antibodies are very few. Here we review 3 groups of monoclonal antibodies: antibodies which only bind to peptide portion, antibodies which only bind to sugar portion, and antibodieswhich bind to both peptide and sugar portions. The antigenicity of peptide and sugar portions of glyco-MUC1 tandemrepeat were analyzed according to available biochem. and structural data, esp. the GSTA and GVTS motifs independent from the most studied PDTR. Tn is focused as a peptide-modifying residue in vaccine design, to induce glycopeptide-binding antibodies with cross reactivity to Tn-related tumor glycans, but not glycans of healthy cells. The unique requirement for the designs of antibody in antibody-drug conjugate, bi-specific antibodies, and chimeric antigen receptors are also discussed.
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    46. 46
      Posey, A. D.; Schwab, R. D.; Boesteanu, A. C.; Steentoft, C.; Mandel, U.; Engels, B.; Stone, J. D.; Madsen, T. D.; Schreiber, K.; Haines, K. M.; Cogdill, A. P.; Chen, T. J.; Song, D.; Scholler, J.; Kranz, D. M.; Feldman, M. D.; Young, R.; Keith, B.; Schreiber, H.; Clausen, H.; Johnson, L. A.; June, C. H. Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma. Immunity 2016, 44, 1444– 1454,  DOI: 10.1016/j.immuni.2016.05.014
      46
      Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma
      Posey, Avery D. Jr.; Schwab, Robert D.; Boesteanu, Alina C.; Steentoft, Catharina; Mandel, Ulla; Engels, Boris; Stone, Jennifer D.; Madsen, Thomas D.; Schreiber, Karin; Haines, Kathleen M.; Cogdill, Alexandria P.; Chen, Taylor J.; Song, Decheng; Scholler, John; Kranz, David M.; Feldman, Michael D.; Young, Regina; Keith, Brian; Schreiber, Hans; Clausen, Henrik; Johnson, Laura A.; June, Carl H.
      Immunity (2016), 44 (6), 1444-1454CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)
      Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematol. cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-assocd. Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.
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    47. 47
      Karsten, U. Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1). Glycobiology 2004, 14, 681– 692,  DOI: 10.1093/glycob/cwh090
      47
      Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1)
      Karsten, Uwe; Serttas, Nida; Paulsen, Hans; Danielczyk, Antje; Goletz, Steffen
      Glycobiology (2004), 14 (8), 681-692CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
      Glycosylation dets. essential biol. functions of epithelial mucins in health and disease. We report on the influence of glycosylation of the immunodominant DTR motif of MUC1 on its antigenicity. Sets of novel glycopeptides were synthesized that enabled us to examine sole and combined effects of peptide length (no. of repeats) and O-glycosylation with GalNAc at the DTR motif on the binding patterns of 22 monoclonal antibodies recognizing this motif. In case of unglycosylated peptides almost all antibodies bound better to multiple MUC1 tandem repeats. Glycosylation at the DTR led to enhanced binding in 11 cases, whereas 10 antibodies were not influenced in binding, and one was inhibited. In nine of the former cases both length and DTR glycosylation were additive in their influence on antibody binding, suggesting that both effects are different. Improved binding to the glycosylated DTR motif was exclusively found with antibodies generated against tumor-derived MUC1. Based on these data a tumor-specific MUC1 epitope is defined comprising the ...PDTRP... sequence in a particular conformation essentially detd. by O-glycosylation at its threonine with either GalNAcα1 or a related short glycan. The results can find application in the field of MUC1-based immunotherapy.
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    48. 48
      Brooks, C. L.; Schietinger, A.; Borisova, S. N.; Kufer, P.; Okon, M.; Hirama, T.; Mackenzie, C. R.; Wang, L. X.; Schreiber, H.; Evans, S. V. Antibody recognition of a unique tumor-specific glycopeptide antigen. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10056– 10061,  DOI: 10.1073/pnas.0915176107
      48
      Antibody recognition of a unique tumor-specific glycopeptide antigen
      Brooks, Cory L.; Schietinger, Andrea; Borisova, Svetlana N.; Kufer, Peter; Okon, Mark; Hirama, Tomoko; MacKenzie, C. Roger; Wang, Lai-Xi; Schreiber, Hans; Evans, Stephen V.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (22), 10056-10061, S10056/1-S10056/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Aberrant glycosylation and the over expression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-assocd. oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the "Tn antigen.". Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been obsd. on embryonic and non-malignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide contg. the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no obsd. physiol. cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of antigen.
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    49. 49
      Movahedin, M.; Brooks, T. M.; Supekar, N. T.; Gokanapudi, N.; Boons, G.-J.; Brooks, C. L. Glycosylation of MUC1 influences the binding of a therapeutic antibody by altering the conformational equilibrium of the antigen. Glycobiology 2016, 27, 677– 687,  DOI: 10.1093/glycob/cww131
      There is no corresponding record for this reference.
    50. 50
      Martínez-Sáez, N.; Castro-Lopez, J.; Valero-Gonzalez, J.; Madariaga, D.; Companon, I.; Somovilla, V. J.; Salvado, M.; Asensio, J. L.; Jimenez-Barbero, J.; Avenoza, A.; Busto, J. H.; Bernardes, G. J. L.; Peregrina, J. M.; Hurtado-Guerrero, R.; Corzana, F. Deciphering the non-equivalence of serine and threonine O-glycosylation points: Implications for molecular recognition of the Tn antigen by an anti-MUC1 antibody. Angew. Chem., Int. Ed. 2015, 54, 9830– 9834,  DOI: 10.1002/anie.201502813
      50
      Deciphering the non-equivalence of serine and threonine O-glycosylation points: Implications for molecular recognition of the Tn antigen by an anti-MUC1 antibody
      Martinez-Saez, Nuria; Castro-Lopez, Jorge; Valero-Gonzalez, Jessika; Madariaga, David; Companon, Ismael; Somovilla, Victor J.; Salvado, Miriam; Asensio, Juan L.; Jimenez-Barbero, Jesus; Avenoza, Alberto; Busto, Jesus H.; Bernardes, Goncalo J. L.; Peregrina, Jesus M.; Hurtado-Guerrero, Ramon; Corzana, Francisco
      Angewandte Chemie, International Edition (2015), 54 (34), 9830-9834CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at at. resoln. For the α-O-GalNAc-Ser deriv., the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also obsd. in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the Me group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in mol. recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.
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    51. 51
      Coelho, H.; Matsushita, T.; Artigas, G.; Hinou, H.; Cañada, F. J.; Lo-Man, R.; Leclerc, C.; Cabrita, E. J.; Jiménez-Barbero, J.; Nishimura, S.-I.; Garcia-Martín, F.; Marcelo, F. The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR. J. Am. Chem. Soc. 2015, 137, 12438– 12441,  DOI: 10.1021/jacs.5b06787
      51
      The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR
      Coelho, Helena; Matsushita, Takahiko; Artigas, Gerard; Hinou, Hiroshi; Canada, F. Javier; Lo-Man, Richard; Leclerc, Claude; Cabrita, Eurico J.; Jimenez-Barbero, Jesus; Nishimura, Shin-Ichiro; Garcia-Martin, Fayna; Marcelo, Filipa
      Journal of the American Chemical Society (2015), 137 (39), 12438-12441CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The identification of MUC1 tumor-assocd. Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and satn. transfer difference NMR, the authors have characterized the fine-epitope mapping of a MUC1 chem. library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides contg. the Tn-Ser antigen rather than the Tn-Thr analog, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.
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    52. 52
      Wakui, H.; Tanaka, Y.; Ose, T.; Matsumoto, I.; Kato, K.; Min, Y.; Tachibana, T.; Sato, M.; Naruchi, K.; Martin, F. G.; Hinou, H.; Nishimura, S.-I. A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes. Chem. Sci. 2020, 11, 4999– 5006,  DOI: 10.1039/D0SC00317D
      52
      A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes
      Wakui, Hajime; Tanaka, Yoshikazu; Ose, Toyoyuki; Matsumoto, Isamu; Kato, Koji; Min, Yao; Tachibana, Taro; Sato, Masaharu; Naruchi, Kentaro; Martin, Fayna Garcia; Hinou, Hiroshi; Nishimura, Shin-Ichiro
      Chemical Science (2020), 11 (19), 4999-5006CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Aberrantly truncated immature O-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly assocd. with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immature O-glycans, no anti-MUC1 antibody recognizes carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure anal., epitope mapping and biochem. anal. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognizes specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its soln. NMR structure, an extended conformation induced by site-specific glycosylation.
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    53. 53
      Macias-Leon, J.; Bermejo, I. A.; Asin, A.; Garcia-Garcia, A.; Companon, I.; Jimenez-Moreno, E.; Coelho, H.; Mangini, V.; Albuquerque, I. S.; Marcelo, F.; Asensio, J. L.; Bernardes, G. J. L.; Joshi, H. J.; Fiammengo, R.; Blixt, O.; Hurtado-Guerrero, R.; Corzana, F. Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody. Chem. Commun. 2020, 56, 15137– 15140,  DOI: 10.1039/D0CC06349E
      53
      Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody
      Macias-Leon, Javier; Bermejo, Iris A.; Asin, Alicia; Garcia-Garcia, Ana; Companon, Ismael; Jimenez-Moreno, Ester; Coelho, Helena; Mangini, Vincenzo; Albuquerque, Ines S.; Marcelo, Filipa; Asensio, Juan L.; Bernardes, Goncalo J. L.; Joshi, Hiren J.; Fiammengo, Roberto; Blixt, Ola; Hurtado-Guerrero, Ramon; Corzana, Francisco
      Chemical Communications (Cambridge, United Kingdom) (2020), 56 (96), 15137-15140CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      The mol. basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chem. design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.
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    54. 54
      Yoshimura, Y.; Denda-Nagai, K.; Takahashi, Y.; Nagashima, I.; Shimizu, H.; Kishimoto, T.; Noji, M.; Shichino, S.; Chiba, Y.; Irimura, T. Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies. Sci. Rep. 2019, 9, 16641,  DOI: 10.1038/s41598-019-53052-1
      54
      Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies
      Yoshimura Yayoi; Kishimoto Toshimitsu; Yoshimura Yayoi; Takahashi Yoshie; Chiba Yasunori; Denda-Nagai Kaori; Noji Miki; Irimura Tatsuro; Nagashima Izuru; Shimizu Hiroki; Shichino Shigeyuki
      Scientific reports (2019), 9 (1), 16641 ISSN:.
      Anti-mucin1 (MUC1) antibodies have long been used clinically in cancer diagnosis and therapy and specific bindings of some of them are known to be dependent on the differential glycosylation of MUC1. However, a systematic comparison of the binding specificities of anti-MUC1 antibodies was not previously conducted. Here, a total of 20 glycopeptides including the tandem repeat unit of MUC1, APPAHGVTSAPDTRPAPGSTAPPAHGV with GalNAc (Tn-antigen), Galβ1-3GalNAc (T-antigen), NeuAcα2-3Galβ1-3GalNAc (sialyl-T-antigen), or NeuAcα2-6GalNAc (sialyl-Tn-antigen) at each threonine or serine residue were prepared by a combination of chemical glycopeptide synthesis and enzymatic extension of carbohydrate chains. These glycopeptides were tested by the enzyme-linked immunosorbent assay (ELISA) for their capacity to bind 13 monoclonal antibodies (mAbs) known to be specific for MUC1. The results indicated that anti-MUC1 mAbs have diverse specificities but can be classified into a few characteristic groups based on their binding pattern toward glycopeptides in some cases having a specific glycan at unique glycosylation sites. Because the clinical significance of some of these antibodies was already established, the structural features identified by these antibodies as revealed in the present study should provide useful information relevant to their further clinical use and the biological understanding of MUC1.
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    55. 55
      Corzana, F.; Busto, J. H.; Jiménez-Osés, G.; García De Luis, M.; Asensio, J. L.; Jiménez-Barbero, J.; Peregrina, J. M.; Avenoza, A. Serine versus Threonine Glycosylation: The Methyl Group Causes a Drastic Alteration on the Carbohydrate Orientation and on the Surrounding Water Shell. J. Am. Chem. Soc. 2007, 129, 9458– 9467,  DOI: 10.1021/ja072181b
      55
      Serine versus Threonine Glycosylation: The Methyl Group Causes a Drastic Alteration on the Carbohydrate Orientation and on the Surrounding Water Shell
      Corzana, Francisco; Busto, Jesus H.; Jimenez-Oses, Gonzalo; Garcia de Luis, Marisa; Asensio, Juan L.; Jimenez-Barbero, Jesus; Peregrina, Jesus M.; Avenoza, Alberto
      Journal of the American Chemical Society (2007), 129 (30), 9458-9467CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Different behavior has been obsd. for the ψ torsion angle of the glycosidic linkages of D-GalNAc-Ser and D-GalNAc-Thr motifs, allowing the carbohydrate moiety to adopt a completely different orientation. In addn., the fact that the water pockets found in α-D-GalNAc-Thr differ from those obtained for its serine analog could be related to the different capability that the two model glycopeptides have to structure the surrounding water. This fact could have important biol. inferences (i.e., antifreeze activity).
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    56. 56
      (a) Clark, D.; Mao, L. Cancer biomarker discovery: lectin-based strategies targeting glycoproteins. Dis. Markers 2012, 33, 1– 10,  DOI: 10.1155/2012/308738
      56a
      Cancer biomarker discovery: Lectin-based strategies targeting glycoproteins
      Clark, David; Mao, Li
      Disease Markers (2012), 33 (1), 1-10CODEN: DMARD3; ISSN:0278-0240. (IOS Press)
      A review. Biomarker discovery can identify mol. markers in various cancers that can be used for detection, screening, diagnosis, and monitoring of disease progression. Lectin-affinity is a technique that can be used for the enrichment of glycoproteins from a complex sample, facilitating the discovery of novel cancer biomarkers assocd. with a disease state.
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      (b) Lastovickova, M.; Strouhalova, D.; Bobalova, J. Use of Lectin-based Affinity Techniques in Breast Cancer Glycoproteomics: A Review. J. Proteome Res. 2020, 19, 1885– 1899,  DOI: 10.1021/acs.jproteome.9b00818
      56b
      Use of Lectin-based Affinity Techniques in Breast Cancer Glycoproteomics: A Review
      Lastovickova, Marketa; Strouhalova, Dana; Bobalova, Janette
      Journal of Proteome Research (2020), 19 (5), 1885-1899CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      A review. Changes in glycoprotein content, altered glycosylations, and aberrant glycan structures are increasingly recognized as cancer hallmarks. Because breast cancer is one of the most common causes of cancer deaths in the world, it is highly urgent to find other reliable biomarkers for its initial diagnosis and to learn as much as possible about this disease. In this Review, the applications of lectins to a screening of potential breast cancer biomarkers published during recent years are overviewed. These data provide a deeper insight into the use of modern strategies, technologies, and scientific knowledge in glycoproteomic breast cancer research. Particular attention is concd. on the use of lectin-based affinity techniques, applied independently or most frequently in combination with mass spectrometry, as an effective tool for the targeting, sepn., and reliable identification of glycoprotein mols. Individual procedures and lectins used in published glycoproteomic studies of breast-cancer-related glycoproteins are discussed. The summarized approaches have the potential for use in diagnostic and predictive applications. Finally, the use of lectins is briefly discussed from the view of their future applications in the anal. of glycoproteins in cancer.
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    57. 57
      de Oliveira Figueirôa, E.; Albuquerque da Cunha, C. R.; Albuquerque, P. B. S.; de Paula, R. A.; Aranda-Souza, M. A.; Alves, M. S.; Zagmignan, A.; Carneiro-da-Cunha, M. G.; Nascimento da Silva, L. C.; dos Santos Correia, M. T. Lectin-carbohydrate interactions: Implications for the development of new anticancer agents. Curr. Med. Chem. 2017, 24, 3667– 3680,  DOI: 10.2174/0929867324666170523110400
      57
      Lectin-Carbohydrate Interactions: Implications for the Development of New Anticancer Agents
      de Oliveira Figueiroa, Evellyne; Albuquerque da Cunha, Cassia Regina; Albuquerque, Priscilla B. S.; de Paula, Raiana Apolinario; Aranda-Souza, Mary Angela; Alves, Matheus Silva; Zagmignan, Adrielle; Carneiro-da-Cunha, Maria G.; Nascimento da Silva, Luis Claudio; dos Santos Correia, Maria Tereza
      Current Medicinal Chemistry (2017), 24 (34), 3667-3680CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
      Lectins are a large group of proteins found in animals, plants, fungi, and bacteria that recognize specific carbohydrate targets and play an important role in cell recognition and communication, host-pathogen interactions, embryogenesis, and tissue development. Recently, lectins have emerged as important biomedical tools that have been used in the development of immunomodulatory, antipathogenic, and anticancer agents. Several lectins have been shown to have the ability to discriminate between normal cells and tumor cells as a result of their different glycosylation patterns. Furthermore, the specific binding of lectins to cancer cells has been shown to trigger mechanisms that can promote the death of these abnormal cells. Here, we review the importance of lectins-carbohydrates interactions in cancer therapy and diagnosis. We examine the use of lectins in the modification of nanoparticles (liposomes, solid lipid nanoparticles and other polymers) for anticancer drug delivery. The development of drug delivery systems (liposomes, alginate/chitosan microcapsules, alginate beads) carrying some antitumor lectins is also discussed. In these cases, the processes of cell death induced by these antitumor lectins were also showed (if available). In both cases (lectin-conjugated polymers or encapsulated lectins), these new pharmaceutical prepns. showed improved intracellular delivery, bioavailability and targetability leading to enhanced therapeutic index and significantly less side effects.
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    58. 58
      Gabba, A.; Bogucka, A.; Luz, J. G.; Diniz, A.; Coelho, H.; Corzana, F.; Cañada, F. J.; Marcelo, F.; Murphy, P. V.; Birrane, G. Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen. Biochemistry 2021, 60, 1327– 1336,  DOI: 10.1021/acs.biochem.1c00009
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      Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen
      Gabba, Adele; Bogucka, Agnieszka; Luz, John G.; Diniz, Ana; Coelho, Helena; Corzana, Francisco; Canada, Francisco Javier; Marcelo, Filipa; Murphy, Paul V.; Birrane, Gabriel
      Biochemistry (2021), 60 (17), 1327-1336CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The human macrophage galactose lectin (MGL) is an endocytic type ii transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N-acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins were identified, the mol. basis for substrate recognition has remained elusive due to the lack of high-resoln. structures. Here the authors present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-assocd. Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, addnl. calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the mol. basis for preferential binding of N-acetylgalactosamine over galactose and prompted the reevaluation of the binding modes previously proposed in soln. Satn. transfer difference NMR data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in soln. Models of MGL1 and MGL2, the mouse homologs of MGL, explain how these proteins might recognize LewisX and GalNAc, resp.
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    59. 59
      Lescar, J.; Sanchez, J.-F.; Audfray, A.; Coll, J.-L.; Breton, C.; Mitchell, E. P.; Imberty, A. Structural basis for recognition of breast and colon cancer epitopes Tn antigen and Forssman disaccharide by Helix pomatia lectin. Glycobiology 2007, 17, 1077– 1083,  DOI: 10.1093/glycob/cwm077
      59
      Structural basis for recognition of breast and colon cancer epitopes Tn antigen and Forssman disaccharide by Helix pomatia lectin
      Lescar, Julien; Sanchez, Jean-Frederic; Audfray, Aymeric; Coll, Jean-Luc; Breton, Christelle; Mitchell, Edward P.; Imberty, Anne
      Glycobiology (2007), 17 (10), 1077-1083CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
      Helix pomatia agglutinin (HPA) is a lectin that has been used extensively in histopathol., since its binding to tissue sections from breast and colon cancers is correlated with the worst prognosis for the patients. The lectin recognizes α-D-N-acetylgalactosamine (αGalNAc) contg. epitopes which are only present in cancer cell lines having a high likelihood to undergo metastasis, such as the HT29 cancer colon cell line. Several breast cancer cell lines have also been shown to be labeled, although IGROV1, an ovarian cancer cell line, is not. Inhibition studies, using GalNAc monosaccharides, are reported here, showing that the labeling is dependent upon the presence of carbohydrate epitopes. The crystal structures of the lectin complexed with two GalNAc contg. epitopes assocd. with cancer, the Tn (αGalNAc-Ser) and Forssman (αGalNAc1-3GalNAc) antigens, show the lectin's specificity for GalNAc is due to a particular network of hydrogen bonds. A histidine residue makes hydrophobic contact with the aglycon, rationalizing the preference for GalNAc bearing an addnl. sugar or amino acid in the α position. These structures provide the mol. basis for the use of HPA in metastasis research.
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    60. 60
      Maveyraud, L.; Niwa, H.; Guillet, V.; Svergun, D. I.; Konarev, P. V.; Palmer, R. A.; Peumans, W. J.; Rougé, P.; Van Damme, E. J.; Reynolds, C. D.; Mourey, L. Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra. Proteins 2009, 75, 89– 103,  DOI: 10.1002/prot.22222
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      Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra
      Maveyraud, Laurent; Niwa, Hideaki; Guillet, Valerie; Svergun, Dmitri I.; Konarev, Peter V.; Palmer, Rex A.; Peumans, Willy J.; Rouge, Pierre; Van Damme, Els J. M.; Reynolds, Colin D.; Mourey, Lionel
      Proteins: Structure, Function, and Bioinformatics (2009), 75 (1), 89-103CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional x-ray structure of SNA-II has been detd. in two distinct crystal forms, hexagonal and tetragonal, at 1.90 Å and 1.35 Å, resp. In both crystal forms, the SNA-II mol. folds into two linked β-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is obsd. at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resoln. structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, α1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were detd. at 1.55 Å resoln. or better. Binding is obsd. in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, addnl. interactions with the protein involve the O6 atom. Anal. gel filtration, small angle x-ray scattering studies and crystal packing anal. indicate that, although some oligomeric species are present, the monomeric species predominate in soln.
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    61. 61
      Kulkarni, K. A.; Sinha, S.; Katiyar, S.; Surolia, A.; Vijayan, M.; Suguna, K. Structural basis for the specificity of basic winged bean lectin for the Tn-antigen: A crystallographic, thermodynamic and modelling study. FEBS Lett. 2005, 579, 6775– 6780,  DOI: 10.1016/j.febslet.2005.11.011
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      Structural basis for the specificity of basic winged bean lectin for the Tn-antigen: A crystallographic, thermodynamic and modelling study
      Kulkarni, Kiran A.; Sinha, Sharmistha; Katiyar, Samiksha; Surolia, Avadhesha; Vijayan, Mamannamana; Suguna, Kaza
      FEBS Letters (2005), 579 (30), 6775-6780CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
      The crystal structure of winged bean basic agglutinin in complex with GalNAc-α-O-Ser (Tn-antigen) has been elucidated at 2.35 Å resoln. in order to characterize the mode of binding of Tn-antigen with the lectin. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its Me group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qual. explanation for the thermodn. parameters of the complexation of the lectin with Tn-antigen. Modeling studies indicate the possibility of an addnl. hydrogen bond with the lectin when the antigen is part of a glycoprotein.
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    62. 62
      Sousa, B. L.; Silva Filho, J. C.; Kumar, P.; Pereira, R. I.; Łyskowski, A.; Rocha, B. A.; Delatorre, P.; Bezerra, G. A.; Nagano, C. S.; Gruber, K.; Cavada, B. S. High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins. Int. J. Biochem. Cell Biol. 2015, 59, 103– 110,  DOI: 10.1016/j.biocel.2014.12.002
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      High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins
      Sousa, Bruno Lopes; Silva Filho, Jose Caetano; Kumar, Prashant; Pereira, Ronniery Ilario; Lyskowski, Andrzej; Rocha, Bruno Anderson Matias; Delatorre, Plinio; Bezerra, Gustavo Arruda; Nagano, Celso Shiniti; Gruber, Karl; Cavada, Benildo Sousa
      International Journal of Biochemistry & Cell Biology (2015), 59 (), 103-110CODEN: IJBBFU; ISSN:1357-2725. (Elsevier Ltd.)
      Plant lectins have been studied as histol. markers and promising antineoplastic mols. for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been detd. at the resoln. of 1.4 Å and 1.7 Å, resp. Mol. docking anal. of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addn. to the specific biol. effects of VML, the structural and binding similarities between it and other lectins commonly used as histol. markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.
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    63. 63
      Lubkowski, J.; Durbin, S. V.; Silva, M. C. C.; Farnsworth, D.; Gildersleeve, J. C.; Oliva, M. L. V.; Wlodawer, A. Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth. FEBS J. 2017, 284, 429– 450,  DOI: 10.1111/febs.13989
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      Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth
      Lubkowski, Jacek; Durbin, Sarah V.; Silva, Mariana C. C.; Farnsworth, David; Gildersleeve, Jeffrey C.; Oliva, Maria Luiza V.; Wlodawer, Alexander
      FEBS Journal (2017), 284 (3), 429-450CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      Lectins have been used at length for basic research and clin. applications. New insights into the mol. recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallog. to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in soln. and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have esp. strong binding (apparent Kd = 0.5-1.0 nM) to the tumor-assocd. Tn antigen. High-resoln. crystal structures were detd. for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive anal. of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the mol. recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. Database : Structural data are available in the PDB under the accession nos. , , , , , , , and .
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    64. 64
      Madariaga, D.; Martínez-Sáez, N.; Somovilla, V. J.; Coelho, H.; Valero-González, J.; Castro-López, J.; Asensio, J. L.; Jiménez-Barbero, J.; Busto, J. H.; Avenoza, A.; Marcelo, F.; Hurtado-Guerrero, R.; Corzana, F.; Peregrina, J. M. Detection of tumor-associated glycopeptides by lectins: the peptide context modulates carbohydrate recognition. ACS Chem. Biol. 2015, 10, 747– 756,  DOI: 10.1021/cb500855x
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      Detection of Tumor-Associated Glycopeptides by Lectins: The Peptide Context Modulates Carbohydrate Recognition
      Madariaga, David; Martinez-Saez, Nuria; Somovilla, Victor J.; Coelho, Helena; Valero-Gonzalez, Jessika; Castro-Lopez, Jorge; Asensio, Juan L.; Jimenez-Barbero, Jesus; Busto, Jesus H.; Avenoza, Alberto; Marcelo, Filipa; Hurtado-Guerrero, Ramon; Corzana, Francisco; Peregrina, Jesus M.
      ACS Chemical Biology (2015), 10 (3), 747-756CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is obsd. when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide-lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.
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    65. 65
      Madariaga, D.; Martínez-Sáez, N.; Somovilla, V. J.; García-García, L.; Berbis, M. Á.; Valero-Gónzalez, J.; Martín-Santamaría, S.; Hurtado-Guerrero, R.; Asensio, J. L.; Jiménez-Barbero, J.; Avenoza, A.; Busto, J. H.; Corzana, F.; Peregrina, J. M. Serine versus Threonine Glycosylation with α-O-GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins. Chem. Eur. J. 2014, 20, 12616– 12627,  DOI: 10.1002/chem.201403700
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      Serine versus Threonine Glycosylation with α-O-GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins
      Madariaga, David; Martinez-Saez, Nuria; Somovilla, Victor J.; Garcia-Garcia, Laura; Berbis, M. Alvaro; Valero-Gonzalez, Jessika; Martin-Santamaria, Sonsoles; Hurtado-Guerrero, Ramon; Asensio, Juan L.; Jimenez-Barbero, Jesus; Avenoza, Alberto; Busto, Jesus H.; Corzana, Francisco; Peregrina, Jesus M.
      Chemistry - A European Journal (2014), 20 (39), 12616-12627CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The mol. recognition of several glycopeptides bearing Tn antigen (α-O-GalNAc-Ser or α-O-GalNAc-Thr) in their structure by three lectins with affinity for this determinant has been analyzed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the mol. recognition. In fact, while Soybean agglutinin (SBA) and Vicia villosa agglutinin (VVA) lectins prefer Tn-threonine, Helix pomatia agglutinin (HPA) shows a higher affinity for the glycopeptides carrying Tn-serine. The different conformational behavior of the two Tn biol. entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topol. of the binding site of the lectins are at the origin of these differences.
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    66. 66
      Gibadullin, R.; Farnsworth, D. W.; Barchi, J. J.; Gildersleeve, J. C. GalNAc-Tyrosine Is a Ligand of Plant Lectins, Antibodies, and Human and Murine Macrophage Galactose-Type Lectins. ACS Chem. Biol. 2017, 12, 2172– 2182,  DOI: 10.1021/acschembio.7b00471
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      GalNAc-Tyrosine Is a Ligand of Plant Lectins, Antibodies, and Human and Murine Macrophage Galactose-Type Lectins
      Gibadullin, Ruslan; Farnsworth, David Wayne; Barchi, Joseph J.; Gildersleeve, Jeffrey C.
      ACS Chemical Biology (2017), 12 (8), 2172-2182CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      In 2011, a new type of protein O-glycosylation was discovered in which N-acetylgalactosamine is attached to the side chain of tyrosine (GalNAc-Tyr). While present on dozens of proteins, the biol. roles of GalNAc-Tyr are unknown. To gain insight into this new type of modification, we synthesized a group of GalNAc-Tyr glycopeptides, constructed microarrays, and evaluated potential recognition of GalNAc-Tyr by a series of glycan-binding proteins. Through a series of >150 microarray expts., we assessed binding properties of a variety of plant lectins, monoclonal antibodies, and endogenous lectins. VVL, HPA, and SBA were all found to bind tightly to GalNAc-Tyr, and several Tn binding antibodies and blood group A antibodies were found to cross-react with GalNAc-Tyr. Thus, detection of GalNAc-Tyr modified proteins is an important consideration when analyzing results from these reagents. Addnl., we evaluated potential recognition by two mammalian lectins, human (hMGL) and murine (mMGL-2) macrophage galactose type C-type lectins. Both hMGL and mMGL-2 bound tightly to GalNAc-Tyr determinants. The apparent Kd values (∼1-40 nM) were on par with some of the best known ligands for MGL, such as the Tn antigen. hMGL also bound the natural beta-amyloid peptide contg. a GalNAc-Tyr epitope. STD NMR expts. provided structural insights into the mol. basis of recognition. Finally, GalNAc-Tyr was selectively captured by mMGL-2 pos. dendritic cells. These results provide the first evidence that GalNAc-Tyr modified proteins and/or peptides may be ligands for hMGL and mMGL-2 and offer unique structures for the design of MGL targeting agents.
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    67. 67
      Pluvinage, B.; Ficko-Blean, E.; Noach, I.; Stuart, C.; Thompson, N.; McClure, H.; Buenbrazo, N.; Wakarchuk, W.; Boraston, A. B. Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis. Proc. Natl. Acad. Sci. U. S. A. 2021, 118, e2019220118  DOI: 10.1073/pnas.2019220118
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      Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis
      Pluvinage, Benjamin; Ficko-Blean, Elizabeth; Noach, Ilit; Stuart, Christopher; Thompson, Nicole; McClure, Hayden; Buenbrazo, Nakita; Wakarchuk, Warren; Boraston, Alisdair B.
      Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (10), e2019220118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      A challenge faced by peptidases is the recognition of highly diverse substrates. A feature of some peptidase families is the capacity to specifically use post-translationally added glycans present on their protein substrates as a recognition determinant. This is ultimately crit. to enabling peptide bond hydrolysis. This class of enzyme is also frequently large and architecturally sophisticated. However, the mol. details underpinning glycan recognition by these O-glycopeptidases, the importance of these interactions, and the functional roles of their ancillary domains remain unclear. Here, using the Clostridium perfringens ZmpA, ZmpB, and ZmpC M60 peptidases as model proteins, we provide structural and functional insight into how these intricate proteins recognize glycans as part of catalytic and noncatalytic substrate recognition. Structural, kinetic, and mutagenic analyses support the key role of glycan recognition within the M60 domain catalytic site, though they point to ZmpA as an apparently inactive enzyme. Wider examn. of the Zmp domain content reveals noncatalytic carbohydrate binding as a feature of these proteins. The complete three-dimensional structure of ZmpB provides rare insight into the overall mol. organization of a highly multimodular enzyme and reveals how the interplay of individual domain function may influence biol. activity. O-glycopeptidases frequently occur in host-adapted microbes that inhabit or attack mucus layers. Therefore, we anticipate that these results will be fundamental to informing more detailed models of how the glycoproteins that are abundant in mucus are destroyed as part of pathogenic processes or liberated as energy sources during normal commensal lifestyles.
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    68. 68
      Noach, I.; Ficko-Blean, E.; Pluvinage, B.; Stuart, C.; Jenkins, M. L.; Brochu, D.; Buenbrazo, N.; Wakarchuk, W.; Burke, J. E.; Gilbert, M.; Boraston, A. B. Recognition of protein-linked glycans as a determinant of peptidase activity. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E679  DOI: 10.1073/pnas.1615141114
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      Recognition of protein-linked glycans as a determinant of peptidase activity
      Noach, Ilit; Ficko-Blean, Elizabeth; Pluvinage, Benjamin; Stuart, Christopher; Jenkins, Meredith L.; Brochu, Denis; Buenbrazo, Nakita; Wakarchuk, Warren; Burke, John E.; Gilbert, Michel; Boraston, Alisdair B.
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (5), E679-E688CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The vast majority of proteins are post-translationally altered, with the addn. of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate post-translational modifications, including glycosylation, has not been addressed. Here, through biochem. analyses and x-ray crystallog. structures, the authors show that to hydrolyze their substrates, 3 structurally related metallopeptidases (BT4244, IMPa, and ZmpB) require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The 3 peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight built upon the paradigm of how peptidases recognize substrates and provides a mol. understanding of glycoprotein degrdn.
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    69. 69
      Somovilla, V. J.; Bermejo, I. A.; Albuquerque, I. S.; Martinez-Saez, N.; Castro-Lopez, J.; Garcia-Martin, F.; Companon, I.; Hinou, H.; Nishimura, S.-I.; Jimenez-Barbero, J.; Asensio, J. L.; Avenoza, A.; Busto, J. H.; Hurtado-Guerrero, R.; Peregrina, J. M.; Bernardes, G. J. L.; Corzana, F. The use of fluoroproline in MUC1 antigen enables efficient detection of antibodies in patients with prostate cancer. J. Am. Chem. Soc. 2017, 139, 18255– 18261,  DOI: 10.1021/jacs.7b09447
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      The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer
      Somovilla, Victor J.; Bermejo, Iris A.; Albuquerque, Ines S.; Martinez-Saez, Nuria; Castro-Lopez, Jorge; Garcia-Martin, Fayna; Companon, Ismael; Hinou, Hiroshi; Nishimura, Shin-Ichiro; Jimenez-Barbero, Jesus; Asensio, Juan L.; Avenoza, Alberto; Busto, Jesus H.; Hurtado-Guerrero, Ramon; Peregrina, Jesus M.; Bernardes, Goncalo J. L.; Corzana, Francisco
      Journal of the American Chemical Society (2017), 139 (50), 18255-18261CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A structure-based design of a new generation of tumor-assocd. glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-L-proline or 4,4-difluoro-L-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallog. and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-assocd. anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivs. can be considered better diagnostic tools than the natural antigen for prostate cancer.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvVGgtr7M&md5=16fc13a7f085e53143d161609f54dad7
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      Companon, I.; Guerreiro, A.; Mangini, V.; Castro-Lopez, J.; Escudero-Casao, M.; Avenoza, A.; Busto, J. H.; Castillon, S.; Jimenez-Barbero, J.; Asensio, J. L.; Jimenez-Oses, G.; Boutureira, O.; Peregrina, J. M.; Hurtado-Guerrero, R.; Fiammengo, R.; Bernardes, G. J. L.; Corzana, F. Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage. J. Am. Chem. Soc. 2019, 141, 4063– 4072,  DOI: 10.1021/jacs.8b13503
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      Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage
      Companon, Ismael; Guerreiro, Ana; Mangini, Vincenzo; Castro-Lopez, Jorge; Escudero-Casao, Margarita; Avenoza, Alberto; Busto, Jesus H.; Castillon, Sergio; Jimenez-Barbero, Jesus; Asensio, Juan L.; Jimenez-Oses, Gonzalo; Boutureira, Omar; Peregrina, Jesus M.; Hurtado-Guerrero, Ramon; Fiammengo, Roberto; Bernardes, Goncalo J. L.; Corzana, Francisco
      Journal of the American Chemical Society (2019), 141 (9), 4063-4072CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-assocd. antigens. α-O-glycosylation forces the peptide to adopt an extended conformation in soln., which is far from the structure obsd. in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chem. modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-assocd. MUC1 antigen mimics, the deriv. bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiol. context. Clin., the exploitation of tumor-assocd. antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodol. presented here is of general interest for applications because it may be extended to modulate the affinity of biol. relevant glycopeptides toward their receptors.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisFyjtr0%253D&md5=fd760988cb0f5b5399f0a0c0baebdcb5