Rationally Designed Short Polyisoprenol-Linked PglB Substrates for Engineered Polypeptide and Protein N-Glycosylation
- Feng Liu
- Balakumar Vijayakrishnan
- Amirreza Faridmoayer
- Thomas A. Taylor
- Thomas B. Parsons
- Gonçalo J.L. Bernardes
- Michael Kowarik
- Benjamin G. Davis
View Author Information
† Department of Chemistry, Chemistry Research Laboratory, Oxford University, Oxford OX1 3TA United Kingdom
‡ GlycoVaxyn AG, 8952 Schlieren, Switzerland
Copyright © 2013 American Chemical Society
ACS AuthorChoice with CC-BY license
Abstract
The lipid carrier specificity of the protein N-glycosylation enzyme C. jejuni PglB was tested using a logical, synthetic array of natural and unnatural C10, C20, C30, and C40 polyisoprenol sugar pyrophosphates, including those bearing repeating cis-prenyl units. Unusual, short, synthetically accessible C20 prenols (nerylnerol 1d and geranylnerol 1e) were shown to be effective lipid carriers for PglB sugar substrates. Kinetic analyses for PglB revealed clear KM-only modulation with lipid chain length, thereby implicating successful in vitro application at appropriate concentrations. This was confirmed by optimized, efficient in vitro synthesis allowing >90% of Asn-linked β-N-GlcNAc-ylated peptide and proteins. This reveals a simple, flexible biocatalytic method for glycoconjugate synthesis using PglB N-glycosylation machinery and varied chemically synthesized glycosylation donor precursors.
Protein glycosylation is a vital co- or post-translational modification that links glycans to proteins typically through N- or O- linkages. Such modifications exist widely in eukaryotic and archaeal organisms and greatly expand the diversity of the proteome.(1-3) While N-linked glycosylation had been believed to be absent in prokaryotic systems, the discovery of the protein N-glycosyltransferase PglB in Gram-negative bacterium Campylobacter jejuni highlighted greater diversity; PglB is responsible for glycosylating over 50 different proteins.(4-7) Since then PglB orthologues have been found in Campylobacter lari,(8) the Helicobacter genus (H. pullorum, H. canadensis, H. winghamensis)(9) as well as Desulfovibrio desulfuricans(10) and, very recently, Methanococcus voltae.(11)In vivo PglB uses a C55 undecaprenylpyrophosphate-linked oligosaccharide as its substrate and glycosylates the primary amide nitrogen of the asparagine side chain in a D/E-X-N-X-T/S consensus sequence (where X can be any amino acid except proline, Figure 1). In contrast to its eukaryotic counterpart Stt3p, PglB does not require other proteins/subunits and can apparently catalyze this N-glycosylation in vivo alone.(12) Moreover, in vivo biosynthetic studies that allowed exposure of PglB to other lipid-linked oligosaccharides have suggested a possibly relaxed specificity toward the nature of glycan substrate, as compared to the relatively highly specific eukaryotic N-glycosylation enzymes.(13) Recent elegant approaches flexibly using PglB in vivo have been developed to prepare glycoproteins.(14, 15) These distinct characteristics of PglB, as well as the fact that C. jejuni PglB can be readily overexpressed in functional form in Escherichia coli,(16) highlight PglB’s potential as a synthetic biocatalyst. They suggest it as a potentially ideal model from which to generate a ready synthetic system for in vitro protein glycosylation. However, the donor substrates used normally by PglB in vivo (C55 lipid pyrophosphoryl-linked oligosaccharides containing the rare, bacterial sugar bacillosamine (Bac)) would restrict this system (both in substrate accessibility and product relevance).
Figure 1
Figure 1. Bacterial N-linked glycosylation and designed unnatural candidate polyprenols 1a–g as alternative short lipid carriers for PglB-catalyzed glycosylation. The polyprenols were synthesized from building blocks 2a–d and 3a and 3b.
In an attempt to optimize the PglB protein N-glycosylation platform for practical, synthetic (and hence in vitro) use, we designed an array of chemically generated polyisoprenol variants to find those simpler and shorter than the natural undecaprenol (Figure 1) and that might serve as alternative lipid carriers that could be recognized by PglB. Insightful prior work has elucidated some aspects of the polyisoprenol specificity of PglB.(17, 18) However, estimated conversions for these reactions were ≤20%. In addition, many of these prior substrates were prepared enzymatically from polyisoprenols isolated from natural sources, enabling only nmol analysis of lipid pyrophosphates containing the natural Campylobacter (GalNAc-GalNAc-BacdiNAc) glycans. This revealed tantalizing activity for two shorter (mixed cis/trans prenol-9 and prenol-8) variants. However, despite some individual pioneering examples(19-28) synthetic access to other prenols has been rare, and homologated families of lipids have not yet been probed. Therefore, with the intention of probing fuller lipid and sugar substrate plasticity and breadth in PglB and with the intention of creating synthetically accessible substrates, we first systematically varied the lipid carriers (Figure 1).
Scheme 1
Scheme 1. Syntheses of Polyprenol Variantsa
a(i) BuLi, THF, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMTP), −78 °C. Yield: 4a = 92%, 4b = 93%, 4c = 83%, 4d = 63%, 4e = 67%, 4f = 74%. (ii) (1) TBAF, THF, RT, 4 h; (2) LiEt3BH, (dppp)PdCl2, THF, 0 °C–RT. Yield: 1a= 65%, 1b = 57%, 1c = 50%, 1d = 70%, 1e = 60%, 1f = 54%.
As a starting point for lipid variation, we speculated that the use of repeating cis-prenyl units at the hydroxyl terminus would confer binding affinity due to a resemblance to the alcohol-terminus that bears the glycan-pyrophosphate found in the natural substrate and likely enters a binding pocket in PglB.(29) This notion is supported by a crystal structure which has a Mg2+ bound at the active site.(29) Subsequent modeling suggested that the lipid carrier would likely locate first in a narrow pocket that would tightly accommodate two isoprenyl units (and thus impose tighter stereochemical requirements) and then along a broader hydrophobic groove with potentially more relaxed requirements (Figure S12). Thus, compounds 1a–g, all bearing one or more terminal repeating cis-prenyl units were designed and synthesized as primary candidate lipid carriers (Figure 1). In brief (see SI for further details), compounds 1a–f were synthesized from head (compound 3a and 3b) and tail building blocks (compound 2a–d). The requisite building blocks were prepared and elongated using coupling between appropriate sulfones and allyl chloride. Utilization of a convergent synthetic strategy in the case of long lipids (Scheme 1) allowed useful flexibility in the generation of lipids with different stereochemistry. Removal of sulfonyl groups in one step using LiEt3BH/(dppp)PdCl2 valuably reduced both the total number of synthetic steps and the formation of isomeric products resulting from double-bond migration and basic conditions.(23, 25) The resulting, pure synthetic polyprenols were shown to have a d.r. (E/Z) > 95% according to the characteristic allylic methyl group signals in 1H NMR (see Figure S1); no unwanted isomeric product (<2%, 1H NMR signals at δ 2.7, 5.9 ppm)(30) was detected.
These polyprenols were readily phosphorylated using mono(tetrabutylammonium) phosphate (TBAP) activated with trichloroacetonitrile (TCA).(31) We next attached just a single residue atypical glycan to provide a stringent test of the catalytic activity of PglB. Importantly, we used the sugar that would be found as the first N-linked residue in eukaryotic glycoproteins (but not prokaryotic and so not that normally employed by Campylobacter): GlcNAc. The resulting polyprenyl monophosphates 5a–g were therefore coupled to 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-d-glucopyranose 1-phosphate (6) using carbonyldiimidazole (CDI)-mediated phosphoesterification.(32, 33) Deacetylation with catalytic NaOMe in MeOH (Zemplén conditions) gave the lipid pyrophosphate-linked saccharides (LPPS) 8a–g in 9–38% yield over three steps (Scheme 2).
Scheme 2
Scheme 2. Syntheses of Lipid-linked Oligosaccharides and use in Glycopeptide Synthesisa
a(i) 5a–g, TBAP, TCA, DCM, RT; 5h, POCl3, Et3N, 2h. (ii) (1) CDI, DMF; (2) MeOH; (3) 6, DMF, RT, 3–5 days. (iii) NaOMe, MeOH. Yield over 3 steps: 8a = 17%, 8b = 23%, 8c = 9%, 8d = 17%, 8e = 9%, 8f = 38%, 8g = 29%, 8h = 32%, 13 = 22%, 14 = 30%. Extension reactions (blue arrows, 5–94%); see SI for further details.
With these first synthetic LPPS’s in hand, 8a–h were then tested as substrates of PglB. The glycosylation of peptides bearing the required D/E-X-N-X-S/T consensus motif was determined by examining the increase of molecular weight (16%/6 M urea tricine-SDS-PAGE)(34) that corresponds to the addition of GlcNAc (Figure 2). An extract from E. coli LPPS containing C. jejuni heptasaccharide-linked undecaprenyl pyrophosphate was used as a positive control. Excitingly, 8a–e were active substrates for PglB-catalyzed N-glycosylation of the fluorescent peptide Tamra-DANYTK as indicated by the appearance of glycopeptide product bands with higher molecular weight consistent with the addition of GlcNAc. This importantly revealed that the lipid carrier for the substrate of PglB can contain as few as two cis-head repeating units. The activity of 8c and 8e as substrates for PglB also indicated that PglB can tolerate lipid carriers bearing trans geometry close to the hydroxyl terminus. However, activity was lost as the length of lipid became shorter than four prenyl units: nerol (1g)-PP-GlcNAc (8g), cis,cis-farnesol (1f)-PP-GlcNAc conjugate (8f), and citronellol-PP-GlcNAc conjugate (8h)(32, 33) did not show any detectable activity in the in vitro assay. These activities ‘mapped’ tight proximal-site activity and relaxed distal-site activity consistent with model shown in Figure S12.
Figure 2
Figure 2. Peptide and protein glycosylation. (a) Fluorescent electrophoretic analysis of peptide glycosylation with lipids (8a–h) (− = no lipid; + = lipid extracted and enriched from E. coli cells producing C. jejuni heptasaccharide-linked undecaprenyl pyrophosphate); [glycolipid] = [peptide] 20 μM; [PglB] = 0.44 μM. (b) ES-MS of in vitro N-linked protein (AcrA) glycosylation with 8c; >95% diglycosylation.
These first kinetic parameters (Table 1) for PglB suggested key features. In particular, the variation of activity with lipid length in the substrate is strikingly only dependent on KM; kcat remains essentially unaltered. This suggests that the lipid may not play a primary role in catalytic turnover but is a key regulator of substrate uptake. This suggested too that in vitro reactions conducted at sufficiently high concentrations >KM would allow transfer efficiencies equal to those found for full length lipid substrates. This was valuably confirmed in synthetic reactions that allowed the synthesis of GlcNAc-ylated glycopeptide in yields >90% using 0.1 mM glycosyl donor substrate 8c with 20 μM of acceptor peptide. These >90% reactions usefully extend(17, 18) the synthetic utility of PglB.
Table 1. Kinetic Parameters for PglB with GlcNAc Lipidsa
| glycolipid substrate | kcat [min–1] | KM [mM] | kcat/KM [min–1mM–1] |
|---|---|---|---|
| 8d GlcNAc-PP-ωZ3 | 0.0234 ± 0.0021 | 0.077 ± 0.016 | 0.30 |
| 8c GlcNAc-PP-ωEZ4 | 0.0231 ± 0.0016 | 0.055 ± 0.01 | 0.42 |
| 8a GlcNAc-PP-prenol8 | 0.0225 ± 0.0021 | 0.034 ± 0.01 | 0.66 |
a
UPLC using TAMRA-fluorescence intensity; substrate concentrations: [glycolipid] = 1, 5, 10, 50, 100, 200 μM, [peptide] = 20 μM; enzyme concentration [PglB] = 0.44 μM; reaction time <2h, 30 °C; all conducted in duplicate.
Having elucidated valuable plasticity toward unnatural lipid-variant substrates, we next examined glycan breadth beyond the atypical monosaccharide GlcNAc already demonstrated. Conjugates (13 and 14) that contain both unnatural sugar and lipid carrier (Scheme 2) were prepared by coupling nerylnerylphosphate (5d) with the 6-azido-GlcNAc (9) and 2-azidoGlcNAc (10, GlcNAz). These compounds would allow subsequent flexible postexpressional modifications on proteins that contain D/E-X-N-X-S/T tag via reaction of the introduced azide with a number of compatible methods. Both azido analogues (13 and 14) failed to undergo glycosylation with the peptide in the presence of PglB. The failure of these glycolipids to act as substrates may be explained by the use of nonpreferred moieties in both halves of these unnatural sugar-unnatural lipid conjugates. It may also suggest a particular lack of plasticity by PglB toward alterations at sugar sites C-2 or C-6; glycolipids 13 and 14 did not inhibit PglB glycosylations using 8c (see SI).
GlcNAc-ylated peptide could then be extended (Scheme 2) with the use of endoglycosidases(35, 36) (EndoS,(37) EndoA,(15, 38, 39) Scheme 2) and glycosyltransferases (β-1,4-GalT).(40) This allowed ready access to differently elaborated glycopeptides bearing, e.g., LacNAc (94%), or the eukaryotic N-glycan core-pentasaccharide (64%). Finally, we then tested the ability of this system to glycosylate proteins. Using short GlcNAc-lipids 8c and 8e, we could effect in vitro glycosylation >95% at the two consensus Asn sites in the C. jejuni AcrA(14) protein.
In conclusion, a wide variety of cis-polyisoprenol variants were chemically synthesized and studied for their binding specificity against PglB. For the first time, LPPS’s with only a single sugar and lipid chain lengths as short as C20 and C30 have been shown to be effective substrates for PglB in glycosylating specific peptide motifs. This reveals unexpected breadth for PglB beyond the minimal C40 lipid-trisaccharide substrate determined previously.(17) Our experimental catalytic data are consistent with previous crystallographic and modeling analyses. A closer examination of the lipid-binding pocket also reveals a relationship between the PglB structure and the chain length requirements that we have discovered here. The narrow pocket that precedes the hydrophobic groove is surrounded by polar residues Ser198, Ser201, Arg375. Longer lipid chains beyond the third isoprenyl unit may be required for increased affinity, by favorable interaction with the hydrophobic groove. This explains the observation that 8e was an active substrate, but 8f was not. Similar studies on the lipid carrier specificity of MurG have however shown a quite different trend, in which nerol (1g) and nerylnerol (1d) conjugates were much better substrates than those of longer lipids bearing repeating cis units.(24, 41, 42) Investigation of the crystal structures of MurG and/or PglB with lipid carrier bound, once available, would shed light on these clear differences in lipid carrier specificities.
The discovery of the breadth of PglB and these accessible lipid carriers now effectively enables the synthesis of lipid-pyrophosphate-linked substrates suitable for the in vitro generation of tailor-made glycoproteins. Importantly, to our knowledge, this currently represents the only in vitro biocatalytic system for the formation of the vital GlcNAc-β-1-N-Asn linkage (and importantly can be driven to >95% on proteins, here for AcrA); the recently discovered Methanococcus AglB, e.g.,(11) does not transfer GlcNAc and requires an unusual disaccharide. This discovery complements prior GalNAc transfer and confirms a predicted activity.(18) Notably, WecA,(43) the enzyme that would generate PP-linked glycolipid substrates for PglB, is membrane-associated and cannot be readily exploited in vitro. In vitro biocatalytic installation of GlcNAc, shown here, also creates a useful precursor sugar site for carbohydrate-processing enzyme-mediated extension, as shown here (Scheme 2). The >90% in vitro efficiencies shown here therefore make PglB a highly viable synthetic biocatalyst for varied glycopolypeptides, coupled with substrate accessibility and potential for further enzymatic transformation.
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§Author Contributions
These authors contributed equally.
The authors declare the following competing financial interest(s): M.K. and A.F. are employees of Glycovaxyn. A patent has been filed and will afford inventors royalties, if licensed, in line with university guidelines.
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Acknowledgment
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We thank the BBSRC (T.A.T.), Glycovaxyn (F.L., B.V., B.G.D.) for funding, Drs P. Carranza, C. Bich for method development and glycopeptide analysis, and Drs L. Gong, J. Wickens for HRMS analysis. B.G.D. is a Royal Society Wolfson Merit Research Award Recipient.
References
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This article references 43 other publications.
- 1Spiro, R. G. Glycobiology 2002, 12, 43R[ Crossref], [ PubMed], [ CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XltVyhtrY%253D&md5=b9c8933df399c8128ff275614d7ca7acProtein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bondsSpiro, Robert G.Glycobiology (2002), 12 (4), 43R-56RCODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)A review. Formation of the sugar-amino acid linkage is a crucial event in the biosynthesis of the carbohydrate units of glycoproteins. It sets into motion a complex series of posttranslational enzymic steps that lead to the formation of a host of protein-bound oligosaccharides with diverse biol. functions. These reactions occur throughout the entire phylogenetic spectrum, ranging from archaea and eubacteria to eukaryotes. It is the aim of this review to describe the glycopeptide linkages that have been found to date and specify their presence on well-characterized glycoproteins. A survey is also made of the enzymes involved in the formation of the various glycopeptide bonds as well as the site of their intracellular action and their affinity for particular peptide domains is evaluated. This examn. indicates that 13 different monosaccharides and 8 amino acids are involved in glycoprotein linkages leading to a total of at least 41 bonds, if the anomeric configurations, the phosphoglycosyl linkages, as well as the GPI (glycophosphatidylinositol) phosphoethanolamine bridge are also considered. These bonds represent the products of N- and O-glycosylation, C-mannosylation, phosphoglycation, and glypiation. Currently at least 16 enzymes involved in their formation have been identified and in many cases cloned. Their intracellular site of action varies and includes the endoplasmic reticulum, Golgi app., cytosol, and nucleus. With the exception of the Asn-linked carbohydrate and the GPI anchor, which are transferred to the polypeptide en bloc, the sugar-amino acid linkages are formed by the enzymic transfer of an activated monosaccharide directly to the protein. This review also deals briefly with glycosidases, which are involved in physiol. important cleavages of glycopeptide bonds in higher organisms, and with a no. of human disease states in which defects in enzymic transfer of saccharides to protein have been implicated.
- 2Walsh, C. T.; Garneau-Tsodikova, S.; Gatto, G. J. Angew. Chem., Int. Ed. 2005, 44, 7342[ Crossref], [ PubMed], [ CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlSms77N&md5=1e1ccf345916c39631a1cc83093de9b1Protein posttranslational modifications: The chemistry of proteome diversificationsWalsh, Christopher T.; Garneau-Tsodikova, Sylvie; Gatto, Gregory J., Jr.Angewandte Chemie, International Edition (2005), 44 (45), 7342-7372CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the no. of proteins predicted by DNA coding capacities owing to directed post-translational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitination, can be classified according to the type of amino acid side-chain modified, the category of the modifying enzyme, and the extent of reversibility. Chem. events such as protein splicing, green fluorescent protein (GFP) maturation, and proteasome autoactivation also represent post-translational modifications. An understanding of the scope and pattern of the many post-translational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compns.
- 3Ohtsubo, K.; Marth, J. D. Cell 2006, 126, 855[ Crossref], [ PubMed], [ CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpvVKitbo%253D&md5=959a24814730df3802c399ba78f7cd0fGlycosylation in cellular mechanisms of health and diseaseOhtsubo, Kazuaki; Marth, Jamey D.Cell (Cambridge, MA, United States) (2006), 126 (5), 855-867CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Glycosylation produces an abundant, diverse, and highly regulated repertoire of cellular glycans that are frequently attached to proteins and lipids. The past decade of research on glycan function has revealed that the enzymes responsible for glycosylation-the glycosyl-transferases and glycosidases-are essential in the development and physiol. of living organisms. Glycans participate in many key biol. processes including cell adhesion, mol. trafficking and clearance, receptor activation, signal transduction, and endocytosis. This review discusses the increasingly sophisticated mol. mechanisms being discovered by which mammalian glycosylation governs physiol. and contributes to disease.
- 4Szymanski, C. M.; Yao, R.; Ewing, C. P.; Trust, T. J.; Guerry, P. Mol. Microbiol. 1999, 32, 1022[ Crossref], [ PubMed], [ CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvVSmsLw%253D&md5=34430a0fdd62a3238e74ec487a72095bEvidence for a system of general protein glycosylation in Campylobacter jejuniSzymanski, Christine M.; Yao, Ruijin; Ewing, Cheryl P.; Trust, Trevor J.; Guerry, PatriciaMolecular Microbiology (1999), 32 (5), 1022-1030CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)A genetic locus from Campylobacter jejuni 81-176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5α contg. some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site-specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site-specific mutation of each of the seven genes in 81-176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild-type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chem. deglycosylation of protein fractions from the 81-176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.
- 5Young, N. M.; Brisson, J. R.; Kelly, J.; Watson, D. C.; Tessier, L.; Lanthier, P. H.; Jarrell, H. C.; Cadotte, N.; St Michael, F.; Aberg, E.; Szymanski, C. M. J. Biol. Chem. 2002, 277, 42530[ Crossref], [ PubMed], [ CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xos1ensLs%253D&md5=0359877f22079bf195c91b931751f69eStructure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuniYoung, N. Martin; Brisson, Jean-Robert; Kelly, John; Watson, David C.; Tessier, Luc; Lanthier, Patricia H.; Jarrell, Harold C.; Cadotte, Nicolas; St. Michael, Frank; Aberg, Erika; Szymanski, Christine M.Journal of Biological Chemistry (2002), 277 (45), 42530-42539CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatog., a mixt. of glycoproteins was obtained from a glycine ext., and two-dimensional gel proteomics anal. led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepd. from the glycoprotein mixt. by Pronase digestion and gel filtration. The structure of the glycan was detd. by using nano-NMR techniques to be GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3-]GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.
- 6Linton, D.; Allan, E.; Karlyshev, A. V.; Cronshaw, A. D.; Wren, B. W. Mol. Microbiol. 2002, 43, 497[ Crossref], [ PubMed], [ CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xhtlajsb4%253D&md5=7e2b9ade854de0e58de9652aadd51dffIdentification of N-acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuniLinton, Dennis; Allan, Elaine; Karlyshev, Andrey V.; Cronshaw, Andrew D.; Wren, Brendan W.Molecular Microbiology (2002), 43 (2), 497-508CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)There is a system of general protein glycosylation in the human enteropathogen Campylobacter jejuni. To characterize such glycoproteins, we identified a lectin, soybean agglutinin (SBA), which binds to multiple C. jejuni proteins on Western blots. Binding of lectin SBA was disrupted by mutagenesis of genes within the previously identified protein glycosylation locus. This lectin was used to purify putative glycoproteins selectively and, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie-stained bands were cut from the gels. The bands were digested with trypsin, and peptides were identified by mass spectrometry and database searching. A 28 kDa band was identified as PEB3, a previously characterized immunogenic cell surface protein. Bands of 32 and 34 kDa were both identified as a putative periplasmic protein encoded by the C. jejuni NCTC 11168 coding sequence Cj1670c. We have named this putative glycoprotein CgpA. We constructed insertional knockout mutants of both the peb3 and cgpA genes, and surface protein exts. from mutant and wild-type strains were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). In this way, we were able to identify the PEB3 protein as a 28 kDa SBA-reactive and immunoreactive glycoprotein. The cgpA gene encoded SBA-reactive and immunoreactive proteins of 32 and 34 kDa. By using specific exoglycosidases, we demonstrated that the SBA binding property of acid-glycine extractable C. jejuni glycoproteins, including PEB3 and CgpA, is a result of the presence of α-linked N-acetylgalactosamine residues. These data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni. Furthermore, we have identified two such glycoproteins, the first non-flagellin Campylobacter glycoproteins to be identified, and demonstrated that their glycan components contain α-linked N-acetylgalactosamine residues.
- 7Scott, N. E.; Parker, B. L.; Connolly, A. M.; Paulech, J.; Edwards, A. V. G.; Crossett, B.; Falconer, L.; Kolarich, D.; Djordjevic, S. P.; Højrup, P.; Packer, N. H.; Larsen, M. R.; Cordwell, S. J. Mol. Cell. Proteomics 2011, 10, M000031
- 8Schwarz, F.; Lizak, C.; Fan, Y.-Y.; Fleurkens, S.; Kowarik, M.; Aebi, M. Glycobiology 2011, 21, 45[ Crossref], [ PubMed], [ CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFGru73F&md5=66b94c57da11b068925fd1a61a2bdef2Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivoSchwarz, Flavio; Lizak, Christian; Fan, Yao-Yun; Fleurkens, Susanna; Kowarik, Michael; Aebi, MarkusGlycobiology (2011), 21 (1), 45-54CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)A no. of proteobacteria carry the genetic information to perform N-linked glycosylation, but only the protein glycosylation (pgl) pathway of Campylobacter jejuni has been studied to date. Here, we report that the pgl gene cluster of Campylobacter lari encodes for a functional glycosylation machinery that can be reconstituted in Escherichia coli. We detd. that the N-glycan produced in this system consisted of a linear hexasaccharide. We found that the oligosaccharyltransferase (OST) of C. lari conserved a predominant specificity for the primary sequence D/E-X-1-N-X+1-S/T (where X-1 and X+1 can be any amino acid but proline). At the same time, we obsd. that this enzyme exhibited a relaxed specificity toward the acceptor site and modified asparagine residues of a protein at sequences DASG and NNST. Moreover, C. lari pgl glycosylated a native E. coli protein. Bacterial N-glycosylation appears as a useful tool to establish a mol. description of how single-subunit OSTs perform selection of glycosyl acceptor sites.
- 9Jervis, A. J.; Langdon, R.; Hitchen, P.; Lawson, A. J.; Wood, A.; Fothergill, J. L.; Morris, H. R.; Dell, A.; Wren, B.; Linton, D. J. Bacteriol. 2010, 192, 5228[ Crossref], [ PubMed], [ CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFCitbjJ&md5=1ba8109b717bc3ced101eb31e10e065dCharacterization of N-linked protein glycosylation in Helicobacter pullorumJervis, Adrian J.; Langdon, Rebecca; Hitchen, Paul; Lawson, Andrew J.; Wood, Alison; Fothergill, Joanne L.; Morris, Howard R.; Dell, Anne; Wren, Brendan; Linton, DennisJournal of Bacteriology (2010), 192 (19), 5228-5236CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologs are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation expts., the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Anal. of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was obsd. This reaction required an acidic residue at the -2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.
- 10Ielmini, M. V.; Feldman, M. F. Glycobiology 2011, 21, 734[ Crossref], [ PubMed], [ CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmt1ajtbs%253D&md5=a1f953b18b18b0f07d352435b6ea44a7Desulfovibrio desulfuricans PglB homolog possesses oligosaccharyltransferase activity with relaxed glycan specificity and distinct protein acceptor sequence requirementsIelmini, Maria V.; Feldman, Mario F.Glycobiology (2011), 21 (6), 734-742CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)Oligosaccharyltransferases (OTases) are responsible for the transfer of carbohydrates from lipid carriers to acceptor proteins and are present in all domains of life. In bacteria, the most studied member of this family is PglB from Campylobacter jejuni (PglBCj). This enzyme is functional in Escherichia coli and, contrary to its eukaryotic counterparts, has the ability to transfer a variety of oligo- and polysaccharides to protein carriers in vivo. Phylogenetic anal. revealed that in the delta proteobacteria Desulfovibrio sp., the PglB homolog is more closely related to eukaryotic and archaeal OTases than to its Campylobacter counterparts. Genetic anal. revealed the presence of a putative operon that might encode all enzymes required for N-glycosylation in Desulfovibrio desulfuricans. D. desulfuricans PglB (PglBDd) was cloned and successfully expressed in E. coli, and its activity was confirmed by transferring the C. jejuni heptasaccharide onto the model protein acceptor AcrA. In contrast to PglBCj, which adds two glycan chains to AcrA, a single oligosaccharide was attached to the protein by PglBDd. Site-directed mutagenesis of the five putative N-X-S/T glycosylation sites in AcrA and mass spectrometry anal. showed that PglBDd does not recognize the "conventional bacterial glycosylation sequon" consisting of the sequence D/E-X1-N-X2-S/T (where X1 and X2 are any amino acid except proline), and instead used a different site for the attachment of the oligosaccharide than PglBCj. Furthermore, PglBDd exhibited relaxed glycan specificity, being able to transfer mono- and polysaccharides to AcrA. Our anal. constitutes the first characterization of an OTase from delta-proteobacteria involved in N-linked protein glycosylation.
- 11Larkin, A.; Chang, M. M.; Whitworth, G. E.; Imperiali, B. Nat. Chem. Biol. 2013, 9, 367[ Crossref], [ PubMed], [ CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1Wms70%253D&md5=8002ef3e92ef581f18013004cee47d42Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesisLarkin, Angelyn; Chang, Michelle M.; Whitworth, Garrett E.; Imperiali, BarbaraNature Chemical Biology (2013), 9 (6), 367-373CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro anal. of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homol. to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochem. evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.
- 12Glover, K. J.; Weerapana, E.; Numao, S.; Imperiali, B. Chem. Biol. 2005, 12, 1311[ Crossref], [ PubMed], [ CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlWktLjK&md5=60291c5dce3c72c700292b1a5fde74b7Chemoenzymatic Synthesis of Glycopeptides with PglB, a Bacterial Oligosaccharyl Transferase from Campylobacter jejuniGlover, Kerney Jebrell; Weerapana, Eranthie; Numao, Shin; Imperiali, BarbaraChemistry & Biology (Cambridge, MA, United States) (2005), 12 (12), 1311-1315CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)The gram-neg. bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PglB, is thought to be the oligosaccharyl transferase due to its significant homol. to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PglB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GalNAc-α1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PglB in vitro. Furthermore, the prepn. of addnl. undecaprenyl-linked glycan variants reveals the ability of PglB to transfer a wide variety of saccharides. With the demonstration of PglB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.
- 13Wacker, M.; Feldman, M. F.; Callewaert, N.; Kowarik, M.; Clarke, B. R.; Pohl, N. L.; Hernandez, M.; Vines, E. D.; Valvano, M. A.; Whitfield, C.; Aebi, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7088[ Crossref], [ PubMed], [ CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XkslCkt7k%253D&md5=ee2bf4bcd1ef2cba7c3226eae5bf9db3Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systemsWacker, Michael; Feldman, Mario F.; Callewaert, Nico; Kowarik, Michael; Clarke, Bradley R.; Pohl, Nicola L.; Hernandez, Marcela; Vines, Enrique D.; Valvano, Miguel A.; Whitfield, Chris; Aebi, MarkusProceedings of the National Academy of Sciences of the United States of America (2006), 103 (18), 7088-7093CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The PglB oligosaccharyltransferase (OTase) of Campylobacter jejuni can be functionally expressed in Escherichia coli, and its relaxed oligosaccharide substrate specificity allows the transfer of different glycans from the lipid carrier undecaprenyl pyrophosphate to an acceptor protein. To investigate the substrate specificity of PglB, we tested the transfer of a set of lipid-linked polysaccharides in E. coli and Salmonella enterica serovar Typhimurium. A hexose linked to the C-6 of the monosaccharide at the reducing end did not inhibit the transfer of the O antigen to the acceptor protein. However, PglB required an acetamido group at the C-2. A model for the mechanism of PglB involving this functional group was proposed. Previous expts. have shown that eukaryotic OTases have the same requirement, suggesting that eukaryotic and prokaryotic OTases catalyze the transfer of oligosaccharides by a conserved mechanism. Moreover, we demonstrated the functional transfer of the C. jejuni glycosylation system into S. enterica. The elucidation of the mechanism of action and the substrate specificity of PglB represents the foundation for engineering glycoproteins that will have an impact on biotechnol.
- 14Feldman, M. F.; Wacker, M.; Hernandez, M.; Hitchen, P. G.; Marolda, C. L.; Kowarik, M.; Morris, H. R.; Dell, A.; Valvano, M. A.; Aebi, M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3016[ Crossref], [ PubMed], [ CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXitVSktL4%253D&md5=2d348d3f2cb30206eb22d6e47ad00410Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coliFeldman, Mario F.; Wacker, Michael; Hernandez, Marcela; Hitchen, Paul G.; Marolda, Cristina L.; Kowarik, Michael; Morris, Howard R.; Dell, Anne; Valvano, Miguel A.; Aebi, MarkusProceedings of the National Academy of Sciences of the United States of America (2005), 102 (8), 3016-3021CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures contg. two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo prodn. of O polysaccharides-protein conjugates for use as antibacterial vaccines.
- 15Schwarz, F.; Huang, W.; Li, C.; Schulz, B. L.; Lizak, C.; Palumbo, A.; Numao, S.; Neri, D.; Aebi, M.; Wang, L.-X. Nat. Chem. Biol. 2010, 6, 264[ Crossref], [ PubMed], [ CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXisFeksro%253D&md5=20276ab4992696b1ed34ac1cbd436ab3A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylationSchwarz, Flavio; Huang, Wei; Li, Cishan; Schulz, Benjamin L.; Lizak, Christian; Palumbo, Alessandro; Numao, Shin; Neri, Dario; Aebi, Markus; Wang, Lai-XiNature Chemical Biology (2010), 6 (4), 264-266CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)We describe a new method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the Campylobacter jejuni glycosylation machinery in Escherichia coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins.
- 16Wacker, M.; Linton, D.; Hitchen, P. G.; Nita-Lazar, M.; Haslam, S. M.; North, S. J.; Panico, M.; Morris, H. R.; Dell, A.; Wren, B. W.; Aebi, M. Science 2002, 298, 1790[ Crossref], [ PubMed], [ CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XovFKmsL0%253D&md5=cbdd0efcd7491b2ceb610f2ba5bc115aN-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coliWacker, Michael; Linton, Dennis; Hitchen, Paul G.; Nita-Lazar, Mihai; Haslam, Stuart M.; North, Simon J.; Panico, Maria; Morris, Howard R.; Dell, Anne; Wren, Brendan W.; Aebi, MarkusScience (Washington, DC, United States) (2002), 298 (5599), 1790-1793CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)N-linked protein glycosylation is the most abundant posttranslation modification of secretory proteins in eukaryotes. A wide range of functions are attributed to glycan structures covalently linked to asparagine residues within the asparagine-X-serine/threonine consensus sequence (Asn-Xaa-Ser/Thr). We found an N-linked glycosylation system in the bacterium Campylobacter jejuni and demonstrate that a functional N-linked glycosylation pathway could be transferred into Escherichia coli. Although the bacterial N-glycan differs structurally from its eukaryotic counterparts, the cloning of a universal N-linked glycosylation cassette in E. coli opens up the possibility of engineering permutations of recombinant glycan structures for research and industrial applications.
- 17Chen, M. M.; Weerapana, E.; Ciepichal, E.; Stupak, J.; Reid, C. W.; Swiezewska, E.; Imperiali, B. Biochemistry 2007, 46, 14342[ ACS Full Text
], [ CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlCqtLfM&md5=e2eb24e5972b49be62536cffcbc85c2bPolyisoprenol Specificity in the Campylobacter jejuni N-Linked Glycosylation PathwayChen, Mark M.; Weerapana, Eranthie; Ciepichal, Ewa; Stupak, Jacek; Reid, Christopher W.; Swiezewska, Ewa; Imperiali, BarbaraBiochemistry (2007), 46 (50), 14342-14348CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in common amphiphilic membrane-bound polyisoprenyl-linked substrates. Herein, we examine the potential role of the polyisoprene component of the substrates by investigating the relative substrate efficiencies of polyisoprene-modified analogs in individual steps of the pathway. Chem. defined substrates for PglC, PglJ, and PglB are prepd. via semisynthetic approaches. The substrates included polyisoprenols of varying length, double bond geometry, and degree of satn. for probing the role of the hydrophobic polyisoprene in substrate specificity. Kinetic anal. reveals that all three enzymes exhibit distinct preferences for the polyisoprenyl carrier whereby cis-double bond geometry and α-unsatn. of the native substrate are important features, while the precise polyisoprene length may be less crit. These findings suggest that the polyisoprenyl carrier plays a specific role in the function of these enzymes beyond a purely phys. role as a membrane anchor. These studies underscore the potential of the C. Jejuni N-linked glycosylation pathway as a system for investigating the biochem. and biophys. roles of polyisoprenyl carriers common to prokaryotic and eukaryotic glycosylation. - 18Li, L.; Woodward, R.; Ding, Y.; Liu, X.-w.; Yi, W.; Bhatt, V. S.; Chen, M.; Zhang, L.-w.; Wang, P. G. Biochem. Biophys. Res. Commun. 2010, 394, 1069[ Crossref], [ PubMed], [ CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkvFChurg%253D&md5=a37f5912e81bc4c395b69176fdb9d7efOverexpression and topology of bacterial oligosaccharyltransferase PglBLi, Lei; Woodward, Robert; Ding, Yan; Liu, Xian-wei; Yi, Wen; Bhatt, Veer S.; Chen, Min; Zhang, Lian-wen; Wang, Peng GeorgeBiochemical and Biophysical Research Communications (2010), 394 (4), 1069-1074CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homolog, PglB, functions as the oligosaccharyltransferase. Here, the authors established a method for obtaining relatively large quantities of homogeneous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chem. synthesized sugar donor: N-acetylgalactosamine-diphosphoundecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirmed that PglB is solely responsible for the oligosaccharyltransferase activity and complemented the finding that PglB exhibits relaxed sugar substrate specificity. In addn., the performed the topol. mapping of PglB using the PhoA/LacZ fusion method. The topol. model showed that PglB possesses 11 transmembrane segments and 2 relatively large periplasmic regions other than the C-terminal domain, which was consistent with the proposal of the common Ncyt-Cperi topol. with 11 transmembrane segments for the STT3 family proteins.
- 19Sato, K.; Inoue, S.; Onishi, A.; Uchida, N.; Minowa, N. J. Chem. Soc., Perkin Trans. 1 1981, 761[ Crossref], [ CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXls12msbk%253D&md5=f56064750bb2250c63da73e31af6e691Stereoselective synthesis of solanesol and all-trans-decaprenolSato, Kikumasa; Inoue, Seiichi; Onishi, Akira; Uchida, Nobuhiko; Minowa, NobutoJournal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999) (1981), (3), 761-9CODEN: JCPRB4; ISSN:0300-922X.Stereochem. pure 1,5-dienes were prepd. in moderate to good yields by coupling of allylic p-tolyl sulfones with an allylic bromide. E.g., coupling reaction of all-trans-bromogeranyl acetate (I) with the sulfone II and with higher isoprene analogs, followed by reductive elimination of p-MeC6H4SO2, gave all-trans-polyprenols and decaprenol (III; n = 10) stereoselectively. Solanesol (III; n = 9) was similarly prepd. through coupling of ClCH2CMe:CHCH2OAc.
- 20Sato, K.; Miyamoto, O.; Inoue, S.; Furusawa, F.; Matsuhashi, Y. Chem. Lett. 1983, 12, 725
- 21Jaenicke, L.; Siegmund, H. Biol. Chem. Hoppe-Seyler 1986, 367, 787[ Crossref], [ PubMed], [ CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXmt1ei&md5=718b7df70e43b18a736eef6e013ce33bTotal synthesis of chain-length-uniform dolichyl phosphates and their fitness to accept hexoses in the enzymic formation of lipoglycansJaenicke, Lothar; Siegmund, Hans UlrichBiological Chemistry Hoppe-Seyler (1986), 367 (8), 787-95CODEN: BCHSEI; ISSN:0177-3593.Dolichols I (R = H, n = 3, 5, 7) of defined uniform chain length (C35, C45, and C55) and geometry were prepd. from (E,E)-farnesol, activated as its 4-tolyl sulfone via condensation with 8-chloroneryl benzyl ether, conversion to THF 4-tolyl sulfone, and after several cycles of this C10-elongation sequence ending with 8-chlorocitronellyl benzyl ether to introduce the satd. α-isoprene unit. I (R = H, n = 3, 5, 7) were phosphorylated (POCl3/Et3N) and assayed relative to the natural dolichyl phosphate mixt. from pig liver as acceptors for transglycosylation from nucleoside diphosphate sugars (glucose, mannose) by standardized membrane vesicle prepns. from plants (Volvox) and animals (liver). Even I [R = P(O)(OH)2, n = 3] has full activity in this lipoglycan-forming reaction.
- 22Inoue, S.; Kaneko, T.; Takahashi, Y.; Miyamoto, O.; Sato, K. J. Chem. Soc., Chem. Commun. 1987, 1036[ Crossref], [ CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXit1Wqs7w%253D&md5=90fd51a141a4c69aea6875126f6c23c8Stereoselective total synthesis of (S)-(-)-dolichol-20Inoue, Seiichi; Kaneko, Toshihiko; Takahashi, Yuichi; Miyamoto, Osamu; Sato, KikumasaJournal of the Chemical Society, Chemical Communications (1987), (13), 1036-7CODEN: JCCCAT; ISSN:0022-4936.(S)-(-)-Dolichol-20 was prepd. stereoselectively using (Z,Z,Z,Z,Z,Z,Z,Z,E,E)-undecaprenol, ClCH2CMe:CHCH2CH2CMe:CHCH(SO2C6H4Me-p)-(CH2CMe:CHCH2)2-OCH2Ph, and (S)-Cl-(CH2CMe:CHCH2)2-CH2CMe:CHCH(SO2C6H4Me-p)CH2CMe:CHCH2CH2CHMeCH2CH2OCH2Ph as key intermediates.
- 23Grassi, D.; Lippuner, V.; Aebi, M.; Brunner, J.; Vasella, A. J. Am. Chem. Soc. 1997, 119, 10992[ ACS Full Text], [ CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnt1equr8%253D&md5=28b0e8245e7c9f27347889b3cfc38d0aSynthesis and Enzymic Phosphorylation of a Photoactivatable Dolichol AnalogGrassi, D.; Lippuner, V.; Aebi, M.; Brunner, J.; Vasella, A.Journal of the American Chemical Society (1997), 119 (45), 10992-10999CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The synthesis of the photochem. probes I [R = H, PO3H2, R1 = (10Z,14Z,18Z,22Z,26Z,30Z,34E,38E)-Me2C:CHCH2(CH2CMe:CHCH2)2(CH2CMe:CHCH2)6] is described. These photoprobes are analogs of dolichol and dolichol phosphate, obligatory intermediates in the N-linked glycosylation pathway in the endoplasmic reticulum. The synthesis of I follows a new strategy. It involves the sequential alkylation of a monoterpenoid hydroxysulfonyl dianion with allyl chlorides. The photoreactive group, a 3-(trifluoromethyl)-3-aryldiazirine, was connected to the hydroxylated Me group of the β-prenyl unit of the fully assembled polyprenyl chain. The photoactivatable dolichol analog I (R = H) is a substrate for dolichol kinase from yeast membranes, an essential enzyme involved in the N-linked glycosylation pathway.
- 24Chen, L.; Men, H.; Ha, S.; Ye, X.-Y.; Brunner, L.; Hu, Y.; Walker, S. Biochemistry 2002, 41, 6824[ ACS Full Text], [ CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1KrsL4%253D&md5=da1a3d4b4c1b2f059ca32422feb95f16Intrinsic Lipid Preferences and Kinetic Mechanism of Escherichia coli MurGChen, Lan; Men, Hongbin; Ha, Sha; Ye, Xiang-Yang; Brunner, Livia; Hu, Yanan; Walker, SuzanneBiochemistry (2002), 41 (21), 6824-6833CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)MurG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-assocd. glycosyltransferase that couples N-acetyl glucosamine to the C4 hydroxyl of a lipid-linked N-acetyl muramic acid deriv. (lipid I) to form the β-linked disaccharide (lipid II) that is the minimal subunit of peptidoglycan. Lipid I is anchored to the bacterial membrane by a 55 carbon undecaprenyl chain. Because this long lipid chain impedes kinetic anal. of MurG, we have been investigating alternative substrates contg. shortened lipid chains. We now describe the intrinsic lipid preferences of MurG and show that the optimal substrate for MurG in the absence of membranes is not the natural substrate. Thus, while the undecaprenyl carrier lipid may be crit. for certain steps in the biosynthetic pathway to peptidoglycan, it is not required-in fact, is not preferred-by MurG. Using synthetic substrate analogs and products contg. different length lipid chains, as well as a synthetic dead-end acceptor analog, we have also shown that MurG follows a compulsory ordered Bi Bi mechanism in which the donor sugar binds first. This information should facilitate obtaining crystals of MurG with substrates bound, an important goal because MurG belongs to a major superfamily of NDP-glycosyltransferases for which no structures contg. intact substrates have yet been solved.
- 25Chang, Y.-F.; Liu, C.-Y.; Guo, C.-W.; Wang, Y.-C.; Fang, J.-M.; Cheng, W.-C. J. Org. Chem. 2008, 73, 7197[ ACS Full Text], [ CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVWmu7vJ&md5=780b589134ee2bee5e0cdd20aa442d7fSolid-Phase Organic Synthesis of Polyisoprenoid Alcohols with Traceless Sulfone LinkerChang, Yi-Fan; Liu, Chen-Yu; Guo, Chih-Wei; Wang, Yen-Chih; Fang, Jim-Min; Cheng, Wei-ChiehJournal of Organic Chemistry (2008), 73 (18), 7197-7203CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)Solid-phase org. synthesis of polyprenols with a traceless sulfone linker is described. The polymer-bound benzenesulfinate is first linked with the "tail" building blocks of isoprenyl chlorides via S-alkylation. With use of dimsyl anion as an appropriate base, the polymer-bound α-sulfonyl carbanion is generated and coupled with other "body" building blocks in an efficient manner. After repeated processes and a global palladium-catalyzed desulfonation with LiEt3BH as the reducing agent, the desired polyprenols with various chain lengths and geometrical configurations are obtained in 32-59% overall yields. The solid-phase synthesis offers the advantage in facile isolation of polyprenols without tedious operation or time-consuming purifn.
- 26Meng, F.-C.; Chen, K.-T.; Huang, L.-Y.; Shih, H.-W.; Chang, H.-H.; Nien, F.-Y.; Liang, P.-H.; Cheng, T.-J. R.; Wong, C.-H.; Cheng, W.-C. Org. Lett. 2011, 13, 5306[ ACS Full Text], [ CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFOmsbvM&md5=52415914ff4884e2d08cbba6a5b45ab6Total Synthesis of Polyprenyl N-Glycolyl Lipid II as a Mycobacterial Transglycosylase SubstrateMeng, Fan-Chun; Chen, Kuo-Ting; Huang, Lin-Ya; Shih, Hao-Wei; Chang, Han-Hui; Nien, Fu-Yao; Liang, Pi-Hui; Cheng, Ting-Jen R.; Wong, Chi-Huey; Cheng, Wei-ChiehOrganic Letters (2011), 13 (19), 5306-5309CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)A feasible synthetic approach toward the Mycobacterium tuberculosis (Mtb) N-glycolyl lipid II-like mol. is described. The title compd. bears pendant undecaprenol and L-lysin moieties instead of the naturally occurring decaprenol and meso-diaminopimelic acid, which are not readily available. Functionalization of with a fluorophore on the peptide side chain gave a deriv. which was found to be recognized as an Mtb TGase substrate. This result suggests it has tremendous utility for mechanistic studies, the characterization of mycobacterial enzymes, and mycobacterial TGase inhibitor evaluation.
- 27Hesek, D.; Lee, M.; Zajíček, J.; Fisher, J. F.; Mobashery, S. J. Am. Chem. Soc. 2012, 134, 13881[ ACS Full Text], [ CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFClu77L&md5=794b00f4c83e2f75e816b3d867609b8aSynthesis and NMR Characterization of (Z,Z,Z,Z,E,E,ω)-HeptaprenolHesek, Dusan; Lee, Mijoon; Zajicek, Jaroslav; Fisher, Jed F.; Mobashery, ShahriarJournal of the American Chemical Society (2012), 134 (33), 13881-13888CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We describe a practical, multigram synthesis of (2Z,6Z,10Z,14Z,18E,22E)-3,7,11,15,19,23,27-heptamethyl-2,6,10,14,18,22,26-octacosaheptaen-1-ol [(Z4,E2,ω)-heptaprenol] (I) using the nerol-derived sulfone II as the key intermediate. Sulfone II is prepd. by a literature route and is converted in five addnl. steps (18% yield from II) to (Z4,E2,ω)-heptaprenol I. The use of Eu(hfc)3 as an NMR shift reagent not only enabled confirmation of the structure and stereochem. of I, but further enabled the structural assignment to a major side product from a failed synthetic connection. The availability by this synthesis of (Z4,E2,ω)-heptaprenol I in gram quantities will enable preparative access to key reagents for the study of the biosynthesis of the bacterial cell envelope.
- 28Shih, H.-W.; Chang, Y.-F.; Li, W.-J.; Meng, F.-C.; Huang, C.-Y.; Ma, C.; Cheng, T.-J. R.; Wong, C.-H.; Cheng, W.-C. Angew. Chem., Int. Ed. 2012, 51, 10123[ Crossref], [ PubMed], [ CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlSgu7jP&md5=597407837689d8debb954fa22d1acb6eEffect of the Peptide Moiety of Lipid II on Bacterial TransglycosylaseShih, Hao-Wei; Chang, Yi-Fan; Li, Wei-Jing; Meng, Fan-Chun; Huang, Chia-Ying; Ma, Che; Cheng, Ting-Jen R.; Wong, Chi-Huey; Cheng, Wei-ChiehAngewandte Chemie, International Edition (2012), 51 (40), 10123-10126, S10123/1-S10123/26CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A variety of lipid II analogs with different peptide moieties were synthesized and evaluated for binding with transglycosylase (TGase). The results demonstrate that the first two positions of lipid II, D-lactate and L-alanine, esp. the Me groups, are essential for substrate binding and activity toward TGase. This D-Lac-L-Ala moiety in lipid II greatly contributes to the interaction with TGase, perhaps enabling a proper conformation for enzyme recognition. The last two amino acids (D-Ala-D-Ala) do not contribute to the interaction between lipid II and TGase, and the fluorophore tag at the ε-NH2 group of the lysine residue does not affect the binding affinity.
- 29Lizak, C.; Gerber, S.; Numao, S.; Aebi, M.; Locher, K. P. Nature 2011, 474, 350[ Crossref], [ PubMed], [ CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnsFOku7c%253D&md5=55c847719d235c92046d4e58cbdccfd4X-ray structure of a bacterial oligosaccharyltransferaseLizak, Christian; Gerber, Sabina; Numao, Shin; Aebi, Markus; Locher, Kaspar P.Nature (London, United Kingdom) (2011), 474 (7351), 350-355CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Asparagine-linked glycosylation is a post-translational modification of proteins contg. conserved sequence motif Asn-X-Ser/Thr. The attachment of oligosaccharides is implicated in diverse processes such as protein folding and quality control, organism development or host-pathogen interactions. The reaction is catalyzed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The central, catalytic enzyme of OST is the STT3 subunit, which has homologs in bacteria and archaea. Here, the authors report the x-ray structure of a bacterial OST, the PglB protein of Campylobacter lari, in complex with an acceptor peptide. The structure defined the fold of STT3 proteins and provided insight into glycosylation sequon recognition and amide N atom activation, both of which are prerequisites for the formation of the N-glycosidic linkage. The authors also identified and validated catalytically important, acidic amino acid residues. These results provide the mol. basis for understanding the mechanism of N-linked glycosylation.
- 30Jaenicke, L.; Siegmund, H.-U. Chem. Phys. Lipids 1989, 51, 159[ Crossref], [ CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXkslyksbo%253D&md5=b46624ac2cc77f3cd7cf1f90ee1212b7Synthesis and characterization of dolichols and polyprenols of designed geometry and chain lengthJaenicke, Lothar; Siegmund, Hans UlrichChemistry and Physics of Lipids (1989), 51 (3-4), 159-70CODEN: CPLIA4; ISSN:0009-3084.Dolichols of defined uniform chain length (C20 to C55) and geometry were prepd. by total synthesis according to the following principle: (E,E)-farnesol, activated as its p-tolylsulfone, was condensed with 8-chloro-(6Z)-neryl benzyl ether, the sulfonyl group removed and the ether linkage cleaved by Li/Et3N. After several cycles of this C10-elongaton sequence the synthesis was completed in the same way but using 8-chloro-citronellyl benzyl ether as building block to introduce the satd. α-isoprene unit. Polyprenols with an even no. of isoprene units are obtained by coupling activated geraniol with 8-chloro-(6E)-neryl benzyl ether in the first step. 1H- and 13C-NMR data were recorded for qual. and stereochem. comparison with natural dolichols. The versatility of this design makes it possible to synthesize dolichols with different geometry and double bond pattern.
- 31Danilov, L. L.; Druzhinina, T. N.; Kalinchuk, N. A.; Maltsev, S. D.; Shibaev, V. N. Chem. Phys. Lipids 1989, 51, 191[ Crossref], [ PubMed], [ CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXktF2qtrw%253D&md5=34934a274daf5ade435238fece1eab85Polyprenyl phosphates: synthesis and structure-activity relationship for a biosynthetic system of Salmonella anatum O-specific polysaccharideDanilov, L. L.; Druzhinina, T. N.; Kalinchuk, N. A.; Mal'tsev, S. D.; Shibaev, V. N.Chemistry and Physics of Lipids (1989), 51 (3-4), 191-203CODEN: CPLIA4; ISSN:0009-3084.A series of polyprenyl phosphates with modified structure of the polyprenyl residue was prepd. through phosphorylation of polyprenyl trichloroacetimidates with phosphoric acid. Interaction of polyprenols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile represented a very efficient, simple, and general method for the synthesis of polyprenyl phosphates. A procedure was developed for smooth conversion of polyprenyl pyrophosphates into the monophosphates through hydrolysis in the presence of 4-dimethylaminopyridine. The polyprenyl phosphates prepd. were studied as substrates for the enzymes of S. anatum O-specific polysaccharide biosynthesis. Correct stereochem. of α- and β-isoprenic units was essential for substrate efficiency. At the more remote positions of the hydrocarbon chain just the presence of isoprenic units of any configuration seems necessary. Some changes in position of the phosphate group may be permissible without significant loss of substrate properties.
- 32Bernardes, G. J. L.; Davis, B. G. unpublished results, University of Oxford, 2007.Google ScholarThere is no corresponding record for this reference.
- 33Bernardes, G. J. L.; Kikkeri, R.; Maglinao, M.; Laurino, P.; Collot, M.; Hong, S. Y.; Lepenies, B.; Seeberger, P. H. Org. Biomol. Chem. 2010, 8, 4987[ Crossref], [ PubMed], [ CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht1OmsLzE&md5=ba806c8a9538b0a3eaf75738d9e25eeaDesign, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targetingBernardes, Goncalo J. L.; Kikkeri, Raghavendra; Maglinao, Maha; Laurino, Paola; Collot, Mayeul; Hong, Sung You; Lepenies, Bernd; Seeberger, Peter H.Organic & Biomolecular Chemistry (2010), 8 (21), 4987-4996CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The C-type lectin asialo-glycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an efficient synthesis of carbohydrate-functionalized β-cyclodextrins (βCDs) and liposomes for hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded βCDs were functionalized with glycodendrimers. Liposomes were equipped with synthetic glycolipids contg. a terminal d-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular carcinoma cell line HepG2 demonstrated that βCDs and liposomes displaying terminal d-Gal/d-GalNAc residues were preferentially endocytosed. In contrast, uptake of βCDs and liposomes with terminal d-Man or D-GlcNAc residues was markedly reduced. The d-Gal/d-GalNAc-functionalized βCDs and liposomes presented here enable hepatocyte-specific targeting. Gal-functionalized βCDs are efficient mol. carriers to deliver doxorubicin in vitro into hepatocytes and induce apoptosis.
- 34Schagger, H. Nat. Protoc. 2006, 1, 16[ Crossref], [ PubMed], [ CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2s3gsVSmsA%253D%253D&md5=0846bad5701202cba5395129c11168edTricine-SDS-PAGESchagger HermannNature protocols (2006), 1 (1), 16-22 ISSN:.Tricine-SDS-PAGE is commonly used to separate proteins in the mass range 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine-SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1-2 d.
- 35Wang, L.-X. Carbohydr. Res. 2008, 343, 1509[ Crossref], [ PubMed], [ CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnt1yisrk%253D&md5=a5fdbc6e98a7927f571eec06592af1acChemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylationWang, Lai-XiCarbohydrate Research (2008), 343 (10-11), 1509-1522CODEN: CRBRAT; ISSN:0008-6215. (Elsevier Ltd.)A review. Homogeneous glycopeptides and glycoproteins are indispensable for detailed structural and functional studies of glycoproteins. It is also fundamentally important to correct glycosylation patterns for developing effective glycoprotein-based therapeutics. This review discusses a useful chemoenzymic method that takes advantage of the endoglycosidase-catalyzed transglycosylation to attach an intact oligosaccharide to a polypeptide in a single step, without the need for any protecting groups. The exploration of sugar oxazolines (enzymic reaction intermediates) as donor substrates has not only expanded substrate availability, but also has significantly enhanced the enzymic transglycosylation efficiency. Moreover, the discovery of a novel mutant with glycosynthase-like activity has made it possible to synthesize homogeneous glycoproteins with full-size natural N-glycans. Recent advances in this highly convergent chemoenzymic approach and its application for glycopeptide and glycoprotein synthesis are highlighted.
- 36Rich, J. R.; Withers, S. G. Nat. Chem. Biol. 2009, 5, 206[ Crossref], [ PubMed], [ CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtlWmtbg%253D&md5=009fa4833a435582001aabbc71f7e600Emerging methods for the production of homogeneous human glycoproteinsRich, Jamie R.; Withers, Stephen G.Nature Chemical Biology (2009), 5 (4), 206-215CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. Most circulating human proteins exist as heterogeneously glycosylated variants (glycoforms) of an otherwise homogeneous polypeptide. Though glycan heterogeneity is most likely important to glycoprotein function, the prepn. of homogeneous glycoforms is important both for the study of the consequences of glycosylation and for therapeutic purposes. This review details selected approaches to the prodn. of homogeneous human N- and O-linked glycoproteins with human-type glycans. Particular emphasis is placed on recent developments in the engineering of glycosylation pathways within yeast and bacteria for in vivo prodn., and on the in vitro remodeling of glycoproteins by enzymic means. The future of this field is very exciting.
- 37Goodfellow, J. J.; Baruah, K.; Yamamoto, K.; Bonomelli, C.; Krishna, B.; Harvey, D. J.; Crispin, M.; Scanlan, C. N.; Davis, B. G. J. Am. Chem. Soc. 2012, 134, 8030[ ACS Full Text], [ CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmtlSmtr0%253D&md5=3c4af24f272d75afe68d77dcf5bfc881An Endoglycosidase with Alternative Glycan Specificity Allows Broadened Glycoprotein RemodellingGoodfellow, Jonathan J.; Baruah, Kavitha; Yamamoto, Keisuke; Bonomelli, Camille; Krishna, Benjamin; Harvey, David J.; Crispin, Max; Scanlan, Christopher N.; Davis, Benjamin G.Journal of the American Chemical Society (2012), 134 (19), 8030-8033CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein endoglycosidases are useful for biocatalytic alteration of glycans on protein surfaces, but the currently limited selectivity of endoglycosidases has prevented effective manipulation of certain N-linked glycans widely found in nature. Here we reveal that a bacterial endoglycosidase from Streptococcus pyogenes, EndoS, is complementary to other known endoglycosidases (EndoA, EndoH) used for current protein remodeling. It allows processing of complex-type N-linked glycans +/- core fucosylation but does not process oligomannose- or hybrid-type glycans. This biocatalytic activity now addresses previously refractory antibody glycoforms.
- 38Fernández-González, M.; Boutureira, O.; Bernardes, G. J.; Chalker, J. M.; Young, M.; Errey, J.; Davis, B. G. Chem. Sci. 2010, 1, 709[ Crossref], [ CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtl2nsLnE&md5=613c61342fa65cd1273e79866d7bead4Site-selective chemoenzymatic construction of synthetic glycoproteins using endoglycosidasesFernandez-Gonzalez, Marta; Boutureira, Omar; Bernardes, Goncalo J. L.; Chalker, Justin M.; Young, Matthew A.; Errey, James C.; Davis, Benjamin G.Chemical Science (2010), 1 (6), 709-715CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Combined chem. tagging followed by Endo-A catalyzed elongation allows access to homogeneous, elaborated glycoproteins. A survey of different linkages and sugars demonstrated not only that unnatural linkages can be tolerated but they can provide insight into the scope of Endo-A transglycosylation activity. S-linked GlcNAc-glycoproteins are useful substrates for Endo-A extensions and display enhanced stability to hydrolysis at exposed sites. O-CH2-triazole-linked GlcNAc-glycoproteins derived from azidohomoalanine-tagged protein precursors were found to be optimal at sterically demanding sites.
- 39Lomino, J. V.; Naegeli, A.; Orwenyo, J.; Amin, M. N.; Aebi, M.; Wang, L.-X. Bioorg. Med. Chem. 2013, 21, 2262[ Crossref], [ PubMed], [ CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktVOltLk%253D&md5=f556d1063e655a9590088cecb56ed5c8A two-step enzymatic glycosylation of polypeptides with complex N-glycansLomino, Joseph V.; Naegeli, Andreas; Orwenyo, Jared; Amin, Mohammed N.; Aebi, Markus; Wang, Lai-XiBioorganic & Medicinal Chemistry (2013), 21 (8), 2262-2270CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)A chemoenzymatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our expts. demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc-Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc-Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addn., the Glc-Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc-Asn linked glycopeptide. The chemoenzymic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery.
- 40Gamblin, D. P.; Garnier, P.; van Kasteren, S.; Oldham, N. J.; Fairbanks, A. J.; Davis, B. G. Angew. Chem., Int. Ed. 2004, 43, 828[ Crossref], [ PubMed], [ CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhsFyhu70%253D&md5=47923ea045eb426755a91944c12e6d5dGlyco-SeS: selenenyl-sulfide-mediated protein glycoconjugation - A new strategy in post-translational modificationGamblin, David P.; Garnier, Philippe; van Kasteren, Sander; Oldham, Neil J.; Fairbanks, Antony J.; David, Benjamin G.Angewandte Chemie, International Edition (2004), 43 (7), 828-833CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Site-selective glycosylation by Se-S-mediated ligation has led to the efficient formation of a wide variety of conjugates without the need for a large excess of the carbohydrate reagent. By this convergent method it was possible to introduce a heptasaccharide glycan selectively, and to perform a multiple site-selective chem. glycosylation of protein. A chem. Cys-glycosylated glycoprotein was elaborated enzymically.
- 41Ye, X.-Y.; Lo, M.-C.; Brunner, L.; Walker, D.; Kahne, D.; Walker, S. J. Am. Chem. Soc. 2001, 123, 3155[ ACS Full Text], [ CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhs1ymtrg%253D&md5=d1392c2a50d8f06924254982ad3d1059Better Substrates for Bacterial TransglycosylasesYe, Xiang-Yang; Lo, Mei-Chu; Brunner, Livia; Walker, Deborah; Kahne, Daniel; Walker, SuzanneJournal of the American Chemical Society (2001), 123 (13), 3155-3156CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The enzymes that synthesize the peptidoglycan layers surrounding bacterial cell membranes have received special attention because many known antibiotics function by blocking peptidoglycan synthesis. Among these enzymes, the bacterial transglycosylases (TGases) represent some of the most promising targets. TGases are located on the external surface of the bacterial membrane where they polymerize Lipid II, a disaccharide anchored to the membrane by a 55 carbon undecaprenyl chain. Although the TGases were first identified decades ago, their structures and mechanisms are not well understood. Some of the difficulties in studying TGases are related to problems obtaining and handling Lipid II. Because the 55 carbon chain aggregates, assays utilizing Lipid II, which can be isolated only in small quantities from bacterial membranes, must include org. solvents, detergents, and other additives. Results can be variable, and it is difficult to det. whether problems are due to the enzymes or to the substrate. Better substrates would facilitate the study of TGases. To identify better TGase substrates, the authors have synthesized natural Lipid II as well as a set of analogs contg. different lipid chains. These compds. have been tested for their ability to function as TGase substrates. The results show that bacterial TGases have clear preferences with regard to the structure of the lipid chain, but they do not require the 55 carbon undecaprenyl moiety. In fact, the authors have identified a compd. with a shorter lipid chain that is a much better TGase substrate than natural Lipid II.
- 42Liu, C.-Y.; Guo, C.-W.; Chang, Y.-F.; Wang, J.-T.; Shih, H.-W.; Hsu, Y.-F.; Chen, C.-W.; Chen, S.-K.; Wang, Y.-C.; Cheng, T.-J.; Ma, C.; Wong, C.-H.; Fang, J.-M.; Cheng, W.-C. Org. Lett. 2010, 12, 1608[ ACS Full Text], [ CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXisVOqsrY%253D&md5=36aa529a757a7a6c1159f55fdc7fc7e8Synthesis and Evaluation of a New Fluorescent Transglycosylase Substrate: Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl MoietyLiu, Chen-Yu; Guo, Chih-Wei; Chang, Yi-Fan; Wang, Jen-Tsung; Shih, Hao-Wei; Hsu, Yu-Fang; Chen, Chia-Wei; Chen, Shao-Kang; Wang, Yen-Chih; Cheng, Ting-Jen R.; Ma, Che; Wong, Chi-Huey; Fang, Jim-Min; Cheng, Wei-ChiehOrganic Letters (2010), 12 (7), 1608-1611CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)The prepn. of a novel fluorescent lipid II-based substrate for transglycosylases (TGases) is described. This substrate has characteristic structural features including a shorter lipid chain, a fluorophore tag at the end of the lipid chain rather than on the peptide chain, and no labeling with a radioactive atom. This fluorescent substrate is readily utilized in TGase activity assays to characterize TGases and also to evaluate the activities of TGase inhibitors.
- 43Lehrer, J.; Vigeant, K. A.; Tatar, L. D.; Valvano, M. A. J. Bacteriol. 2007, 189, 2618[ Crossref], [ PubMed], [ CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjvVOjtLs%253D&md5=a2b85e8d730703628f81465c7149276fFunctional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharideLehrer, Jason; Vigeant, Karen A.; Tatar, Laura D.; Valvano, Miguel A.Journal of Bacteriology (2007), 189 (7), 2618-2628CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examd. the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also detd. the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent Km and Vmax values for UDP-GlcNAc, Mg2+, and Mn2+ suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg2+, possibly playing a role in orienting the substrates. Topol. anal. using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topol. map of WecA. Also, we show that cells expressing a WecA deriv. C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.
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- Maja Napiórkowska, Jérémy Boilevin, Tamis Darbre, Jean-Louis Reymond, Kaspar P. Locher. Structure of bacterial oligosaccharyltransferase PglB bound to a reactive LLO and an inhibitory peptide. Scientific Reports 2018, 8 (1) DOI: 10.1038/s41598-018-34534-0.
- , , Aravind Natarajan, Thapakorn Jaroentomeechai, Mingji Li, Cameron J. Glasscock, Matthew P. DeLisa. Metabolic engineering of glycoprotein biosynthesis in bacteria. Emerging Topics in Life Sciences 2018, 2 (3) , 419-432. DOI: 10.1042/ETLS20180004.
- Yin Yao Dong, Hua Wang, Ashley C. W. Pike, Stephen A. Cochrane, Sadra Hamedzadeh, Filip J. Wyszyyski, Simon R. Bushell, Sylvain F. Royer, David A. Widdick, Andaleeb Sajid, Helena I. Boshoff, Ricardo Lucas, Wei-Min Liu, Seung Seo Lee, Takuya Machida, Shahid Mehmood, Katsiaryna Belaya, Wei-Wei Liu, Amy Chu, Leela Shrestha, Shubhashish M. M. Mukhopadhyay, Nicola A. Burgess-Brown, Mervyn J. Bibb, Clifton E. Barry III, Carol V. Robinson, David Beeson, Benjamin G. Davis, Elisabeth P. Carpenter. Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design. SSRN Electronic Journal 2018, DOI: 10.2139/ssrn.3188395.
- Maja Napiórkowska, Jérémy Boilevin, Tina Sovdat, Tamis Darbre, Jean-Louis Reymond, Markus Aebi, Kaspar P Locher. Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase. Nature Structural & Molecular Biology 2017, 24 (12) , 1100-1106. DOI: 10.1038/nsmb.3491.
- Shiteshu Shrimal, Natalia A Cherepanova, Reid Gilmore. One flexible loop in OST lassos both substrates. Nature Structural & Molecular Biology 2017, 24 (12) , 1009-1010. DOI: 10.1038/nsmb.3508.
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Abstract
Figure 1
Figure 1. Bacterial N-linked glycosylation and designed unnatural candidate polyprenols 1a–g as alternative short lipid carriers for PglB-catalyzed glycosylation. The polyprenols were synthesized from building blocks 2a–d and 3a and 3b.
Scheme 1
Scheme 1. Syntheses of Polyprenol Variantsaa(i) BuLi, THF, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMTP), −78 °C. Yield: 4a = 92%, 4b = 93%, 4c = 83%, 4d = 63%, 4e = 67%, 4f = 74%. (ii) (1) TBAF, THF, RT, 4 h; (2) LiEt3BH, (dppp)PdCl2, THF, 0 °C–RT. Yield: 1a= 65%, 1b = 57%, 1c = 50%, 1d = 70%, 1e = 60%, 1f = 54%.
Scheme 2
Scheme 2. Syntheses of Lipid-linked Oligosaccharides and use in Glycopeptide Synthesisaa(i) 5a–g, TBAP, TCA, DCM, RT; 5h, POCl3, Et3N, 2h. (ii) (1) CDI, DMF; (2) MeOH; (3) 6, DMF, RT, 3–5 days. (iii) NaOMe, MeOH. Yield over 3 steps: 8a = 17%, 8b = 23%, 8c = 9%, 8d = 17%, 8e = 9%, 8f = 38%, 8g = 29%, 8h = 32%, 13 = 22%, 14 = 30%. Extension reactions (blue arrows, 5–94%); see SI for further details.
Figure 2
Figure 2. Peptide and protein glycosylation. (a) Fluorescent electrophoretic analysis of peptide glycosylation with lipids (8a–h) (− = no lipid; + = lipid extracted and enriched from E. coli cells producing C. jejuni heptasaccharide-linked undecaprenyl pyrophosphate); [glycolipid] = [peptide] 20 μM; [PglB] = 0.44 μM. (b) ES-MS of in vitro N-linked protein (AcrA) glycosylation with 8c; >95% diglycosylation.
References
ARTICLE SECTIONSThis article references 43 other publications.
- 1Spiro, R. G. Glycobiology 2002, 12, 43R[ Crossref], [ PubMed], [ CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XltVyhtrY%253D&md5=b9c8933df399c8128ff275614d7ca7acProtein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bondsSpiro, Robert G.Glycobiology (2002), 12 (4), 43R-56RCODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)A review. Formation of the sugar-amino acid linkage is a crucial event in the biosynthesis of the carbohydrate units of glycoproteins. It sets into motion a complex series of posttranslational enzymic steps that lead to the formation of a host of protein-bound oligosaccharides with diverse biol. functions. These reactions occur throughout the entire phylogenetic spectrum, ranging from archaea and eubacteria to eukaryotes. It is the aim of this review to describe the glycopeptide linkages that have been found to date and specify their presence on well-characterized glycoproteins. A survey is also made of the enzymes involved in the formation of the various glycopeptide bonds as well as the site of their intracellular action and their affinity for particular peptide domains is evaluated. This examn. indicates that 13 different monosaccharides and 8 amino acids are involved in glycoprotein linkages leading to a total of at least 41 bonds, if the anomeric configurations, the phosphoglycosyl linkages, as well as the GPI (glycophosphatidylinositol) phosphoethanolamine bridge are also considered. These bonds represent the products of N- and O-glycosylation, C-mannosylation, phosphoglycation, and glypiation. Currently at least 16 enzymes involved in their formation have been identified and in many cases cloned. Their intracellular site of action varies and includes the endoplasmic reticulum, Golgi app., cytosol, and nucleus. With the exception of the Asn-linked carbohydrate and the GPI anchor, which are transferred to the polypeptide en bloc, the sugar-amino acid linkages are formed by the enzymic transfer of an activated monosaccharide directly to the protein. This review also deals briefly with glycosidases, which are involved in physiol. important cleavages of glycopeptide bonds in higher organisms, and with a no. of human disease states in which defects in enzymic transfer of saccharides to protein have been implicated.
- 2Walsh, C. T.; Garneau-Tsodikova, S.; Gatto, G. J. Angew. Chem., Int. Ed. 2005, 44, 7342[ Crossref], [ PubMed], [ CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlSms77N&md5=1e1ccf345916c39631a1cc83093de9b1Protein posttranslational modifications: The chemistry of proteome diversificationsWalsh, Christopher T.; Garneau-Tsodikova, Sylvie; Gatto, Gregory J., Jr.Angewandte Chemie, International Edition (2005), 44 (45), 7342-7372CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the no. of proteins predicted by DNA coding capacities owing to directed post-translational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitination, can be classified according to the type of amino acid side-chain modified, the category of the modifying enzyme, and the extent of reversibility. Chem. events such as protein splicing, green fluorescent protein (GFP) maturation, and proteasome autoactivation also represent post-translational modifications. An understanding of the scope and pattern of the many post-translational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compns.
- 3Ohtsubo, K.; Marth, J. D. Cell 2006, 126, 855[ Crossref], [ PubMed], [ CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpvVKitbo%253D&md5=959a24814730df3802c399ba78f7cd0fGlycosylation in cellular mechanisms of health and diseaseOhtsubo, Kazuaki; Marth, Jamey D.Cell (Cambridge, MA, United States) (2006), 126 (5), 855-867CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Glycosylation produces an abundant, diverse, and highly regulated repertoire of cellular glycans that are frequently attached to proteins and lipids. The past decade of research on glycan function has revealed that the enzymes responsible for glycosylation-the glycosyl-transferases and glycosidases-are essential in the development and physiol. of living organisms. Glycans participate in many key biol. processes including cell adhesion, mol. trafficking and clearance, receptor activation, signal transduction, and endocytosis. This review discusses the increasingly sophisticated mol. mechanisms being discovered by which mammalian glycosylation governs physiol. and contributes to disease.
- 4Szymanski, C. M.; Yao, R.; Ewing, C. P.; Trust, T. J.; Guerry, P. Mol. Microbiol. 1999, 32, 1022[ Crossref], [ PubMed], [ CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvVSmsLw%253D&md5=34430a0fdd62a3238e74ec487a72095bEvidence for a system of general protein glycosylation in Campylobacter jejuniSzymanski, Christine M.; Yao, Ruijin; Ewing, Cheryl P.; Trust, Trevor J.; Guerry, PatriciaMolecular Microbiology (1999), 32 (5), 1022-1030CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)A genetic locus from Campylobacter jejuni 81-176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5α contg. some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site-specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site-specific mutation of each of the seven genes in 81-176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild-type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chem. deglycosylation of protein fractions from the 81-176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.
- 5Young, N. M.; Brisson, J. R.; Kelly, J.; Watson, D. C.; Tessier, L.; Lanthier, P. H.; Jarrell, H. C.; Cadotte, N.; St Michael, F.; Aberg, E.; Szymanski, C. M. J. Biol. Chem. 2002, 277, 42530[ Crossref], [ PubMed], [ CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xos1ensLs%253D&md5=0359877f22079bf195c91b931751f69eStructure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuniYoung, N. Martin; Brisson, Jean-Robert; Kelly, John; Watson, David C.; Tessier, Luc; Lanthier, Patricia H.; Jarrell, Harold C.; Cadotte, Nicolas; St. Michael, Frank; Aberg, Erika; Szymanski, Christine M.Journal of Biological Chemistry (2002), 277 (45), 42530-42539CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatog., a mixt. of glycoproteins was obtained from a glycine ext., and two-dimensional gel proteomics anal. led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepd. from the glycoprotein mixt. by Pronase digestion and gel filtration. The structure of the glycan was detd. by using nano-NMR techniques to be GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3-]GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.
- 6Linton, D.; Allan, E.; Karlyshev, A. V.; Cronshaw, A. D.; Wren, B. W. Mol. Microbiol. 2002, 43, 497[ Crossref], [ PubMed], [ CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xhtlajsb4%253D&md5=7e2b9ade854de0e58de9652aadd51dffIdentification of N-acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuniLinton, Dennis; Allan, Elaine; Karlyshev, Andrey V.; Cronshaw, Andrew D.; Wren, Brendan W.Molecular Microbiology (2002), 43 (2), 497-508CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)There is a system of general protein glycosylation in the human enteropathogen Campylobacter jejuni. To characterize such glycoproteins, we identified a lectin, soybean agglutinin (SBA), which binds to multiple C. jejuni proteins on Western blots. Binding of lectin SBA was disrupted by mutagenesis of genes within the previously identified protein glycosylation locus. This lectin was used to purify putative glycoproteins selectively and, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie-stained bands were cut from the gels. The bands were digested with trypsin, and peptides were identified by mass spectrometry and database searching. A 28 kDa band was identified as PEB3, a previously characterized immunogenic cell surface protein. Bands of 32 and 34 kDa were both identified as a putative periplasmic protein encoded by the C. jejuni NCTC 11168 coding sequence Cj1670c. We have named this putative glycoprotein CgpA. We constructed insertional knockout mutants of both the peb3 and cgpA genes, and surface protein exts. from mutant and wild-type strains were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). In this way, we were able to identify the PEB3 protein as a 28 kDa SBA-reactive and immunoreactive glycoprotein. The cgpA gene encoded SBA-reactive and immunoreactive proteins of 32 and 34 kDa. By using specific exoglycosidases, we demonstrated that the SBA binding property of acid-glycine extractable C. jejuni glycoproteins, including PEB3 and CgpA, is a result of the presence of α-linked N-acetylgalactosamine residues. These data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni. Furthermore, we have identified two such glycoproteins, the first non-flagellin Campylobacter glycoproteins to be identified, and demonstrated that their glycan components contain α-linked N-acetylgalactosamine residues.
- 7Scott, N. E.; Parker, B. L.; Connolly, A. M.; Paulech, J.; Edwards, A. V. G.; Crossett, B.; Falconer, L.; Kolarich, D.; Djordjevic, S. P.; Højrup, P.; Packer, N. H.; Larsen, M. R.; Cordwell, S. J. Mol. Cell. Proteomics 2011, 10, M000031
- 8Schwarz, F.; Lizak, C.; Fan, Y.-Y.; Fleurkens, S.; Kowarik, M.; Aebi, M. Glycobiology 2011, 21, 45[ Crossref], [ PubMed], [ CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFGru73F&md5=66b94c57da11b068925fd1a61a2bdef2Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivoSchwarz, Flavio; Lizak, Christian; Fan, Yao-Yun; Fleurkens, Susanna; Kowarik, Michael; Aebi, MarkusGlycobiology (2011), 21 (1), 45-54CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)A no. of proteobacteria carry the genetic information to perform N-linked glycosylation, but only the protein glycosylation (pgl) pathway of Campylobacter jejuni has been studied to date. Here, we report that the pgl gene cluster of Campylobacter lari encodes for a functional glycosylation machinery that can be reconstituted in Escherichia coli. We detd. that the N-glycan produced in this system consisted of a linear hexasaccharide. We found that the oligosaccharyltransferase (OST) of C. lari conserved a predominant specificity for the primary sequence D/E-X-1-N-X+1-S/T (where X-1 and X+1 can be any amino acid but proline). At the same time, we obsd. that this enzyme exhibited a relaxed specificity toward the acceptor site and modified asparagine residues of a protein at sequences DASG and NNST. Moreover, C. lari pgl glycosylated a native E. coli protein. Bacterial N-glycosylation appears as a useful tool to establish a mol. description of how single-subunit OSTs perform selection of glycosyl acceptor sites.
- 9Jervis, A. J.; Langdon, R.; Hitchen, P.; Lawson, A. J.; Wood, A.; Fothergill, J. L.; Morris, H. R.; Dell, A.; Wren, B.; Linton, D. J. Bacteriol. 2010, 192, 5228[ Crossref], [ PubMed], [ CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFCitbjJ&md5=1ba8109b717bc3ced101eb31e10e065dCharacterization of N-linked protein glycosylation in Helicobacter pullorumJervis, Adrian J.; Langdon, Rebecca; Hitchen, Paul; Lawson, Andrew J.; Wood, Alison; Fothergill, Joanne L.; Morris, Howard R.; Dell, Anne; Wren, Brendan; Linton, DennisJournal of Bacteriology (2010), 192 (19), 5228-5236CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologs are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation expts., the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Anal. of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was obsd. This reaction required an acidic residue at the -2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.
- 10Ielmini, M. V.; Feldman, M. F. Glycobiology 2011, 21, 734[ Crossref], [ PubMed], [ CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmt1ajtbs%253D&md5=a1f953b18b18b0f07d352435b6ea44a7Desulfovibrio desulfuricans PglB homolog possesses oligosaccharyltransferase activity with relaxed glycan specificity and distinct protein acceptor sequence requirementsIelmini, Maria V.; Feldman, Mario F.Glycobiology (2011), 21 (6), 734-742CODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)Oligosaccharyltransferases (OTases) are responsible for the transfer of carbohydrates from lipid carriers to acceptor proteins and are present in all domains of life. In bacteria, the most studied member of this family is PglB from Campylobacter jejuni (PglBCj). This enzyme is functional in Escherichia coli and, contrary to its eukaryotic counterparts, has the ability to transfer a variety of oligo- and polysaccharides to protein carriers in vivo. Phylogenetic anal. revealed that in the delta proteobacteria Desulfovibrio sp., the PglB homolog is more closely related to eukaryotic and archaeal OTases than to its Campylobacter counterparts. Genetic anal. revealed the presence of a putative operon that might encode all enzymes required for N-glycosylation in Desulfovibrio desulfuricans. D. desulfuricans PglB (PglBDd) was cloned and successfully expressed in E. coli, and its activity was confirmed by transferring the C. jejuni heptasaccharide onto the model protein acceptor AcrA. In contrast to PglBCj, which adds two glycan chains to AcrA, a single oligosaccharide was attached to the protein by PglBDd. Site-directed mutagenesis of the five putative N-X-S/T glycosylation sites in AcrA and mass spectrometry anal. showed that PglBDd does not recognize the "conventional bacterial glycosylation sequon" consisting of the sequence D/E-X1-N-X2-S/T (where X1 and X2 are any amino acid except proline), and instead used a different site for the attachment of the oligosaccharide than PglBCj. Furthermore, PglBDd exhibited relaxed glycan specificity, being able to transfer mono- and polysaccharides to AcrA. Our anal. constitutes the first characterization of an OTase from delta-proteobacteria involved in N-linked protein glycosylation.
- 11Larkin, A.; Chang, M. M.; Whitworth, G. E.; Imperiali, B. Nat. Chem. Biol. 2013, 9, 367[ Crossref], [ PubMed], [ CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1Wms70%253D&md5=8002ef3e92ef581f18013004cee47d42Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesisLarkin, Angelyn; Chang, Michelle M.; Whitworth, Garrett E.; Imperiali, BarbaraNature Chemical Biology (2013), 9 (6), 367-373CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro anal. of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homol. to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochem. evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.
- 12Glover, K. J.; Weerapana, E.; Numao, S.; Imperiali, B. Chem. Biol. 2005, 12, 1311[ Crossref], [ PubMed], [ CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlWktLjK&md5=60291c5dce3c72c700292b1a5fde74b7Chemoenzymatic Synthesis of Glycopeptides with PglB, a Bacterial Oligosaccharyl Transferase from Campylobacter jejuniGlover, Kerney Jebrell; Weerapana, Eranthie; Numao, Shin; Imperiali, BarbaraChemistry & Biology (Cambridge, MA, United States) (2005), 12 (12), 1311-1315CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)The gram-neg. bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PglB, is thought to be the oligosaccharyl transferase due to its significant homol. to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PglB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GalNAc-α1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PglB in vitro. Furthermore, the prepn. of addnl. undecaprenyl-linked glycan variants reveals the ability of PglB to transfer a wide variety of saccharides. With the demonstration of PglB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.
- 13Wacker, M.; Feldman, M. F.; Callewaert, N.; Kowarik, M.; Clarke, B. R.; Pohl, N. L.; Hernandez, M.; Vines, E. D.; Valvano, M. A.; Whitfield, C.; Aebi, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7088[ Crossref], [ PubMed], [ CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XkslCkt7k%253D&md5=ee2bf4bcd1ef2cba7c3226eae5bf9db3Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systemsWacker, Michael; Feldman, Mario F.; Callewaert, Nico; Kowarik, Michael; Clarke, Bradley R.; Pohl, Nicola L.; Hernandez, Marcela; Vines, Enrique D.; Valvano, Miguel A.; Whitfield, Chris; Aebi, MarkusProceedings of the National Academy of Sciences of the United States of America (2006), 103 (18), 7088-7093CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The PglB oligosaccharyltransferase (OTase) of Campylobacter jejuni can be functionally expressed in Escherichia coli, and its relaxed oligosaccharide substrate specificity allows the transfer of different glycans from the lipid carrier undecaprenyl pyrophosphate to an acceptor protein. To investigate the substrate specificity of PglB, we tested the transfer of a set of lipid-linked polysaccharides in E. coli and Salmonella enterica serovar Typhimurium. A hexose linked to the C-6 of the monosaccharide at the reducing end did not inhibit the transfer of the O antigen to the acceptor protein. However, PglB required an acetamido group at the C-2. A model for the mechanism of PglB involving this functional group was proposed. Previous expts. have shown that eukaryotic OTases have the same requirement, suggesting that eukaryotic and prokaryotic OTases catalyze the transfer of oligosaccharides by a conserved mechanism. Moreover, we demonstrated the functional transfer of the C. jejuni glycosylation system into S. enterica. The elucidation of the mechanism of action and the substrate specificity of PglB represents the foundation for engineering glycoproteins that will have an impact on biotechnol.
- 14Feldman, M. F.; Wacker, M.; Hernandez, M.; Hitchen, P. G.; Marolda, C. L.; Kowarik, M.; Morris, H. R.; Dell, A.; Valvano, M. A.; Aebi, M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3016[ Crossref], [ PubMed], [ CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXitVSktL4%253D&md5=2d348d3f2cb30206eb22d6e47ad00410Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coliFeldman, Mario F.; Wacker, Michael; Hernandez, Marcela; Hitchen, Paul G.; Marolda, Cristina L.; Kowarik, Michael; Morris, Howard R.; Dell, Anne; Valvano, Miguel A.; Aebi, MarkusProceedings of the National Academy of Sciences of the United States of America (2005), 102 (8), 3016-3021CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures contg. two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo prodn. of O polysaccharides-protein conjugates for use as antibacterial vaccines.
- 15Schwarz, F.; Huang, W.; Li, C.; Schulz, B. L.; Lizak, C.; Palumbo, A.; Numao, S.; Neri, D.; Aebi, M.; Wang, L.-X. Nat. Chem. Biol. 2010, 6, 264[ Crossref], [ PubMed], [ CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXisFeksro%253D&md5=20276ab4992696b1ed34ac1cbd436ab3A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylationSchwarz, Flavio; Huang, Wei; Li, Cishan; Schulz, Benjamin L.; Lizak, Christian; Palumbo, Alessandro; Numao, Shin; Neri, Dario; Aebi, Markus; Wang, Lai-XiNature Chemical Biology (2010), 6 (4), 264-266CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)We describe a new method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the Campylobacter jejuni glycosylation machinery in Escherichia coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins.
- 16Wacker, M.; Linton, D.; Hitchen, P. G.; Nita-Lazar, M.; Haslam, S. M.; North, S. J.; Panico, M.; Morris, H. R.; Dell, A.; Wren, B. W.; Aebi, M. Science 2002, 298, 1790[ Crossref], [ PubMed], [ CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XovFKmsL0%253D&md5=cbdd0efcd7491b2ceb610f2ba5bc115aN-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coliWacker, Michael; Linton, Dennis; Hitchen, Paul G.; Nita-Lazar, Mihai; Haslam, Stuart M.; North, Simon J.; Panico, Maria; Morris, Howard R.; Dell, Anne; Wren, Brendan W.; Aebi, MarkusScience (Washington, DC, United States) (2002), 298 (5599), 1790-1793CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)N-linked protein glycosylation is the most abundant posttranslation modification of secretory proteins in eukaryotes. A wide range of functions are attributed to glycan structures covalently linked to asparagine residues within the asparagine-X-serine/threonine consensus sequence (Asn-Xaa-Ser/Thr). We found an N-linked glycosylation system in the bacterium Campylobacter jejuni and demonstrate that a functional N-linked glycosylation pathway could be transferred into Escherichia coli. Although the bacterial N-glycan differs structurally from its eukaryotic counterparts, the cloning of a universal N-linked glycosylation cassette in E. coli opens up the possibility of engineering permutations of recombinant glycan structures for research and industrial applications.
- 17Chen, M. M.; Weerapana, E.; Ciepichal, E.; Stupak, J.; Reid, C. W.; Swiezewska, E.; Imperiali, B. Biochemistry 2007, 46, 14342[ ACS Full Text], [ CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlCqtLfM&md5=e2eb24e5972b49be62536cffcbc85c2bPolyisoprenol Specificity in the Campylobacter jejuni N-Linked Glycosylation PathwayChen, Mark M.; Weerapana, Eranthie; Ciepichal, Ewa; Stupak, Jacek; Reid, Christopher W.; Swiezewska, Ewa; Imperiali, BarbaraBiochemistry (2007), 46 (50), 14342-14348CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in common amphiphilic membrane-bound polyisoprenyl-linked substrates. Herein, we examine the potential role of the polyisoprene component of the substrates by investigating the relative substrate efficiencies of polyisoprene-modified analogs in individual steps of the pathway. Chem. defined substrates for PglC, PglJ, and PglB are prepd. via semisynthetic approaches. The substrates included polyisoprenols of varying length, double bond geometry, and degree of satn. for probing the role of the hydrophobic polyisoprene in substrate specificity. Kinetic anal. reveals that all three enzymes exhibit distinct preferences for the polyisoprenyl carrier whereby cis-double bond geometry and α-unsatn. of the native substrate are important features, while the precise polyisoprene length may be less crit. These findings suggest that the polyisoprenyl carrier plays a specific role in the function of these enzymes beyond a purely phys. role as a membrane anchor. These studies underscore the potential of the C. Jejuni N-linked glycosylation pathway as a system for investigating the biochem. and biophys. roles of polyisoprenyl carriers common to prokaryotic and eukaryotic glycosylation.
- 18Li, L.; Woodward, R.; Ding, Y.; Liu, X.-w.; Yi, W.; Bhatt, V. S.; Chen, M.; Zhang, L.-w.; Wang, P. G. Biochem. Biophys. Res. Commun. 2010, 394, 1069[ Crossref], [ PubMed], [ CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkvFChurg%253D&md5=a37f5912e81bc4c395b69176fdb9d7efOverexpression and topology of bacterial oligosaccharyltransferase PglBLi, Lei; Woodward, Robert; Ding, Yan; Liu, Xian-wei; Yi, Wen; Bhatt, Veer S.; Chen, Min; Zhang, Lian-wen; Wang, Peng GeorgeBiochemical and Biophysical Research Communications (2010), 394 (4), 1069-1074CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homolog, PglB, functions as the oligosaccharyltransferase. Here, the authors established a method for obtaining relatively large quantities of homogeneous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chem. synthesized sugar donor: N-acetylgalactosamine-diphosphoundecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirmed that PglB is solely responsible for the oligosaccharyltransferase activity and complemented the finding that PglB exhibits relaxed sugar substrate specificity. In addn., the performed the topol. mapping of PglB using the PhoA/LacZ fusion method. The topol. model showed that PglB possesses 11 transmembrane segments and 2 relatively large periplasmic regions other than the C-terminal domain, which was consistent with the proposal of the common Ncyt-Cperi topol. with 11 transmembrane segments for the STT3 family proteins.
- 19Sato, K.; Inoue, S.; Onishi, A.; Uchida, N.; Minowa, N. J. Chem. Soc., Perkin Trans. 1 1981, 761[ Crossref], [ CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXls12msbk%253D&md5=f56064750bb2250c63da73e31af6e691Stereoselective synthesis of solanesol and all-trans-decaprenolSato, Kikumasa; Inoue, Seiichi; Onishi, Akira; Uchida, Nobuhiko; Minowa, NobutoJournal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999) (1981), (3), 761-9CODEN: JCPRB4; ISSN:0300-922X.Stereochem. pure 1,5-dienes were prepd. in moderate to good yields by coupling of allylic p-tolyl sulfones with an allylic bromide. E.g., coupling reaction of all-trans-bromogeranyl acetate (I) with the sulfone II and with higher isoprene analogs, followed by reductive elimination of p-MeC6H4SO2, gave all-trans-polyprenols and decaprenol (III; n = 10) stereoselectively. Solanesol (III; n = 9) was similarly prepd. through coupling of ClCH2CMe:CHCH2OAc.
- 20Sato, K.; Miyamoto, O.; Inoue, S.; Furusawa, F.; Matsuhashi, Y. Chem. Lett. 1983, 12, 725
- 21Jaenicke, L.; Siegmund, H. Biol. Chem. Hoppe-Seyler 1986, 367, 787[ Crossref], [ PubMed], [ CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXmt1ei&md5=718b7df70e43b18a736eef6e013ce33bTotal synthesis of chain-length-uniform dolichyl phosphates and their fitness to accept hexoses in the enzymic formation of lipoglycansJaenicke, Lothar; Siegmund, Hans UlrichBiological Chemistry Hoppe-Seyler (1986), 367 (8), 787-95CODEN: BCHSEI; ISSN:0177-3593.Dolichols I (R = H, n = 3, 5, 7) of defined uniform chain length (C35, C45, and C55) and geometry were prepd. from (E,E)-farnesol, activated as its 4-tolyl sulfone via condensation with 8-chloroneryl benzyl ether, conversion to THF 4-tolyl sulfone, and after several cycles of this C10-elongation sequence ending with 8-chlorocitronellyl benzyl ether to introduce the satd. α-isoprene unit. I (R = H, n = 3, 5, 7) were phosphorylated (POCl3/Et3N) and assayed relative to the natural dolichyl phosphate mixt. from pig liver as acceptors for transglycosylation from nucleoside diphosphate sugars (glucose, mannose) by standardized membrane vesicle prepns. from plants (Volvox) and animals (liver). Even I [R = P(O)(OH)2, n = 3] has full activity in this lipoglycan-forming reaction.
- 22Inoue, S.; Kaneko, T.; Takahashi, Y.; Miyamoto, O.; Sato, K. J. Chem. Soc., Chem. Commun. 1987, 1036[ Crossref], [ CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXit1Wqs7w%253D&md5=90fd51a141a4c69aea6875126f6c23c8Stereoselective total synthesis of (S)-(-)-dolichol-20Inoue, Seiichi; Kaneko, Toshihiko; Takahashi, Yuichi; Miyamoto, Osamu; Sato, KikumasaJournal of the Chemical Society, Chemical Communications (1987), (13), 1036-7CODEN: JCCCAT; ISSN:0022-4936.(S)-(-)-Dolichol-20 was prepd. stereoselectively using (Z,Z,Z,Z,Z,Z,Z,Z,E,E)-undecaprenol, ClCH2CMe:CHCH2CH2CMe:CHCH(SO2C6H4Me-p)-(CH2CMe:CHCH2)2-OCH2Ph, and (S)-Cl-(CH2CMe:CHCH2)2-CH2CMe:CHCH(SO2C6H4Me-p)CH2CMe:CHCH2CH2CHMeCH2CH2OCH2Ph as key intermediates.
- 23Grassi, D.; Lippuner, V.; Aebi, M.; Brunner, J.; Vasella, A. J. Am. Chem. Soc. 1997, 119, 10992[ ACS Full Text], [ CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXnt1equr8%253D&md5=28b0e8245e7c9f27347889b3cfc38d0aSynthesis and Enzymic Phosphorylation of a Photoactivatable Dolichol AnalogGrassi, D.; Lippuner, V.; Aebi, M.; Brunner, J.; Vasella, A.Journal of the American Chemical Society (1997), 119 (45), 10992-10999CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The synthesis of the photochem. probes I [R = H, PO3H2, R1 = (10Z,14Z,18Z,22Z,26Z,30Z,34E,38E)-Me2C:CHCH2(CH2CMe:CHCH2)2(CH2CMe:CHCH2)6] is described. These photoprobes are analogs of dolichol and dolichol phosphate, obligatory intermediates in the N-linked glycosylation pathway in the endoplasmic reticulum. The synthesis of I follows a new strategy. It involves the sequential alkylation of a monoterpenoid hydroxysulfonyl dianion with allyl chlorides. The photoreactive group, a 3-(trifluoromethyl)-3-aryldiazirine, was connected to the hydroxylated Me group of the β-prenyl unit of the fully assembled polyprenyl chain. The photoactivatable dolichol analog I (R = H) is a substrate for dolichol kinase from yeast membranes, an essential enzyme involved in the N-linked glycosylation pathway.
- 24Chen, L.; Men, H.; Ha, S.; Ye, X.-Y.; Brunner, L.; Hu, Y.; Walker, S. Biochemistry 2002, 41, 6824[ ACS Full Text], [ CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1KrsL4%253D&md5=da1a3d4b4c1b2f059ca32422feb95f16Intrinsic Lipid Preferences and Kinetic Mechanism of Escherichia coli MurGChen, Lan; Men, Hongbin; Ha, Sha; Ye, Xiang-Yang; Brunner, Livia; Hu, Yanan; Walker, SuzanneBiochemistry (2002), 41 (21), 6824-6833CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)MurG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-assocd. glycosyltransferase that couples N-acetyl glucosamine to the C4 hydroxyl of a lipid-linked N-acetyl muramic acid deriv. (lipid I) to form the β-linked disaccharide (lipid II) that is the minimal subunit of peptidoglycan. Lipid I is anchored to the bacterial membrane by a 55 carbon undecaprenyl chain. Because this long lipid chain impedes kinetic anal. of MurG, we have been investigating alternative substrates contg. shortened lipid chains. We now describe the intrinsic lipid preferences of MurG and show that the optimal substrate for MurG in the absence of membranes is not the natural substrate. Thus, while the undecaprenyl carrier lipid may be crit. for certain steps in the biosynthetic pathway to peptidoglycan, it is not required-in fact, is not preferred-by MurG. Using synthetic substrate analogs and products contg. different length lipid chains, as well as a synthetic dead-end acceptor analog, we have also shown that MurG follows a compulsory ordered Bi Bi mechanism in which the donor sugar binds first. This information should facilitate obtaining crystals of MurG with substrates bound, an important goal because MurG belongs to a major superfamily of NDP-glycosyltransferases for which no structures contg. intact substrates have yet been solved.
- 25Chang, Y.-F.; Liu, C.-Y.; Guo, C.-W.; Wang, Y.-C.; Fang, J.-M.; Cheng, W.-C. J. Org. Chem. 2008, 73, 7197[ ACS Full Text], [ CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVWmu7vJ&md5=780b589134ee2bee5e0cdd20aa442d7fSolid-Phase Organic Synthesis of Polyisoprenoid Alcohols with Traceless Sulfone LinkerChang, Yi-Fan; Liu, Chen-Yu; Guo, Chih-Wei; Wang, Yen-Chih; Fang, Jim-Min; Cheng, Wei-ChiehJournal of Organic Chemistry (2008), 73 (18), 7197-7203CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)Solid-phase org. synthesis of polyprenols with a traceless sulfone linker is described. The polymer-bound benzenesulfinate is first linked with the "tail" building blocks of isoprenyl chlorides via S-alkylation. With use of dimsyl anion as an appropriate base, the polymer-bound α-sulfonyl carbanion is generated and coupled with other "body" building blocks in an efficient manner. After repeated processes and a global palladium-catalyzed desulfonation with LiEt3BH as the reducing agent, the desired polyprenols with various chain lengths and geometrical configurations are obtained in 32-59% overall yields. The solid-phase synthesis offers the advantage in facile isolation of polyprenols without tedious operation or time-consuming purifn.
- 26Meng, F.-C.; Chen, K.-T.; Huang, L.-Y.; Shih, H.-W.; Chang, H.-H.; Nien, F.-Y.; Liang, P.-H.; Cheng, T.-J. R.; Wong, C.-H.; Cheng, W.-C. Org. Lett. 2011, 13, 5306[ ACS Full Text], [ CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFOmsbvM&md5=52415914ff4884e2d08cbba6a5b45ab6Total Synthesis of Polyprenyl N-Glycolyl Lipid II as a Mycobacterial Transglycosylase SubstrateMeng, Fan-Chun; Chen, Kuo-Ting; Huang, Lin-Ya; Shih, Hao-Wei; Chang, Han-Hui; Nien, Fu-Yao; Liang, Pi-Hui; Cheng, Ting-Jen R.; Wong, Chi-Huey; Cheng, Wei-ChiehOrganic Letters (2011), 13 (19), 5306-5309CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)A feasible synthetic approach toward the Mycobacterium tuberculosis (Mtb) N-glycolyl lipid II-like mol. is described. The title compd. bears pendant undecaprenol and L-lysin moieties instead of the naturally occurring decaprenol and meso-diaminopimelic acid, which are not readily available. Functionalization of with a fluorophore on the peptide side chain gave a deriv. which was found to be recognized as an Mtb TGase substrate. This result suggests it has tremendous utility for mechanistic studies, the characterization of mycobacterial enzymes, and mycobacterial TGase inhibitor evaluation.
- 27Hesek, D.; Lee, M.; Zajíček, J.; Fisher, J. F.; Mobashery, S. J. Am. Chem. Soc. 2012, 134, 13881[ ACS Full Text], [ CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFClu77L&md5=794b00f4c83e2f75e816b3d867609b8aSynthesis and NMR Characterization of (Z,Z,Z,Z,E,E,ω)-HeptaprenolHesek, Dusan; Lee, Mijoon; Zajicek, Jaroslav; Fisher, Jed F.; Mobashery, ShahriarJournal of the American Chemical Society (2012), 134 (33), 13881-13888CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We describe a practical, multigram synthesis of (2Z,6Z,10Z,14Z,18E,22E)-3,7,11,15,19,23,27-heptamethyl-2,6,10,14,18,22,26-octacosaheptaen-1-ol [(Z4,E2,ω)-heptaprenol] (I) using the nerol-derived sulfone II as the key intermediate. Sulfone II is prepd. by a literature route and is converted in five addnl. steps (18% yield from II) to (Z4,E2,ω)-heptaprenol I. The use of Eu(hfc)3 as an NMR shift reagent not only enabled confirmation of the structure and stereochem. of I, but further enabled the structural assignment to a major side product from a failed synthetic connection. The availability by this synthesis of (Z4,E2,ω)-heptaprenol I in gram quantities will enable preparative access to key reagents for the study of the biosynthesis of the bacterial cell envelope.
- 28Shih, H.-W.; Chang, Y.-F.; Li, W.-J.; Meng, F.-C.; Huang, C.-Y.; Ma, C.; Cheng, T.-J. R.; Wong, C.-H.; Cheng, W.-C. Angew. Chem., Int. Ed. 2012, 51, 10123[ Crossref], [ PubMed], [ CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlSgu7jP&md5=597407837689d8debb954fa22d1acb6eEffect of the Peptide Moiety of Lipid II on Bacterial TransglycosylaseShih, Hao-Wei; Chang, Yi-Fan; Li, Wei-Jing; Meng, Fan-Chun; Huang, Chia-Ying; Ma, Che; Cheng, Ting-Jen R.; Wong, Chi-Huey; Cheng, Wei-ChiehAngewandte Chemie, International Edition (2012), 51 (40), 10123-10126, S10123/1-S10123/26CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A variety of lipid II analogs with different peptide moieties were synthesized and evaluated for binding with transglycosylase (TGase). The results demonstrate that the first two positions of lipid II, D-lactate and L-alanine, esp. the Me groups, are essential for substrate binding and activity toward TGase. This D-Lac-L-Ala moiety in lipid II greatly contributes to the interaction with TGase, perhaps enabling a proper conformation for enzyme recognition. The last two amino acids (D-Ala-D-Ala) do not contribute to the interaction between lipid II and TGase, and the fluorophore tag at the ε-NH2 group of the lysine residue does not affect the binding affinity.
- 29Lizak, C.; Gerber, S.; Numao, S.; Aebi, M.; Locher, K. P. Nature 2011, 474, 350[ Crossref], [ PubMed], [ CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnsFOku7c%253D&md5=55c847719d235c92046d4e58cbdccfd4X-ray structure of a bacterial oligosaccharyltransferaseLizak, Christian; Gerber, Sabina; Numao, Shin; Aebi, Markus; Locher, Kaspar P.Nature (London, United Kingdom) (2011), 474 (7351), 350-355CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Asparagine-linked glycosylation is a post-translational modification of proteins contg. conserved sequence motif Asn-X-Ser/Thr. The attachment of oligosaccharides is implicated in diverse processes such as protein folding and quality control, organism development or host-pathogen interactions. The reaction is catalyzed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The central, catalytic enzyme of OST is the STT3 subunit, which has homologs in bacteria and archaea. Here, the authors report the x-ray structure of a bacterial OST, the PglB protein of Campylobacter lari, in complex with an acceptor peptide. The structure defined the fold of STT3 proteins and provided insight into glycosylation sequon recognition and amide N atom activation, both of which are prerequisites for the formation of the N-glycosidic linkage. The authors also identified and validated catalytically important, acidic amino acid residues. These results provide the mol. basis for understanding the mechanism of N-linked glycosylation.
- 30Jaenicke, L.; Siegmund, H.-U. Chem. Phys. Lipids 1989, 51, 159[ Crossref], [ CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXkslyksbo%253D&md5=b46624ac2cc77f3cd7cf1f90ee1212b7Synthesis and characterization of dolichols and polyprenols of designed geometry and chain lengthJaenicke, Lothar; Siegmund, Hans UlrichChemistry and Physics of Lipids (1989), 51 (3-4), 159-70CODEN: CPLIA4; ISSN:0009-3084.Dolichols of defined uniform chain length (C20 to C55) and geometry were prepd. by total synthesis according to the following principle: (E,E)-farnesol, activated as its p-tolylsulfone, was condensed with 8-chloro-(6Z)-neryl benzyl ether, the sulfonyl group removed and the ether linkage cleaved by Li/Et3N. After several cycles of this C10-elongaton sequence the synthesis was completed in the same way but using 8-chloro-citronellyl benzyl ether as building block to introduce the satd. α-isoprene unit. Polyprenols with an even no. of isoprene units are obtained by coupling activated geraniol with 8-chloro-(6E)-neryl benzyl ether in the first step. 1H- and 13C-NMR data were recorded for qual. and stereochem. comparison with natural dolichols. The versatility of this design makes it possible to synthesize dolichols with different geometry and double bond pattern.
- 31Danilov, L. L.; Druzhinina, T. N.; Kalinchuk, N. A.; Maltsev, S. D.; Shibaev, V. N. Chem. Phys. Lipids 1989, 51, 191[ Crossref], [ PubMed], [ CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXktF2qtrw%253D&md5=34934a274daf5ade435238fece1eab85Polyprenyl phosphates: synthesis and structure-activity relationship for a biosynthetic system of Salmonella anatum O-specific polysaccharideDanilov, L. L.; Druzhinina, T. N.; Kalinchuk, N. A.; Mal'tsev, S. D.; Shibaev, V. N.Chemistry and Physics of Lipids (1989), 51 (3-4), 191-203CODEN: CPLIA4; ISSN:0009-3084.A series of polyprenyl phosphates with modified structure of the polyprenyl residue was prepd. through phosphorylation of polyprenyl trichloroacetimidates with phosphoric acid. Interaction of polyprenols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile represented a very efficient, simple, and general method for the synthesis of polyprenyl phosphates. A procedure was developed for smooth conversion of polyprenyl pyrophosphates into the monophosphates through hydrolysis in the presence of 4-dimethylaminopyridine. The polyprenyl phosphates prepd. were studied as substrates for the enzymes of S. anatum O-specific polysaccharide biosynthesis. Correct stereochem. of α- and β-isoprenic units was essential for substrate efficiency. At the more remote positions of the hydrocarbon chain just the presence of isoprenic units of any configuration seems necessary. Some changes in position of the phosphate group may be permissible without significant loss of substrate properties.
- 32Bernardes, G. J. L.; Davis, B. G. unpublished results, University of Oxford, 2007.Google ScholarThere is no corresponding record for this reference.
- 33Bernardes, G. J. L.; Kikkeri, R.; Maglinao, M.; Laurino, P.; Collot, M.; Hong, S. Y.; Lepenies, B.; Seeberger, P. H. Org. Biomol. Chem. 2010, 8, 4987[ Crossref], [ PubMed], [ CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXht1OmsLzE&md5=ba806c8a9538b0a3eaf75738d9e25eeaDesign, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targetingBernardes, Goncalo J. L.; Kikkeri, Raghavendra; Maglinao, Maha; Laurino, Paola; Collot, Mayeul; Hong, Sung You; Lepenies, Bernd; Seeberger, Peter H.Organic & Biomolecular Chemistry (2010), 8 (21), 4987-4996CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The C-type lectin asialo-glycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an efficient synthesis of carbohydrate-functionalized β-cyclodextrins (βCDs) and liposomes for hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded βCDs were functionalized with glycodendrimers. Liposomes were equipped with synthetic glycolipids contg. a terminal d-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular carcinoma cell line HepG2 demonstrated that βCDs and liposomes displaying terminal d-Gal/d-GalNAc residues were preferentially endocytosed. In contrast, uptake of βCDs and liposomes with terminal d-Man or D-GlcNAc residues was markedly reduced. The d-Gal/d-GalNAc-functionalized βCDs and liposomes presented here enable hepatocyte-specific targeting. Gal-functionalized βCDs are efficient mol. carriers to deliver doxorubicin in vitro into hepatocytes and induce apoptosis.
- 34Schagger, H. Nat. Protoc. 2006, 1, 16[ Crossref], [ PubMed], [ CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2s3gsVSmsA%253D%253D&md5=0846bad5701202cba5395129c11168edTricine-SDS-PAGESchagger HermannNature protocols (2006), 1 (1), 16-22 ISSN:.Tricine-SDS-PAGE is commonly used to separate proteins in the mass range 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine-SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1-2 d.
- 35Wang, L.-X. Carbohydr. Res. 2008, 343, 1509[ Crossref], [ PubMed], [ CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnt1yisrk%253D&md5=a5fdbc6e98a7927f571eec06592af1acChemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylationWang, Lai-XiCarbohydrate Research (2008), 343 (10-11), 1509-1522CODEN: CRBRAT; ISSN:0008-6215. (Elsevier Ltd.)A review. Homogeneous glycopeptides and glycoproteins are indispensable for detailed structural and functional studies of glycoproteins. It is also fundamentally important to correct glycosylation patterns for developing effective glycoprotein-based therapeutics. This review discusses a useful chemoenzymic method that takes advantage of the endoglycosidase-catalyzed transglycosylation to attach an intact oligosaccharide to a polypeptide in a single step, without the need for any protecting groups. The exploration of sugar oxazolines (enzymic reaction intermediates) as donor substrates has not only expanded substrate availability, but also has significantly enhanced the enzymic transglycosylation efficiency. Moreover, the discovery of a novel mutant with glycosynthase-like activity has made it possible to synthesize homogeneous glycoproteins with full-size natural N-glycans. Recent advances in this highly convergent chemoenzymic approach and its application for glycopeptide and glycoprotein synthesis are highlighted.
- 36Rich, J. R.; Withers, S. G. Nat. Chem. Biol. 2009, 5, 206[ Crossref], [ PubMed], [ CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtlWmtbg%253D&md5=009fa4833a435582001aabbc71f7e600Emerging methods for the production of homogeneous human glycoproteinsRich, Jamie R.; Withers, Stephen G.Nature Chemical Biology (2009), 5 (4), 206-215CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. Most circulating human proteins exist as heterogeneously glycosylated variants (glycoforms) of an otherwise homogeneous polypeptide. Though glycan heterogeneity is most likely important to glycoprotein function, the prepn. of homogeneous glycoforms is important both for the study of the consequences of glycosylation and for therapeutic purposes. This review details selected approaches to the prodn. of homogeneous human N- and O-linked glycoproteins with human-type glycans. Particular emphasis is placed on recent developments in the engineering of glycosylation pathways within yeast and bacteria for in vivo prodn., and on the in vitro remodeling of glycoproteins by enzymic means. The future of this field is very exciting.
- 37Goodfellow, J. J.; Baruah, K.; Yamamoto, K.; Bonomelli, C.; Krishna, B.; Harvey, D. J.; Crispin, M.; Scanlan, C. N.; Davis, B. G. J. Am. Chem. Soc. 2012, 134, 8030[ ACS Full Text], [ CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmtlSmtr0%253D&md5=3c4af24f272d75afe68d77dcf5bfc881An Endoglycosidase with Alternative Glycan Specificity Allows Broadened Glycoprotein RemodellingGoodfellow, Jonathan J.; Baruah, Kavitha; Yamamoto, Keisuke; Bonomelli, Camille; Krishna, Benjamin; Harvey, David J.; Crispin, Max; Scanlan, Christopher N.; Davis, Benjamin G.Journal of the American Chemical Society (2012), 134 (19), 8030-8033CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein endoglycosidases are useful for biocatalytic alteration of glycans on protein surfaces, but the currently limited selectivity of endoglycosidases has prevented effective manipulation of certain N-linked glycans widely found in nature. Here we reveal that a bacterial endoglycosidase from Streptococcus pyogenes, EndoS, is complementary to other known endoglycosidases (EndoA, EndoH) used for current protein remodeling. It allows processing of complex-type N-linked glycans +/- core fucosylation but does not process oligomannose- or hybrid-type glycans. This biocatalytic activity now addresses previously refractory antibody glycoforms.
- 38Fernández-González, M.; Boutureira, O.; Bernardes, G. J.; Chalker, J. M.; Young, M.; Errey, J.; Davis, B. G. Chem. Sci. 2010, 1, 709[ Crossref], [ CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtl2nsLnE&md5=613c61342fa65cd1273e79866d7bead4Site-selective chemoenzymatic construction of synthetic glycoproteins using endoglycosidasesFernandez-Gonzalez, Marta; Boutureira, Omar; Bernardes, Goncalo J. L.; Chalker, Justin M.; Young, Matthew A.; Errey, James C.; Davis, Benjamin G.Chemical Science (2010), 1 (6), 709-715CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Combined chem. tagging followed by Endo-A catalyzed elongation allows access to homogeneous, elaborated glycoproteins. A survey of different linkages and sugars demonstrated not only that unnatural linkages can be tolerated but they can provide insight into the scope of Endo-A transglycosylation activity. S-linked GlcNAc-glycoproteins are useful substrates for Endo-A extensions and display enhanced stability to hydrolysis at exposed sites. O-CH2-triazole-linked GlcNAc-glycoproteins derived from azidohomoalanine-tagged protein precursors were found to be optimal at sterically demanding sites.
- 39Lomino, J. V.; Naegeli, A.; Orwenyo, J.; Amin, M. N.; Aebi, M.; Wang, L.-X. Bioorg. Med. Chem. 2013, 21, 2262[ Crossref], [ PubMed], [ CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktVOltLk%253D&md5=f556d1063e655a9590088cecb56ed5c8A two-step enzymatic glycosylation of polypeptides with complex N-glycansLomino, Joseph V.; Naegeli, Andreas; Orwenyo, Jared; Amin, Mohammed N.; Aebi, Markus; Wang, Lai-XiBioorganic & Medicinal Chemistry (2013), 21 (8), 2262-2270CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)A chemoenzymatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our expts. demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc-Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc-Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addn., the Glc-Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc-Asn linked glycopeptide. The chemoenzymic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery.
- 40Gamblin, D. P.; Garnier, P.; van Kasteren, S.; Oldham, N. J.; Fairbanks, A. J.; Davis, B. G. Angew. Chem., Int. Ed. 2004, 43, 828[ Crossref], [ PubMed], [ CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhsFyhu70%253D&md5=47923ea045eb426755a91944c12e6d5dGlyco-SeS: selenenyl-sulfide-mediated protein glycoconjugation - A new strategy in post-translational modificationGamblin, David P.; Garnier, Philippe; van Kasteren, Sander; Oldham, Neil J.; Fairbanks, Antony J.; David, Benjamin G.Angewandte Chemie, International Edition (2004), 43 (7), 828-833CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Site-selective glycosylation by Se-S-mediated ligation has led to the efficient formation of a wide variety of conjugates without the need for a large excess of the carbohydrate reagent. By this convergent method it was possible to introduce a heptasaccharide glycan selectively, and to perform a multiple site-selective chem. glycosylation of protein. A chem. Cys-glycosylated glycoprotein was elaborated enzymically.
- 41Ye, X.-Y.; Lo, M.-C.; Brunner, L.; Walker, D.; Kahne, D.; Walker, S. J. Am. Chem. Soc. 2001, 123, 3155[ ACS Full Text], [ CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhs1ymtrg%253D&md5=d1392c2a50d8f06924254982ad3d1059Better Substrates for Bacterial TransglycosylasesYe, Xiang-Yang; Lo, Mei-Chu; Brunner, Livia; Walker, Deborah; Kahne, Daniel; Walker, SuzanneJournal of the American Chemical Society (2001), 123 (13), 3155-3156CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The enzymes that synthesize the peptidoglycan layers surrounding bacterial cell membranes have received special attention because many known antibiotics function by blocking peptidoglycan synthesis. Among these enzymes, the bacterial transglycosylases (TGases) represent some of the most promising targets. TGases are located on the external surface of the bacterial membrane where they polymerize Lipid II, a disaccharide anchored to the membrane by a 55 carbon undecaprenyl chain. Although the TGases were first identified decades ago, their structures and mechanisms are not well understood. Some of the difficulties in studying TGases are related to problems obtaining and handling Lipid II. Because the 55 carbon chain aggregates, assays utilizing Lipid II, which can be isolated only in small quantities from bacterial membranes, must include org. solvents, detergents, and other additives. Results can be variable, and it is difficult to det. whether problems are due to the enzymes or to the substrate. Better substrates would facilitate the study of TGases. To identify better TGase substrates, the authors have synthesized natural Lipid II as well as a set of analogs contg. different lipid chains. These compds. have been tested for their ability to function as TGase substrates. The results show that bacterial TGases have clear preferences with regard to the structure of the lipid chain, but they do not require the 55 carbon undecaprenyl moiety. In fact, the authors have identified a compd. with a shorter lipid chain that is a much better TGase substrate than natural Lipid II.
- 42Liu, C.-Y.; Guo, C.-W.; Chang, Y.-F.; Wang, J.-T.; Shih, H.-W.; Hsu, Y.-F.; Chen, C.-W.; Chen, S.-K.; Wang, Y.-C.; Cheng, T.-J.; Ma, C.; Wong, C.-H.; Fang, J.-M.; Cheng, W.-C. Org. Lett. 2010, 12, 1608[ ACS Full Text], [ CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXisVOqsrY%253D&md5=36aa529a757a7a6c1159f55fdc7fc7e8Synthesis and Evaluation of a New Fluorescent Transglycosylase Substrate: Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl MoietyLiu, Chen-Yu; Guo, Chih-Wei; Chang, Yi-Fan; Wang, Jen-Tsung; Shih, Hao-Wei; Hsu, Yu-Fang; Chen, Chia-Wei; Chen, Shao-Kang; Wang, Yen-Chih; Cheng, Ting-Jen R.; Ma, Che; Wong, Chi-Huey; Fang, Jim-Min; Cheng, Wei-ChiehOrganic Letters (2010), 12 (7), 1608-1611CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)The prepn. of a novel fluorescent lipid II-based substrate for transglycosylases (TGases) is described. This substrate has characteristic structural features including a shorter lipid chain, a fluorophore tag at the end of the lipid chain rather than on the peptide chain, and no labeling with a radioactive atom. This fluorescent substrate is readily utilized in TGase activity assays to characterize TGases and also to evaluate the activities of TGase inhibitors.
- 43Lehrer, J.; Vigeant, K. A.; Tatar, L. D.; Valvano, M. A. J. Bacteriol. 2007, 189, 2618[ Crossref], [ PubMed], [ CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjvVOjtLs%253D&md5=a2b85e8d730703628f81465c7149276fFunctional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharideLehrer, Jason; Vigeant, Karen A.; Tatar, Laura D.; Valvano, Miguel A.Journal of Bacteriology (2007), 189 (7), 2618-2628CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examd. the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also detd. the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent Km and Vmax values for UDP-GlcNAc, Mg2+, and Mn2+ suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg2+, possibly playing a role in orienting the substrates. Topol. anal. using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topol. map of WecA. Also, we show that cells expressing a WecA deriv. C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.
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