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Engineering of Kuma030: A Gliadin Peptidase That Rapidly Degrades Immunogenic Gliadin Peptides in Gastric Conditions

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† Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States
‡ Departments of Chemistry, Biochemistry, and Molecular Medicine, University of California—Davis, Davis, California 95616, United States
§ Institute of Food Science-CNR, Avellino, Italy
∥ Institute of Protein Biochemistry-CNR, Naples, Italy
⊥ Department of Gastroenterology, University of Washington Medical Center, Seattle, Washington 98195, United States
# Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, New Jersey 08901, United States
*[email protected]
Cite this: J. Am. Chem. Soc. 2015, 137, 40, 13106–13113
Publication Date (Web):September 15, 2015
https://doi.org/10.1021/jacs.5b08325

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Abstract

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Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions within physiologically relevant time frames, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease.

Introduction

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Celiac disease is a debilitating illness that afflicts approximately 0.5–1% of the global population. (1, 2) The basis for celiac disease is a Th1-mediated inflammatory immune response directed against peptides derived from gliadin, a protein component that constitutes roughly half of the total protein composition of dietary gluten. Gliadin in highly enriched in the amino acids proline (P) and glutamine (Q), which renders it recalcitrant to degradation by human digestive enzymes. PQ-rich peptide fragments derived from partial digestion of gliadin in the stomach and intestines are deamidated in the intestinal lumen by tissue transglutaminase (TG2), thereby allowing binding to HLA-DQ2 or DQ8, and stimulation of an inflammatory response in people with celiac disease. (3) Chronic inflammation resulting from continuous gluten exposure leads to damaging of the intestinal villi, which promotes malnutrition-related symptoms, and to an increased incidence of developing intestinal lymphomas, (4) among other complications.
Currently, the only treatment for celiac disease is complete elimination of gluten from the diet. The gluten-free diet is very burdensome for patients because it is difficult to strictly achieve, due to the ubiquity of gluten in modern food production. (5-8) Medical treatment that could reduce or eliminate the effects of accidental gluten exposure would significantly benefit the celiac patient population. (9) Oral enzyme therapy, in which orally administered enzymes break down the PQ-rich regions of gliadin in the gastrointestinal tract, has been proposed as a method to treat celiac disease. (10) However, the requirement for proteolytic activity by these enzymes is high, because ingested gluten must be kept at 10 mg or less to prevent intestinal damage. (11, 12) Upon the accidental ingestion of 1 g of gluten (approximately the amount of gluten in 1/4 of a slice of bread), reduction to 10 mg or less would require degradation of immunogenic gluten content by 99+% at physiologically relevant time scales in order to eliminate intestinal damage. Additionally, degradation must take place in the gastric compartment, because immunogenic gliadin peptides initiate the immune response immediately upon entering the duodenum. (13) Finally, an enzyme therapeutic for celiac disease should be specific for the immunogenic fraction of gliadin, so that its activity is not markedly hindered by other proteins present during a meal. While several enzymes have been considered as potential oral enzyme therapeutics for celiac disease, (14, 15) no enzyme has been identified that harbors all of these properties.
Previously, we reported the engineering of a novel gliadin peptidase called KumaMax (hereafter referred to as Kuma010), which demonstrates stability and functionality in postprandial gastric conditions. (16) Kuma010 specifically degrades peptides after the PQ dipeptide motif, which is found throughout the immunogenic fraction of gliadin. In this work, we describe the computational redesign of the active site of Kuma010 in order to achieve the 99% activity threshold. We then assess the potential of Kuma030 as an enzyme therapeutic for celiac disease by quantifying its ability to reduce gluten content in laboratory-simulated digestions of wheat bread and beer and to reduce the immunostimulatory potential of gliadin as determined by T cell assays.

Results

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Alteration of the Kuma010 Active Site by Computational Enzyme Design

To guide the improvement of Kuma010 by computational protein design, we solved the crystal structure of Kuma010 at 2.5 Å resolution (PDB ID 4NE7). On the basis of this structure, we utilized the Rosetta Molecular Modeling Suite (17, 18) to redesign the Kuma010 active site, selecting for mutations that were predicted to increase activity against immunogenic gliadin peptides. Designed mutants were then screened for increased activity on the highly immunogenic 33mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) and 26mer (FLQPQQPFPQQPQQPYPQQPQQPFPQ) gliadin peptides (19, 20) (Supporting Information Table 1). These peptides harbor either the PQL or PQQ tripeptide motif, tripeptides that have been implicated in the immunogenic cores of gliadin T cell epitopes shown to be toxic for celiac patients. (21) Mutations to Kuma010 that conferred increases in activity were combined in an iterative process, and the resulting mutant enzymes were retested for increased activity (Supporting Information Table 2). In this manner, the variant Kuma030 was built. Kuma030 is 44-fold more active against peptides containing PQQ, and 11-fold more active against peptides containing PQL, than Kuma010, as assessed by kcat/KM values for these enzymes on relevant gliadin peptides (Supporting Information Figure 1).
As determined by molecular modeling using Rosetta, the putative S1′ peptide binding interface of Kuma010 consists entirely of hydrophobic residues and should therefore prefer hydrophobic residues such as leucine over polar residues such as glutamine at P1′. The S1′ binding pocket of Kuma030 includes an isoleucine to threonine mutation (I463T), which is predicted to provide a hydrogen bond with a P1′ glutamine, enabling this enzyme to accommodate both leucine and glutamine in the S1′ subsite and thereby target both PQL and PQQ tripeptides (Figure 1). Kuma030 also includes six additional mutations (K262E, E269T, S354Q, G358S, D399Q, A449Q) that are required for the enhanced catalytic efficiency on the 26mer and 33mer peptides (Supporting Information Figure 2). Two of these mutations (K262E and G358S) are substitutions of mutations previously introduced into Kuma010. (16) G358S is thought to stabilize the loop containing a histidine introduced in Kuma010 which is predicted to hydrogen bond to the P1 glutamine residue (Figure 1). The remaining mutations are predicted to stabilize the protein structure as modeled.

Figure 1

Figure 1. Amino acid substitutions in the Kuma030 endopeptidase. Model of a representative PQQP tetrapeptide in the active site of Kuma030. Enzyme backbone is shown in gray, gliadin tetrapeptide is shown in yellow. Green residues were mutated to generate Kuma030. Blue residues are thought to be important in coordinating the tetrapeptide into the active site. Image was created using PyMol.

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Kuma030 Targets Immunodominant Peptides in Wheat, Rye, and Barley

While dozens of PQ-rich epitopes have been linked to celiac disease, several peptides derived from gliadin (wheat), hordein (barley), and secalin (rye) have been shown to account for the vast majority of the immune response in celiac disease and have thus been classified as immunodominant. (21) In wheat, these include the peptides W02-E07 (LQPFPQPQLPYPQPQ), W03-E07 (QPFPQPQQPFPWQP), and the 33mer peptide, which contains the W02-E07 sequence. (19, 22) These peptides harbor several HLA-DQ2 or -DQ8 epitopes shown to be strongly immunogenic. (19, 23) To evaluate the ability of Kuma030 to destroy these epitopes throughout gluten, purified whole gluten was incubated with Kuma030 under simulated postprandial gastric conditions (pH 4.0 at 37 °C with 0.6 mg mL–1 pepsin). (24) The gluten fraction remaining after degradation was quantified using ELISA assays based on either the G12 or R5 antibodies, which recognize the amino acid motifs QPQLPY and QQPFP, respectively. These motifs encompass many immunogenic gliadin epitopes, including the immunodominant peptides mentioned above, (25, 26) and is therefore a reasonable assessment of the immunostimulatory potential of gluten. To compare the activity of Kuma030 to that of published glutenases, we also examined the glutenases EPB2 and SCPEP, which are currently being pioneered at a 1:1 ratio as a combination enzyme therapeutic for celiac disease. The EPB2 and SCPEP enzymes generated in this work were verified to have activities consistent with that of published values (27, 28) (Supporting Information Table 3). Upon incubation with gluten, we observed a dose-dependent reduction in the QQPFP or QPQLPY load using either Kuma030, Kuma010, or a 1:1 combination of EPB2 and SCPEP (Figure 2A and Supporting Information Figure 3). At a 1:25 w:w ratio of enzyme:gluten, EPB2 and SCPEP broke down 84.8% of the gluten present, consistent with previously published reports. (14, 29) Kuma030 was highly effective at eliminating these peptide epitopes throughout gluten, with a 1:400 w:w ratio of enzyme:gluten being sufficient to reduce the immunogenic gluten present by over 99.5% at 60 min, as quantified by both ELISA methods (Figure 2A and Supporting Information Figure 3), and a 1:100 w:w ratio was sufficient to decrease the detected gluten by >99.9% by 30 min (Figure 2B). Kuma030-dependent reduction in immunogenic gluten load was rapid, with >98% degradation achieved by 5 min at a w:w ratio of 1:25 (Figure 2B). HPLC-MS analysis of the Kuma030 cleavage products revealed that Kuma030 cleaved each peptide after the PQ dipeptide motif in the immunodominant epitopes from wheat (33mer, W02-E07, and W03-E07), and also those from barley B08-E2E7 (PQQPIPQQPQPYPQQ) and rye R11-E2E7 (QPFPQQPEQIIPQQP) (22) (Figure 2C, Supporting Information Figure 4). While these intact peptides are highly immunostimulatory, the peptide breakdown products are not predicted to stimulate the immune system, because Kuma030 action results in elimination of the core 9mer epitope required to trigger the immune response. (30) The ability of Kuma030 to cleave peptides containing either a PQL or a PQQ tripeptide motif, and to reduce the immunogenic gluten load as measured by both G12 and R5 antibodies, is consistent with the hypothesis that Kuma030 can bind and cleave peptides with either a leucine or a glutamine in the S1′ binding pocket.

Figure 2

Figure 2. Kuma030 is capable of rapidly and effectively degrading the immunogenic regions of gluten in gastric conditions. (A) concentrations of either EPB2 and SCPEP (at a 1:1 ratio), Kuma010, or Kuma030 in gastric conditions, as measured by ELISA using the G12 antibody. The starting concentration of gluten was 10 mg mL–1 (10 000 ppm). Note that the Y-axis is plotted on a logarithmic scale. (B) The amount of gluten detected at 5 or 30 min after incubation with EPB2 and SCPEP or Kuma030. Shown is the ratio of enzyme:gluten. The starting concentration of gluten was 10 mg mL–1. Samples were normalized to the amount of gluten remaining after incubation with pepsin alone. Asterisk indicates that greater than 99.9% of the gluten was degraded. (C) HPLC trace of full-length peptide (gray dotted lines) or breakdown products (black lines) of the immunodominant peptides from gliadin (W02-E07, W03-E07, 33mer), hordein (B08-E2E7), or secalin (R11-E4E7). The bottom of the figure shows the amino acid sequence of the 33mer peptide, position of known immunogenic epitopes (horizontal lines), location of Kuma030 cleavage site (vertical lines), and elution peaks of the resulting breakdown products (gray arrows). Immunodominant peptides displayed the following breakdown patterns: B08-E2E7: PQQPIPQ∥QPQPYPQ∥Q; R11-E4E5: QPFPQ∥QPEQIIPQ∥QP; W02-E7: LQPFPQPQ∥LPYPQPQ; W03-E7: QPFPQPQ∥QPFPWQP. All peptide masses and elution times were confirmed by LCMS. Note that although the undigested W03-E07 peptide eluted at approximately the same time as a W03-E07 breakdown fragment, these are separate peaks, as determined by LCMS (Supporting Information Figure 4). aAU, arbitrary absorbance units.

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Gliadin Treated with Kuma030 Loses Its Ability To Stimulate a T-Cell Response

The ability of Kuma030 to efficiently degrade immunogenic gliadin epitopes suggests that incubation of gliadin with Kuma030 might reduce its capacity to stimulate a T-cell-mediated immune response. T cell assays utilizing cells derived from the intestinal biopsies of celiac patients represent the gold standard for this evaluation. (31) We performed T cell assays in which cells were exposed to Kuma030-treated gliadin and the resulting T cell reaction was assessed. The gliadin-reactive intestinal CD4+ T cell lines used in this study were previously generated from intestinal mucosa of celiac patients and have been shown to react to a diversity of epitopes across different gliadin families (32) (Figure 3F). Kuma030 was incubated with purified wheat gliadin at enzyme:gliadin ratios spanning 1:10–1:1000 in laboratory-simulated gastric conditions for 60 min. In order to mimic transit into the intestinal compartment, the pH levels of the samples were then increased, and the samples were treated with chymotrypsin and deamidated with tTG to unmask the immunogenic epitopes. The resulting gliadin samples were presented to T cell lines, and stimulation was assessed by measuring IFN-γ production (Figure 3) and T cell proliferation (Supporting Information Figure 5). In all cell lines tested, exposure to pepsin-treated gliadin resulted in stimulation of production of IFN-γ, and treatment of gliadin with Kuma030 reduced this response, eliminating it at higher concentrations of Kuma030. The decreases observed in IFN-γ production were not due to Kuma030 toxicity (Supporting Information Figure 6). Importantly, Kuma030 eliminated the T cell response to gliadin in every T cell line tested regardless of T cell epitope specificity, indicating that Kuma030 is effective against all epitopes recognized by the T cell lines used in this assay. Because these epitopes span the three major gliadin families, α-, ω-, and γ-gliadin, this suggests that Kuma030 is capable of degrading immunogenic epitopes within all relevant regions of gliadin.

Figure 3

Figure 3. Gliadin treated with Kuma030 loses it immunostimulatory potential. (A–E) Purified gliadin was treated with Kuma030 at the specified concentration for 60 min at pH 4.0 at 37 °C in the presence of 0.6 mg mL–1 pepsin. After the gastric phase, the pH of the samples was increased, and samples were treated with chymotrypsin and TG2. Samples were then exposed to T cell lines from patients #1 (A), #2 (B), #3 (C), #4 (D), or #5 (E), in the presence of autologous irradiated B cell lines, and IFN-γ was measured by ELISA. Phytohemagglutenin (PHA) and a peptic-tryptic digest of gliadin (PT-gliadin) were included as positive controls. Incubation of T cell lines with antigen-presenting cells in the absence of antigens (medium) acted as a negative control. (F) T cell stimulatory epitopes recognized by the T cells used in this assay and the predicted Kuma030 cleavage sites within these epitopes. (21) Predicted Kuma030 cleavage sites are shown by a vertical line |. Cleavage sites are predicted based on Kuma030 activity on gliadin peptides as presented in Figure 2.

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Kuma030 Reduces Gluten Load in Laboratory-Simulated Gastric Digestions Representing Ingestion of 4–8 g of Gluten to Below Levels Thought to Be Toxic to Celiac Patients

To evaluate the practical application of Kuma030, we tested the ability of this enzyme to break down gluten present in simulated gastric digests of whole wheat bread and wheat beer. Whole wheat bread was mashed in artificial saliva to simulate mastication. The mixture was then acidified by the addition of HCl and pepsin at a bread concentration representative of that in the stomach after ingestion of 1–2 slices of bread (assuming 4 g of gluten per slice and a stomach volume of 400–800 mL), and glutenases at various concentrations were added. The amount of gluten remaining was then quantified after 30 min of digestion, representing a lag time of food in the stomach before the commencement of ingesta release into the duodenum through the pyloric opening. (33) At the highest concentration of glutenase tested (1:10 w:w ratio), treatment with EPB2 and SCPEP resulted in 84.4% gluten degradation (Figure 4A, Supporting Information Figure 7). This is consistent with published results from a Phase I study showing that EPB2 and SCPEP eliminate 70–79% of gluten in a test meal at a 1:10 enzyme:gluten ratio in the human stomach after a 30 min incubation time. (34) At a 1:40 w/w ratio (250 μg mL–1 enzyme), Kuma030 achieved >99.97% degradation, reaching the limit of quantification by ELISA (3 ppm), which is well below the 20 ppm threshold for “gluten free” labeling in the U.S. (Figure 4A). A 1:320 w:w ratio (31.25 μg mL–1) was sufficient to reduce the level of gluten in the bread by >99%. Finally, the gluten-degrading ability of Kuma030 was also tested directly in a wheat beer, because wheat beers demonstrate higher gluten levels than their barley-made counterparts. (35) Beer was incubated with Kuma030 at 37 or 4 °C at two enzyme concentrations. Samples were taken at various time points and the concentration of remaining gluten was quantified. We found that incubation of the beer, which demonstrated an initial gluten concentration of ∼764 ppm, with Kuma030 decreased the gluten level to below 20 ppm by only 5 min (Figure 4B). Surprisingly, Kuma030 was capable of reaching this 20 ppm threshold in wheat beer even at 4 °C (Figure 4B).

Figure 4

Figure 4. Kuma030 efficiently degrades gluten in complex food matrices. Gluten was detected using G12 ELISA. (A) The amount of gluten remaining in whole wheat bread after a 30 min incubation with either Kuma030 or EPB2/SCPEP at the indicated enzyme concentrations. Dotted lines represent either the initial concentration of gluten (10 000 ppm) or 20 ppm gluten. Note both axes are plotted on a logarithmic scale. (B) The amount of gluten remaining in a wheat beer after incubation with the indicated concentrations of either EPB2/SCPEP or Kuma030 at 37 or 4 °C, for 5, 15, or 60 min. Dotted lines represent either the initial concentration of gluten (764 ppm) or 20 ppm.

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Discussion

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Oral enzyme therapy has been considered an attractive treatment option for celiac disease since the identification of PQ-rich immunogenic gliadin epitopes that stimulate the immune response. (3) An essential characteristic of any enzyme therapeutic for celiac disease is the ability to break down immunogenic gliadin peptides in gastric conditions before these peptides can access the intestinal tract. Gluten challenge studies in celiac patients have shown that the ingested gluten load must be kept at 10 mg or less in order to prevent intestinal damage. (11, 12) Indeed, the U.S. FDA currently mandates that any food labeled as “gluten free” must demonstrate less than 20 ppm gluten, because strict adherence to this standard is predicted to result in a daily ingestion of 10 mg or less. Ingestion of only 1 g of gluten would require a 99% degradation level in order to achieve this threshold; accidental ingestion of a sandwich made with gluten-containing bread (∼8 g of gluten assuming 4 g of gluten per slice) would require a ∼99.9% degradation level. There is therefore a clear need for glutenases that can rapidly and efficiently destroy immunogenic gliadin epitopes in gastric conditions. In a model of gastric digestion of whole wheat bread, Kuma030 degraded >99.97% of the gluten load in 30 min. While oral administration of glutenases may not be a replacement for a gluten-free diet, the ability to provide protection in the event of a large amount of gluten ingestion is important for efficacy. Thus, the fact that Kuma030 is capable of achieving this degradation level suggests its potential for use as a next-generation enzyme therapeutic for celiac disease.
T cell lines derived from celiac patients demonstrate a myriad of responses to different immunogenic epitopes. It is therefore significant that Kuma030, which demonstrates a PQ dipeptide specificity, is effective at decreasing or eliminating gliadin-mediated T cell stimulation in all cell lines tested, which represent reactivity against peptides derived from all major families of gliadin. While many immunogenic gliadin peptides have been identified, virtually all of these harbor the PQ dipeptide motif within the HLA-binding core, and cleavage after each of these motifs is predicted to destroy the HLA-binding region; thus, PQ is a reasonable proteolytic target for therapeutic glutenases. The selection of a dipeptide specificity, rather than a single amino acid specificity, may contribute to the effectiveness of Kuma030, because while this dipeptide is commonly found throughout the immunogenic regions of gluten, it is found much less frequently in the amino acid sequences of other proteins. Because dietary gluten comprises only roughly 20% of the total protein consumed in a Western diet, a dipeptide specificity may allow for the surgical removal of immunogenic epitopes of gliadin even in the presence of large amounts of other dietary protein. This factor alone may provide a significant advantage over proteases with a single amino acid specificity that indiscriminately act upon protein targets.
The ability to design novel therapeutic proteins through computational protein design holds great potential for the engineering of 21st century medicines. Kuma030 represents the first molecule originally designed by the video game Foldit (16) to move through drug development toward clinical trials. These studies demonstrate the power of protein design to incorporate many different beneficial characteristics into a single therapeutic molecule and warrant further work assessing the potential of this enzyme in vitro and in vivo.

Conclusions

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In this work, we describe the engineering of the gliadin endopeptidase Kuma030. Molecular modeling and computational design allowed for the prediction of mutations on the template enzyme Kuma010 that greatly increased proteolytic activity on immunogenic epitopes relevant to individuals with celiac disease. The resulting enzyme, Kuma030, is capable of degrading all immunodominant epitopes from wheat, barley, and rye, indicating that Kuma030 may be able to degrade a broad spectrum of immunogenic targets. Indeed, treatment of wheat gliadin with Kuma030 reduced or eliminated the capacity of gliadin to stimulate T cells that are specific for a variety of immunogenic epitopes. Treatment of foodstuffs with Kuma030 reduced the gliadin load by over 99% in minutes, to below the threshold level shown to be toxic to celiac patients. Future work will determine the potential of Kuma030 as a therapeutic for celiac disease using relevant in vivo models.

Materials and Methods

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Materials

Growth media components were obtained from MP Biomedicals. All reagents for mutagenesis were obtained from New England Biolabs (NEB) with the exception of Pfu Turbo Cx hotstart polymerase which was from Agilent Technologies. Peptides were synthesized by GenScript. Chromogenic substrates were obtained from Bachem. HPLC grade solvents were from Fisher Scientific. Unless otherwise noted, all other reagents were from Sigma-Aldrich.

Crystallization of Kuma010

Purified Kuma010 was concentrated to 9.15 mg mL–1 in buffer containing 5 mM DTT, 100 mM NaCl, 10 mM Tris-HCl pH 7.5, and 0.02% NaN3. The protein was crystallized using the hanging drop vapor evaporation method, at 277 K. The best crystals were obtained at the following condition: 15% PEG 8000, 0.1 M MES pH 6.0, 0.2 M zinc acetate. Diffraction data set to 2.5 Å was collected at the National Synchrotron Light Source, with beamline X4C, and the data were processed with HKL-2000 (HKL Research, Inc.). The structure was determined by molecular replacement using Phaser, (36) with the wild type structure (PDB code 1SN7) as initial search model. The refinement was carried out using Phenix, (37) and model adjusting was done in Coot. (38) The crystal structure was quite close to the original design model for Kuma010, with a Cα RMSD of 0.676 Å.

Computational Enzyme Design

The enzyme design module (17) of the computational protein design software Rosetta was used to suggest mutations that stabilize the PQLP-Kuma010 and PQQP-Kuma010 complexes under the assumption that a higher affinity binding would lead to enhanced catalytic properties. Both the PQLP- and the PQQP-bound models were relaxed in Rosetta using coordinate and catalytic constraints, (39) and then the sequences of the proteins were optimized for peptide–protein interaction energy using fixed-backbone Monte Carlo sequence design. (17) Designable residues were restricted to those within 10 Å of the peptide and were not allowed to mutate to cysteine or proline. Ten to twenty design trajectories were run through design protocols. Mutations were selected for experimental characterization based on occurrence in the designs and in a multiple sequence alignment of Kumamolisin (the Kuma010 template protease), Rosetta, and FoldIt (40) scores, and manual inspection.

Production and Purification of Kuma010 Variants and SCPEP

All mutations to the kuma010 gene were incorporated using the site-directed mutagenesis method PFunkel. (41) Protein was expressed from pET29B plasmids harboring Kuma010 enzyme variants or SCPEP by Escherichia coli BL21(DE3). Enzymes were purified by immobilized-metal affinity chromatography (IMAC) using Ni-NTA Superflow resin (Qiagen) and dialyzed in storage buffer (50 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM β-mercaptoethanol, 10% (v/v) glycerol). Aliquots were stored at −80 °C until use.

EPB2 Expression and Purification

Expression and purification of recombinant EPB2 proenzyme was performed according to published methods. (27, 42) Briefly, EPB2 was expressed from cultures of E. coli BL21(DE3). Cultures were centrifuged, and pellets were resuspended in disruption buffer (200 mM sodium phosphate, pH 7.0, 200 mM NaCl, 2.5 mM DTT, 2.5 mM EDTA, 30% (v/v) glycerol), lysed by sonication, and clarified by centrifugation. Inclusion bodies containing EPB2 were washed twice with water and then dissolved in EPB2 solubilization buffer (50 mM Tris-HCl, pH 8.0, 7 M urea, 2 mM β-mercaptoethanol). The solution was clarified by centrifugation, and the enzyme was purified by IMAC. Fractions containing proEP-B2 were pooled, diluted with refolding buffer (100 mM Tris-HCl, pH 8.0, 5 mM EDTA, 2 mM β-mercaptoethanol, 15% (v/v) glycerol), and stirred overnight. Refolded pro-EPB2 was filtered and concentrated to 15 mg mL–1. Aliquots were stored at −80 °C until use.

KumaMax Kinetic Characterization

Kinetic parameters were assessed using gliadin-derived substrates Glia_α1 (PFPQPQLPY) and Glia_ω1 (PFPQPQQPF). Enzymes were incubated at 37 °C for 10 min and diluted to 100 nM with reaction buffer (100 mM NaOAc, pH 4.0) containing either Glia_α1 or Glia_ω1 at 0–18.4 mM. Degradation was monitored by periodic sample removal and quenching, followed by quantification of the PFPQPQ breakdown product by HPLC/ESI-MS. The kcat/KM value was calculated using the Michaelis–Menten equation in Graphpad Prism.

SCPEP and EPB2 Kinetic Characterization

Kinetic parameters of SCPEP and EPB2 were measured on chromogenic substrates Suc-Ala-Pro-pNA (SCPEP) and Z-Phe-Arg-pNA (EPB2) following previously published protocols. (27, 28) pNA release was spectrophotometrically monitored. Buffers were as follows: 100 mM NaHCO3, pH 6.0, 150 mM NaCl (SCPEP), or 100 mM NaOAc pH 4.0 (EPB2). The kcat/KM value was calculated using the Michaelis–Menten equation in Graphpad Prism.

Peptide Degradation Assays

Lyophilized 33mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), 26mer (FLQPQQPFQQPQQPYPQQPQQPFPQ), W03-E07 ((QPFPQPQQPFPWQP), W02-E7 (LQPFPQPQLPYPQPQ), R11-E4E7 (QPFPQQPEQIIPQQP), and B08-E2E7 (PQQPIPQQPQPYPQQ) peptides were dissolved in reaction buffer (100 mM NaOAc, pH 4.0). Enzymes were incubated at 37 °C for 10 min in reaction buffer and then incubated at 2 μg mL–1 with 1 mg mL–1 substrate (for increased activity screening) or at 500 μg mL–1 with 5 mg mL–1 substrate (for qualitative degradation assessment) at 37 °C for 60 min. Degradation of the gliadin peptides was monitored by analysis by HPLC at Ab215, or by LC/ESI-MS on a TSQ Quantum Access (Thermo Fisher Scientific).

Degradation of Purified Gluten

Purified wheat gluten was suspended in 70% ethanol at 200 mg mL–1. Enzymes were diluted in reaction buffer (100 mM NaOAc, pH 4.0) and incubated at 37 °C for 10 min. Suspended gluten was rapidly diluted 10-fold with reaction buffer and aliquoted into reaction tubes. Pepsin at 0.6 mg mL–1 and enzymes were added, and samples were incubated at 37 °C. The final concentration of gluten in the reaction mixture was 10 mg mL–1. Gluten degradation was halted at ≥95 °C for 10 min, and the gluten level was quantified by competitive ELISA using either the R5 Ridascreen (R-Biopharm) or the G12 GlutenTox kit (Biomedal).

Gluten Degradation in Whole Wheat Bread

Bread slurry was prepared by mashing whole wheat bread (Oroweat: approximately 4 g of protein per slice) with a spatula in artificial saliva (20 mM NaHCO3, 25 mM KCl, 2 mM CaCl2, 0.3 mM MgCl2, pH 7.0), followed by acidification with HCl and blending for 2 min in a Waring blender, at which the pH of the bread mixtures was approximately pH 4.5. Degradation was initiated by the addition of pepsin at 0.6 mg mL–1 and either Kuma030 or a 1:1 mixture of EP-B2 and SC PEP. The final gluten concentration in the bread mixture was 10 mg mL–1 and was verified by ELISA. Samples were incubated at 37 °C for 30 min with periodic agitation. At the end of the degradation period, samples were incubated at ≥95 °C to halt enzyme activity. Gluten levels were quantified by the G12 and R5 ELISA methods described above.

Gluten Degradation in Wheat Beer

Wheat beer (Chainbreaker White IPA, Deschutes Brewery) was aliquoted on ice. Either Kuma030 or a 1:1 mixture of EPB2 and SCPEP was added to the beer and incubated at 37 or 4 °C for 60 min. Samples were incubated at ≥95 °C to halt enzyme activity. The gluten level for each sample was measured by the G12 ELISA method described above.

T Cell Immunogenicity Assays

Gliadin-specific intestinal T cell lines (iTCLs) were previously generated from duodenal biopsies of 5 HLA-DQ2 celiac disease patients. (32) Briefly, mucosal cells were stimulated for two to three cycles with irradiated autologous peripheral blood mononuclear cells (PBMCs) and deamidated peptic-tryptic digest of gliadin extracted from the hexaploid wheat variety Sagittario (PT-Gliadin). Stable TCLs were kept in culture by repeated stimulation with heterologous irradiated PBMCs and PHA, and IL-2 as a growth factor. The peptide specificity of the iTCLs was evaluated by assaying their reactivity toward a panel of immunogenic gluten peptides (32) (and unpublished data), and revealed an oligoclonal pattern of peptide recognition.
The immune response of iTCLs to gliadin treated with Kuma030 at different concentrations was assayed by detecting IFN-γ production and cell proliferation. (32) T cells (3 × 104) were coincubated with irradiated autologous EBV-transformed, B-lymphoblastoid cell lines (B-LCLs, 1 × 105), in 200 μL of complete medium (X-Vivo plus 5% human serum, Lonza-BioWhittaker, Verviers, Belgium) in U-bottom 96-well plates. Gliadin samples, digested with Kuma030 at the specified concentration, were then added at 50 μg mL–1. Cells incubated in absence of antigens acted as negative control while phytohemagglutinin (PHA, 1 μg mL–1; Boehringer; Mannheim, Germany) and PT-gliadin (50 μg mL–1) were included as positive controls. After a 48 h incubation, cell supernatants (50 μL) were collected for IFN-γ determination and T cells were pulsed overnight with [3H]-thymidine (0.5 μCi per well; GE Healthcare; Buc Cedex, France) and therefore harvested for the evaluation of cell proliferation. Each antigen preparation was assayed in duplicate and in at least two independent experiments.
IFN-γ was measured by a classic sandwich ELISA using purified and biotin-conjugated anti-IFN-γ Abs purchased from Mabtech (Nacka Strand, Sweden). The sensitivity of the assay was 32 pg mL–1.
To evaluate possible toxic effects of enzyme-treated gliadins, PBMCs from healthy donors (2 × 105) were stimulated with PHA (1 μg mL–1), or PHA + samples treated with Kuma030 (50 μg mL–1), in 200 μL of complete medium in U-bottom 96-well plates. IFN-γ production and cell proliferation were assayed as described for TCLs.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.5b08325.

  • Lists of mutations on the Kuma010 background and their effects on activity; experimentally determined kinetic parameters for selected enzymes; data supporting conclusions in the main text derived from gluten and gliadin peptide degradation assays; T cell proliferation and toxicity data (PDF)

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  • Corresponding Author
    • Ingrid S. Pultz - Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States;  Email: [email protected]
  • Authors
    • Clancey Wolf - Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States
    • Justin B. Siegel - Departments of Chemistry, Biochemistry, and Molecular Medicine, University of California—Davis, Davis, California 95616, United States
    • Christine Tinberg - Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States
    • Alessandra Camarca - Institute of Food Science-CNR, Avellino, Italy
    • Carmen Gianfrani - Institute of Protein Biochemistry-CNR, Naples, Italy
    • Shirley Paski - Department of Gastroenterology, University of Washington Medical Center, Seattle, Washington 98195, United States
    • Rongjin Guan - Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, New Jersey 08901, United States
    • Gaetano Montelione - Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, New Jersey 08901, United States
    • David Baker - Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States
  • Notes
    The authors declare the following competing financial interest(s): I.S.P., J.S., and D.B. are founders in a company, PvP Biologics, that intends to license the technology described herein. C.W., I.S.P., J.S., D.B., and C.T. are inventors on a patent that describes the technology presented herein.

Acknowledgment

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We thank Jayaraman Seetharaman for data collection during X-ray crystallography. This work was supported by the Life Sciences Discovery Fund of Washington State, the Washington State Research Foundation, the University of Washington, and private philanthropy.

References

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    Gluten is the main storage protein in grains and consists of gliadin and glutenin occurring in the same ratio. Persons suffering from intolerances, including celiac disease. must avoid foods contg. gluten or products contg. wheat, barley. and rye. Accordingly. gluten detection is 01' high interest for the food safety of celiac patients. This study was designed to detennine the concns. of gluten in foods labeled "gluten free" available in the United States. Seventy-eight samples labeled gluten free were collected and analyzed using a gliadin competitive enzyme-linked immunosorbcnt assay. The gluten content was calcd. based on the assumption of the same ratio between gliadin and glutenin. Forty-eight (61.5%) of the 78 samples contained less than the limit of quantification of 10 mg/kg for gluten. In addn. 14(17.9%) of the 78 samples labeled gluten free contained less gluten than the guidelines established by the Codex Alimentarius for gluten-free labeling (20 mg/kg). However, 16 samples (20.5%) did contain gluten levels of ≥20 mg/kg, ranging from 20.3 to 60.3 mg/kg. In particular, five of eight breakfast cereal samples showed gluten contents higher titan 20 mg/kg. These results may be of concern, as gluten sensitivity is known to vary among celiac disease patients.
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    A review. The threshold amt. of gluten in 'gluten-free' products that can be tolerated by people with coeliac disease is unclear. To investigate the threshold amt. of gluten and the threshold concn. of gluten in food products that can be tolerated by people with coeliac disease. Systematic review of studies published between 1966 and May 2007. The data sources used were MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, CINAHL, and ref. lists of retrieved articles. We included studies that evaluated the amt. of dietary gluten or the concns. of gluten in food products that can be tolerated by people with coeliac disease whatever their design, method or language of publication. Thirteen studies (three randomized controlled, one cohort, two crossover, and seven cross-sectional) met the inclusion criteria. The daily amt. of tolerable gluten varied widely between studies. While some patients tolerated an av. of 34-36 mg of gluten per day, other patients who consumed about 10 mg of gluten per day developed mucosal abnormalities. The effect of the consumption of 'gluten-free' products with different degrees of gluten contamination was also inconsistent. The amt. of tolerable gluten varies among people with coeliac disease. Although there is no evidence to suggest a single definitive threshold, a daily gluten intake of <10 mg is unlikely to cause significant histol. abnormalities.
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    Castillo Natalia E; Theethira Thimmaiah G; Leffler Daniel A
    Gastroenterology report (2015), 3 (1), 3-11 ISSN:2052-0034.
    Celiac disease is an autoimmune enteropathy caused by gluten in genetically predisposed individuals. In celiac disease, adaptive and innate immune activation results in intestinal damage and a wide range of clinical manifestations. In the past, celiac disease was thought to result in signs and symptoms solely related to the gastrointestinal tract. Now, more than half of the adult population presents with extra-intestinal manifestations that can also be expected to improve on a gluten-free diet. For this reason, it is recommended that physicians have a low threshold of suspicion for celiac disease. Current knowledge of the immune pathogenesis of this autoimmune disease has served as a catalyst for the development of novel diagnostic tools and therapeutics. Over the years, highly sensitive and specific serological assays, in addition to genetic markers, have been found to target specific steps in the cascade pathway of celiac disease. Also the advent of the gluten challenge has enabled experts to design diagnostic algorithms and monitor clinical responses in clinical trials. The gluten challenge has provided substantial benefit in the advance of novel therapeutics as an adjuvant treatment to the gluten free diet. Generally, a strict gluten-free diet is highly burdensome to patients and can be limited in its efficacy. Alternative therapies-including gluten modification, modulation of intestinal permeability and immune response-could be central to the future treatment of celiac disease.
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    Siegel, M.; Bethune, M. T.; Gass, J.; Ehren, J.; Xia, J.; Johannsen, A.; Stuge, T. B.; Gray, G. M.; Lee, P. P.; Khosla, C. Chem. Biol. 2006, 13, 649 DOI: 10.1016/j.chembiol.2006.04.009
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    Rational Design of Combination Enzyme Therapy for Celiac Sprue
    Siegel, Matthew; Bethune, Michael T.; Gass, Jonathan; Ehren, Jennifer; Xia, Jiang; Johannsen, Alexandre; Stuge, Tor B.; Gray, Gary M.; Lee, Peter P.; Khosla, Chaitan
    Chemistry & Biology (Cambridge, MA, United States) (2006), 13 (6), 649-658CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    Celiac sprue (also known as celiac disease) is an inheritable, gluten-induced enteropathy of the upper small intestine with an estd. prevalence of 0.5%-1% in most parts of the world. The ubiquitous nature of food gluten, coupled with inadequate labeling regulations in most countries, constantly poses a threat of disease exacerbation and relapse for patients. Here, the authors demonstrate that a two-enzyme cocktail comprised of a glutamine-specific cysteine protease (EP-B2) that functions under gastric conditions and a PEP, which acts in concert with pancreatic proteases under duodenal conditions, is a particularly potent candidate for celiac sprue therapy. At a gluten:EP-B2:PEP wt. ratio of 75:3:1, grocery store gluten is fully detoxified within 10 min of simulated duodenal conditions, as judged by chromatog. anal., biopsy-derived T cell proliferation assays, and a com. antigluten antibody test.
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    Mitea, C.; Havenaar, R.; Drijfhout, J. W.; Edens, L.; Dekking, L.; Koning, F. Gut 2008, 57, 25 DOI: 10.1136/gut.2006.111609
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    Gordon, S. R.; Stanley, E. J.; Wolf, S.; Toland, A.; Wu, S. J.; Hadidi, D.; Mills, J. H.; Baker, D.; Pultz, I. S.; Siegel, J. B. J. Am. Chem. Soc. 2012, 134, 20513 DOI: 10.1021/ja3094795
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    Richter, F.; Leaver-Fay, A.; Khare, S. D.; Bjelic, S.; Baker, D. PLoS One 2011, 6, e19230 DOI: 10.1371/journal.pone.0019230
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    Mak, W. S.; Siegel, J. B. Curr. Opin. Struct. Biol. 2014, 27, 87 DOI: 10.1016/j.sbi.2014.05.010
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    Computational enzyme design: Transitioning from catalytic proteins to enzymes
    Mak, Wai Shun; Siegel, Justin B.
    Current Opinion in Structural Biology (2014), 27 (), 87-94CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
    A review. The widespread interest in enzymes stems from their ability to catalyze chem. reactions under mild and ecol. friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biol. catalysts capable of performing the desired chem. transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chem. and force-field based protein mol. modeling have led to the design of novel proteins capable of catalyzing chem. reactions not catalyzed by naturally occurring enzymes. Here, the authors discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward.
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    Shan, L.; Molberg, O.; Parrot, I.; Hausch, F.; Filiz, F.; Gray, G. M.; Sollid, L. M.; Khosla, C. Science 2002, 297, 2275 DOI: 10.1126/science.1074129
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    Shan, L. J. Proteome Res. 2005, 4, 1732 DOI: 10.1021/pr050173t
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    Identification and Analysis of Multivalent Proteolytically Resistant Peptides from Gluten: Implications for Celiac Sprue
    Shan, Lu; Qiao, Shuo-Wang; Arentz-Hansen, Helene; Molberg, Oeyvind; Gray, Gary M.; Sollid, Ludvig M.; Khosla, Chaitan
    Journal of Proteome Research (2005), 4 (5), 1732-1741CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Dietary gluten proteins from wheat, rye, and barely are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent mol. and structural analyses of representative gluten proteins, most notably α- and γ-gliadin proteins from wheat, have improved the authors' understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from α-gliadin under physiol. conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here, the authors report three lines of investigation in support of this hypothesis. First, biochem. and immunol. anal. of deletion mutants of α-2 gliadin confirmed that the DQ2 restricted T cell response to the α-2 gliadin are directed toward the epitopes clustered within the 33-mer. Second, proteolytic anal. of a representative γ-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degrdn., and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico anal. of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiol. generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.
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    Arentz-Hansen, H.; Korner, R.; Molberg, O.; Quarsten, H.; Vader, W.; Kooy, Y. M.; Lundin, K. E.; Koning, F.; Roepstorff, P.; Sollid, L. M.; McAdam, S. N. J. Exp. Med. 2000, 191, 603 DOI: 10.1084/jem.191.4.603
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    Chang, J. H.; Choi, M. G.; Yim, D. S.; Cho, Y. K.; Park, J. M.; Lee, I. S.; Kim, S. W.; Chung, I. S. Dig. Dis. Sci. 2009, 54, 578 DOI: 10.1007/s10620-008-0399-3
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    A novel placement method of the Bravo wireless pH monitoring capsule for measuring intragastric pH
    Chang Jae Hyuck; Choi Myung Gyu; Yim Dong-Seok; Cho Yu Kyung; Park Jae Myung; Lee In Seok; Kim Sang Woo; Chung In Sik
    Digestive diseases and sciences (2009), 54 (3), 578-85 ISSN:.
    PURPOSE: The delivery system of the Bravo capsule was designed for placement on the esophagus. We evaluated the feasibility of our novel placement method of the Bravo capsule using a clip to monitor intragastric pH and to compare the accuracy of the Bravo wireless system to the traditionally used Slimline catheter-Mark III Digitrapper pH monitoring system. METHODS: The Bravo capsule was placed by clip or conventional delivery system using suction on the gastric wall in 20 fasted subjects. A separate group of ten healthy volunteers underwent simultaneous intragastric pH monitoring for comparison of the two systems with meals. RESULTS: Early dislodgment rate of the capsules was lower when placed using clipping (20%) than using conventional delivery system (70%) within 48 h after placement. We observed prominent movement of one catheter in the stomach during the study. Post-test calibration drifts of the catheters at pH 7.01 were significantly greater than those of the Bravo capsules (P = 0.02). CONCLUSION: Our novel clipping method of the Bravo pH capsule placement provided accurate monitoring of intragastric pH with merits of tolerability, acid stability, and fixing position.
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    Lupo, A.; Roebuck, C.; Walsh, A.; Mozola, M.; Abouzied, M. J. AOAC Int. 2013, 96, 121 DOI: 10.5740/jaoacint.12-271
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    Moron, B.; Cebolla, A.; Manyani, H.; Alvarez-Maqueda, M.; Megias, M.; Thomas Mdel, C.; Lopez, M. C.; Sousa, C. Am. J. Clin. Nutr. 2008, 87, 405
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    Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide
    Moron, Belen; Cebolla, Angel; Manyani, Hamid; Alvarez-Maqueda, Moises; Megias, Manuel; del Carmen Thomas, Maria; Lopez, Manuel Carlos; Sousa, Carolina
    American Journal of Clinical Nutrition (2008), 87 (2), 405-414CODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
    Background: Celiac disease is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Immunotoxic gluten peptides that are recalcitrant to degrdn. of digestive enzymes appear to trigger celiac syndromes. A 33-mer peptide from α-2 gliadin has been identified as a principal contributor to gluten immunotoxicity. A gluten-free diet is the usual first therapy for celiac disease patients; therefore, the characterization and quantification of the toxic portion of the gluten in foodstuffs is crucial to avoid celiac damage. Objective: We aimed to develop immunol. assays as a novel food anal. tool for measuring cereal fractions that are immunotoxic to celiac disease patients. Design: The design focused on the prodn. of monoclonal antibodies against the gliadin 33-mer peptide and the development of enzyme-linked immunosorbent assays (ELISAs) and Western blot anal. with the use of novel antibodies. Results: A sandwich ELISA method showed a detection limit for wheat, barley, and rye of <1 ppm prolamine. However, the method required a sample that was ≥1 order of magnitude greater for the detection of low-toxic oats, and there was no signal with the safe cereals maize and rice. A competitive ELISA method was also developed for detection of the toxic peptide in hydrolyzed food, which had a detection limit of <0.5 ppm gliadin. Conclusions: Both ELISAs designed for use with the toxic gliadin 33-mer peptide suggested a high correlation between the presence of the peptide and the amt. of cereal that was toxic to celiac disease patients. The sensitivity was significantly higher than that of equiv. methods recognizing other gluten epitopes.
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    Bethune, M. T.; Strop, P.; Tang, Y.; Sollid, L. M.; Khosla, C. Chem. Biol. 2006, 13, 637 DOI: 10.1016/j.chembiol.2006.04.008
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    27
    Heterologous Expression, Purification, Refolding, and Structural-Functional Characterization of EP-B2, a Self-Activating Barley Cysteine Endoprotease
    Bethune, Michael T.; Strop, Pavel; Tang, Yinyan; Sollid, Ludvig M.; Khosla, Chaitan
    Chemistry & Biology (Cambridge, MA, United States) (2006), 13 (6), 637-647CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/L) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purifn. and refolding. The zymogen was rapidly auto-activated to its mature form under acidic conditions at a rate independent of proEP-B2 concn., suggesting a cis mechanism of auto-activation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, α2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 Å resoln. and provided at. insights into the obsd. subsite specificity of the endoproteases. Our findings suggest that orally administered proEP-B2 may be esp. well suited for treatment of celiac sprue.
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    Ehren, J.; Govindarajan, S.; Moron, B.; Minshull, J.; Khosla, C. Protein Eng., Des. Sel. 2008, 21, 699 DOI: 10.1093/protein/gzn050
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    Protein engineering of improved prolyl endopeptidases for celiac sprue therapy
    Ehren, Jennifer; Govindarajan, Sridhar; Moron, Belen; Minshull, Jeremy; Khosla, Chaitan
    Protein Engineering, Design & Selection (2008), 21 (12), 699-707CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
    Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concn.) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clin. relevance. Through two rounds of iterative mutagenesis and anal., variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, esp. in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and anal. cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacol. significant improvements in gluten detoxification activity.
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    Gass, J.; Bethune, M. T.; Siegel, M.; Spencer, A.; Khosla, C. Gastroenterology 2007, 133, 472 DOI: 10.1053/j.gastro.2007.05.028
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    Petersen, J.; Montserrat, V.; Mujico, J. R.; Loh, K. L.; Beringer, D. X.; van Lummel, M.; Thompson, A.; Mearin, M. L.; Schweizer, J.; Kooy-Winkelaar, Y.; van Bergen, J.; Drijfhout, J. W.; Kan, W. T.; La Gruta, N. L.; Anderson, R. P.; Reid, H. H.; Koning, F.; Rossjohn, J. Nat. Struct. Mol. Biol. 2014, 21, 480 DOI: 10.1038/nsmb.2817
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    T-cell receptor recognition of HLA-DQ2-gliadin complexes associated with celiac disease
    Petersen, Jan; Montserrat, Veronica; Mujico, Jorge R.; Loh, Khai Lee; Beringer, Dennis X.; van Lummel, Menno; Thompson, Allan; Mearin, M. Luisa; Schweizer, Joachim; Kooy-Winkelaar, Yvonne; van Bergen, Jeroen; Drijfhout, Jan W.; Kan, Wan-Ting; La Gruta, Nicole L.; Anderson, Robert P.; Reid, Hugh H.; Koning, Frits; Rossjohn, Jamie
    Nature Structural & Molecular Biology (2014), 21 (5), 480-488CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    Celiac disease is a T cell-mediated disease induced by dietary gluten, a component of which is gliadin. 95% of individuals with celiac disease carry the HLA (human leukocyte antigen)-DQ2 locus. Here we detd. the T-cell receptor (TCR) usage and fine specificity of patient-derived T-cell clones specific for two epitopes from wheat gliadin, DQ2.5-glia-α1a and DQ2.5-glia-α2. We detd. the ternary structures of four distinct biased TCRs specific for those epitopes. All three TCRs specific for DQ2.5-glia-α2 docked centrally above HLA-DQ2, which together with mutagenesis and affinity measurements provided a basis for the biased TCR usage. A non-germline encoded arginine residue within the CDR3β loop acted as the lynchpin within this common docking footprint. Although the TCRs specific for DQ2.5-glia-α1a and DQ2.5-glia-α2 docked similarly, their interactions with the resp. gliadin determinants differed markedly, thereby providing a basis for epitope specificity.
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    Camarca, A.; Anderson, R. P.; Mamone, G.; Fierro, O.; Facchiano, A.; Costantini, S.; Zanzi, D.; Sidney, J.; Auricchio, S.; Sette, A.; Troncone, R.; Gianfrani, C. J. Immunol. 2009, 182, 4158 DOI: 10.4049/jimmunol.0803181
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    Pera, P.; Bucca, C.; Borro, P.; Bernocco, C.; De Lillo, A.; Carossa, S. J. Dent. Res. 2002, 81, 179 DOI: 10.1177/154405910208100307
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    Influence of mastication on gastric emptying
    Pera P; Bucca C; Borro P; Bernocco C; De Lillo A; Carossa S
    Journal of dental research (2002), 81 (3), 179-81 ISSN:0022-0345.
    The role of mastication on digestion efficiency remains to be demonstrated. This study investigates whether masticatory function influences gastric emptying rate. Twelve normal volunteers were studied on two occasions after ingestion of the same test meal containing ham cubes, crackers, and egg (mixed with 13C-octanoic acid), chewed, in random order, either with 50 masticatory cycles or with 25 cycles, swallowing ham cubes whole. Lag phase (Tlag) and gastric half-emptying time (T1/2) were measured by means of the 13C-octanoic acid breath test. Trituration performance was assessed by the sieve test, and was expressed as the percentage of ham particles < or = 1 mm after 50 masticatory cycles. Tlag and T1/2 were significantly shorter when the meal was chewed with 50 cycles than with 25 cycles (Tlag 25.9+/-3.8 vs. 36.4+/-4.1 min, p=0.017; T1/2 49.1+/-5.7 vs. 62.5+/-6 min, p=0.009). Trituration performance was inversely related to both Tlag (r=0.621, p=0.031) and T1/2 (r=0.699, p=0.012). Comminution of food influences significantly gastric emptying rates.
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    Siegel, M.; Garber, M. E.; Spencer, A. G.; Botwick, W.; Kumar, P.; Williams, R. N.; Kozuka, K.; Shreeniwas, R.; Pratha, V.; Adelman, D. C. Dig. Dis. Sci. 2012, 57, 440 DOI: 10.1007/s10620-011-1906-5
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    Safety, Tolerability, and Activity of ALV003: Results from Two Phase 1 Single, Escalating-Dose Clinical Trials
    Siegel, Matthew; Garber, Mitchell E.; Spencer, Andrew G.; Botwick, Wendy; Kumar, Pawan; Williams, Robert N.; Kozuka, Kenji; Shreeniwas, Revati; Pratha, Vijaya; Adelman, Daniel C.
    Digestive Diseases and Sciences (2012), 57 (2), 440-450CODEN: DDSCDJ; ISSN:0163-2116. (Springer)
    Background. Celiac disease is the most common hereditary autoimmune disease in humans. The only treatment option for non-refractory celiac disease patients is adherence to a strict life-long gluten-free diet, which often fails to normalize small bowel histol. ALV003 is a mixt. of two proteases that degrades gluten and is in clin. development as an oral therapy for patients with celiac disease. Aims. The safety, tolerability, and activity of ALV003 were assessed in two phase 1 clin. trials. Methods. In study 1 (N = 28) the study drug was administered in the fasted state; in study 2 (N = 53) the study drug was administered together with a gluten-contg. meal. Both studies were single-dose, single-blind, placebo-controlled, cross-over trials. ALV003 was dosed at escalating dose levels by cohort (100, 300, 900, and 1,800 mg) and gastric samples were aspirated using a nasogastric tube. Adverse events, serum drug levels, and anti-drug antibody titers were measured. Gastric samples were assessed for ALV003 enzymic activity over time (gastric pharmacokinetics) and gluten degrdn. (gastric pharmacodynamics). Results. All doses were well tolerated, and no serious adverse events or allergic reactions were obsd. Gastric aspirates collected 30 min following a meal showed that 100 and 300 mg ALV003 degraded 75 ± 10% (N = 8) and 88 ± 5% (N = 8), resp., of one gram of wheat bread gluten. Conclusions. ALV003 is an orally active protease that appears to be stable in the fed stomach and degrades dietary gluten in this compartment. Single doses of oral ALV003 were not assocd. with serious adverse reactions.
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    Picariello, G.; Mamone, G.; Cutignano, A.; Fontana, A.; Zurlo, L.; Addeo, F.; Ferranti, P. J. Agric. Food Chem. 2015, 63, 3579 DOI: 10.1021/acs.jafc.5b00631
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    McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. J. Appl. Crystallogr. 2007, 40, 658 DOI: 10.1107/S0021889807021206
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    Phaser crystallographic software
    McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Winn, Martyn D.; Storoni, Laurent C.; Read, Randy J.
    Journal of Applied Crystallography (2007), 40 (4), 658-674CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
    Phaser is a program for phasing macromol. crystal structures by both mol. replacement and exptl. phasing methods. The novel phasing algorithms implemented in Phaser have been developed using max. likelihood and multivariate statistics. For mol. replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solns. from noise, and for single-wavelength anomalous dispersion exptl. phasing, the new algorithms, which account for correlations between F+ and F-, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallog. community.
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    Zwart, P. H.; Afonine, P. V.; Grosse-Kunstleve, R. W.; Hung, L. W.; Ioerger, T. R.; McCoy, A. J.; McKee, E.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.; Sauter, N. K.; Storoni, L. C.; Terwilliger, T. C.; Adams, P. D. Methods Mol. Biol. (N. Y., NY, U. S.) 2008, 426, 419 DOI: 10.1007/978-1-60327-058-8_28
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  • Abstract

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    Figure 1

    Figure 1. Amino acid substitutions in the Kuma030 endopeptidase. Model of a representative PQQP tetrapeptide in the active site of Kuma030. Enzyme backbone is shown in gray, gliadin tetrapeptide is shown in yellow. Green residues were mutated to generate Kuma030. Blue residues are thought to be important in coordinating the tetrapeptide into the active site. Image was created using PyMol.

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    Figure 2

    Figure 2. Kuma030 is capable of rapidly and effectively degrading the immunogenic regions of gluten in gastric conditions. (A) concentrations of either EPB2 and SCPEP (at a 1:1 ratio), Kuma010, or Kuma030 in gastric conditions, as measured by ELISA using the G12 antibody. The starting concentration of gluten was 10 mg mL–1 (10 000 ppm). Note that the Y-axis is plotted on a logarithmic scale. (B) The amount of gluten detected at 5 or 30 min after incubation with EPB2 and SCPEP or Kuma030. Shown is the ratio of enzyme:gluten. The starting concentration of gluten was 10 mg mL–1. Samples were normalized to the amount of gluten remaining after incubation with pepsin alone. Asterisk indicates that greater than 99.9% of the gluten was degraded. (C) HPLC trace of full-length peptide (gray dotted lines) or breakdown products (black lines) of the immunodominant peptides from gliadin (W02-E07, W03-E07, 33mer), hordein (B08-E2E7), or secalin (R11-E4E7). The bottom of the figure shows the amino acid sequence of the 33mer peptide, position of known immunogenic epitopes (horizontal lines), location of Kuma030 cleavage site (vertical lines), and elution peaks of the resulting breakdown products (gray arrows). Immunodominant peptides displayed the following breakdown patterns: B08-E2E7: PQQPIPQ∥QPQPYPQ∥Q; R11-E4E5: QPFPQ∥QPEQIIPQ∥QP; W02-E7: LQPFPQPQ∥LPYPQPQ; W03-E7: QPFPQPQ∥QPFPWQP. All peptide masses and elution times were confirmed by LCMS. Note that although the undigested W03-E07 peptide eluted at approximately the same time as a W03-E07 breakdown fragment, these are separate peaks, as determined by LCMS (Supporting Information Figure 4). aAU, arbitrary absorbance units.

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    Figure 3

    Figure 3. Gliadin treated with Kuma030 loses it immunostimulatory potential. (A–E) Purified gliadin was treated with Kuma030 at the specified concentration for 60 min at pH 4.0 at 37 °C in the presence of 0.6 mg mL–1 pepsin. After the gastric phase, the pH of the samples was increased, and samples were treated with chymotrypsin and TG2. Samples were then exposed to T cell lines from patients #1 (A), #2 (B), #3 (C), #4 (D), or #5 (E), in the presence of autologous irradiated B cell lines, and IFN-γ was measured by ELISA. Phytohemagglutenin (PHA) and a peptic-tryptic digest of gliadin (PT-gliadin) were included as positive controls. Incubation of T cell lines with antigen-presenting cells in the absence of antigens (medium) acted as a negative control. (F) T cell stimulatory epitopes recognized by the T cells used in this assay and the predicted Kuma030 cleavage sites within these epitopes. (21) Predicted Kuma030 cleavage sites are shown by a vertical line |. Cleavage sites are predicted based on Kuma030 activity on gliadin peptides as presented in Figure 2.

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    Figure 4

    Figure 4. Kuma030 efficiently degrades gluten in complex food matrices. Gluten was detected using G12 ELISA. (A) The amount of gluten remaining in whole wheat bread after a 30 min incubation with either Kuma030 or EPB2/SCPEP at the indicated enzyme concentrations. Dotted lines represent either the initial concentration of gluten (10 000 ppm) or 20 ppm gluten. Note both axes are plotted on a logarithmic scale. (B) The amount of gluten remaining in a wheat beer after incubation with the indicated concentrations of either EPB2/SCPEP or Kuma030 at 37 or 4 °C, for 5, 15, or 60 min. Dotted lines represent either the initial concentration of gluten (764 ppm) or 20 ppm.

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  • References

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    This article references 42 other publications.

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      The American journal of gastroenterology (2014), 109 (9), 1304-11 ISSN:.
      OBJECTIVES: The only treatment for celiac disease (CD) is life-long adherence to a gluten-free diet (GFD). Noncompliance is associated with signs and symptoms of CD, yet long-term adherence rates are poor. It is not known how the burden of the GFD compares with other medical treatments, and there are limited data on the socioeconomic factors influencing treatment adherence. In this study, we compared treatment burden and health state in CD compared with other chronic illnesses and evaluated the relationship between treatment burden and adherence. METHODS: Survey was mailed to participants with CD, gastroesophageal reflux disease (GERD), irritable bowel syndrome, inflammatory bowel disease, hypertension (HTN), diabetes mellitus (DM), congestive heart failure, and end-stage renal disease (ESRD) on dialysis. Surveys included demographic information and visual analog scales measuring treatment burden, importance of treatment, disease-specific health status, and overall health status. RESULTS: We collected surveys from 341 celiac and 368 non-celiac participants. Celiac participants reported high treatment burden, greater than participants with GERD or HTN and comparable to ESRD. Conversely, patients with CD reported the highest health state of all groups. Factors associated with high treatment burden in CD included poor adherence, concern regarding food cost, eating outside the home, higher income, lack of college education, and time limitations in preparing food. Poor adherence in CD was associated with increased symptoms, income, and low perceived importance of treatment. CONCLUSIONS: Participants with CD have high treatment burden but also excellent overall health status in comparison with other chronic medical conditions. The significant burden of dietary therapy for CD argues for the need for safe adjuvant treatment, as well as interventions designed to lower the perceived burden of the GFD.
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      BACKGROUND & AIMS: The cornerstone of treatment for coeliac disease is a gluten-free diet (GFD). However, adherence to a GFD is variable. Recently investigators have been reporting their preliminary findings using novel therapies. In addition, there is a growing interest in the use of complementary or alternative medicine (CAM) in gastrointestinal illnesses. These observations suggest that patients with coeliac disease may be dissatisfied with a GFD and possibly are seeking/using alternative therapies for their disease. Our aim was to assess the satisfaction levels of adults with coeliac disease towards a GFD, their use of oral CAM and views regarding novel therapies. METHODS: 310 patients with coeliac disease completed a questionnaire survey while attending their out-patient appointment. The control group comprised 477 individuals. RESULTS: Over 40% of patients with coeliac disease were dissatisfied with a GFD. The frequency of CAM use in patients with coeliac disease was 21.6% (67/310) vs 27% in the control group (129/477), p=0.09. All patients expressed an interest in novel therapies, with a vaccine being the first choice in 42% of patients, 35% and 23% for anti- zonulin and peptidases, respectively. Universally, patients placed genetically modified wheat as the lowest preference. CONCLUSIONS: A large proportion of patients with coeliac disease are dissatisfied with a GFD. Coeliac patients are not taking CAM any more than controls, suggesting they do not view CAM as an alternative to a GFD. However, all the patients in this survey were keen to consider novel therapies, with a vaccine being the most preferred option.
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      Gluten is the main storage protein in grains and consists of gliadin and glutenin occurring in the same ratio. Persons suffering from intolerances, including celiac disease. must avoid foods contg. gluten or products contg. wheat, barley. and rye. Accordingly. gluten detection is 01' high interest for the food safety of celiac patients. This study was designed to detennine the concns. of gluten in foods labeled "gluten free" available in the United States. Seventy-eight samples labeled gluten free were collected and analyzed using a gliadin competitive enzyme-linked immunosorbcnt assay. The gluten content was calcd. based on the assumption of the same ratio between gliadin and glutenin. Forty-eight (61.5%) of the 78 samples contained less than the limit of quantification of 10 mg/kg for gluten. In addn. 14(17.9%) of the 78 samples labeled gluten free contained less gluten than the guidelines established by the Codex Alimentarius for gluten-free labeling (20 mg/kg). However, 16 samples (20.5%) did contain gluten levels of ≥20 mg/kg, ranging from 20.3 to 60.3 mg/kg. In particular, five of eight breakfast cereal samples showed gluten contents higher titan 20 mg/kg. These results may be of concern, as gluten sensitivity is known to vary among celiac disease patients.
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      Interest in medical therapy for celiac disease
      Tennyson Christina A; Simpson Suzanne; Lebwohl Benjamin; Lewis Suzanne; Green Peter H R
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      OBJECTIVES: A gluten-free diet is the treatment for celiac disease, but pharmaceutical agents are being developed. The level of interest amongst patients in using a medication to treat celiac disease is unknown. This study examined the level of interest amongst patients in medication to treat celiac disease. METHODS: A questionnaire was distributed to celiac disease patients and data were collected on demographics, presentation, and interest in medication. Three validated celiac disease-specific instruments were incorporated: Celiac Disease Associated Quality of Life, the Celiac Symptom Index, and the Celiac Dietary Adherence Test. RESULTS: Responses were received from 365 individuals with biopsy-proven celiac disease. Respondents were 78% (n = 276) female, 48% (n = 170) over 50 years of age, and experienced a classical (diarrhea predominant) presentation in 44% (n = 154). Of the 339 individuals answering the question regarding use of a medication to treat celiac disease, 66% were interested. Interest was greatest in older individuals (71% >50 years of age versus 60% <50 years of age, p = 0.0415), men (78% men versus 62% women, p = 0.0083), frequent restaurant customers (76% versus 58%, p = 0.0006), those dissatisfied with their weight (73% versus 51%, p = 0.0003) and those concerned with the cost of a gluten-free diet (77% versus 64%, p = 0.0176). Length of time since diagnosis, education, presentation, and symptoms with gluten exposure did not demonstrate any effect. Interest in medication was associated with a worse quality of life (CD-QOL 69.4 versus 80.1, p < 0.0001). CONCLUSIONS: Most individuals with celiac disease are interested in using a medication. Interest was highest among men, older individuals, frequent restaurant customers, individuals dissatisfied with their weight or concerned with the cost of a gluten-free diet, and those with a worse quality of life.
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      Novel treatments for celiac disease: glutenases and beyond
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      Currently, the only effective treatment for celiac disease is a strict lifelong gluten-free diet. However, gluten-free dieting is restrictive, difficult to maintain and nutritionally less than optimal. The improved knowledge on celiac disease pathogenesis has enabled researchers to suggest alternative strategies to treat the disorder. The drug development poses a challenge as any novel drug for celiac disease should be simultaneously effective and as safe as the gluten-free diet. The rationale behind enzyme supplementation therapy as a future treatment option for celiac patients lies in the fact that gluten is only poorly digested by gastrointestinal proteases. Due to incomplete degradation in the gastrointestinal tract, fairly long gluten peptides enter the small-intestinal lumen and come into contact with the mucosal epithelium, and in celiac disease patients this encounter launches deleterious downstream effects. Enzyme supplement therapy using either bacterial or fungal endopeptidases or proteases from germinating cereals has been proposed to promote complete digestion of prolamins and destroy disease-inducing gluten peptides. A major advantage of these glutenases is that they work in the lumen of the small intestine and do not themselves take part in the immunological cascade of events in the lamina propria, thus being unlikely to cause harmful side effects to the host. Studies to test this rationale, e.g. with Aspergillus niger prolyl endoprotease and a combination enzyme product ALV003, are already ongoing. The development of a novel medication for celiac disease is still in its early days, and thus the conventional dietary treatment will hold its place for the time being.
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      Clin. management of celiac disease (CD) is based on avoidance of gluten-contg. foods, but it is not known whether trace amts. of gluten are harmful to treated patients. The safe threshold of prolonged exposure to trace amts. of gluten (contaminating gluten) was studied in 49 adult humans with biopsy-proven CD who were on gluten-free diet (GFD) ≥2 yr. The background gluten intake was maintained at <5 mg/day. After a baseline evaluation (t0), the patients ingested capsules contg. 0, 10, or 50 mg gluten daily for 90 days. Clin., serol., and histol. evaluations of the small intestine were performed at t0 and after the 90-day gluten microchallenge (t1). At t0, the median villous height/crypt depth (Vh/Cd) ratios in the small intestinal mucosa were lower and the intraepithelial lymphocyte (IEL) counts per 100 enterocytes higher in the CD patients (Vh/Cd 2.20; 95% CI 2.11-2.89; IEL 27; 95% CI 23-34) than in 20 non-CD control subjects (Vh/Cd 2.87; 95% CI 2.50-3.09; IEL 22; 95% CI 18-24). One patient challenged with 10 mg gluten developed a clin. relapse. At t1, the % change in Vh/Cd was 9% (95% CI 3-15%) in the placebo group (n = 13), -1% (-18%, 68%) in the 10-mg group (n = 13), and -20% (-22%, -13%) in the 50-mg group (n = 13). No significant differences in the IEL counts were found among the 3 groups. Thus, the ingestion of contaminating gluten should be kept <50 mg/day in the treatment of CD.
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      Akobeng, A. K.; Thomas, A. G.
      Alimentary Pharmacology and Therapeutics (2008), 27 (11), 1044-1052CODEN: APTHEN; ISSN:0269-2813. (Blackwell Publishing Ltd.)
      A review. The threshold amt. of gluten in 'gluten-free' products that can be tolerated by people with coeliac disease is unclear. To investigate the threshold amt. of gluten and the threshold concn. of gluten in food products that can be tolerated by people with coeliac disease. Systematic review of studies published between 1966 and May 2007. The data sources used were MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, CINAHL, and ref. lists of retrieved articles. We included studies that evaluated the amt. of dietary gluten or the concns. of gluten in food products that can be tolerated by people with coeliac disease whatever their design, method or language of publication. Thirteen studies (three randomized controlled, one cohort, two crossover, and seven cross-sectional) met the inclusion criteria. The daily amt. of tolerable gluten varied widely between studies. While some patients tolerated an av. of 34-36 mg of gluten per day, other patients who consumed about 10 mg of gluten per day developed mucosal abnormalities. The effect of the consumption of 'gluten-free' products with different degrees of gluten contamination was also inconsistent. The amt. of tolerable gluten varies among people with coeliac disease. Although there is no evidence to suggest a single definitive threshold, a daily gluten intake of <10 mg is unlikely to cause significant histol. abnormalities.
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      Castillo, N. E.; Theethira, T. G.; Leffler, D. A. Gastroenterol. Rep. (Oxford) 2015, 3, 3 DOI: 10.1093/gastro/gou065
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      The present and the future in the diagnosis and management of celiac disease
      Castillo Natalia E; Theethira Thimmaiah G; Leffler Daniel A
      Gastroenterology report (2015), 3 (1), 3-11 ISSN:2052-0034.
      Celiac disease is an autoimmune enteropathy caused by gluten in genetically predisposed individuals. In celiac disease, adaptive and innate immune activation results in intestinal damage and a wide range of clinical manifestations. In the past, celiac disease was thought to result in signs and symptoms solely related to the gastrointestinal tract. Now, more than half of the adult population presents with extra-intestinal manifestations that can also be expected to improve on a gluten-free diet. For this reason, it is recommended that physicians have a low threshold of suspicion for celiac disease. Current knowledge of the immune pathogenesis of this autoimmune disease has served as a catalyst for the development of novel diagnostic tools and therapeutics. Over the years, highly sensitive and specific serological assays, in addition to genetic markers, have been found to target specific steps in the cascade pathway of celiac disease. Also the advent of the gluten challenge has enabled experts to design diagnostic algorithms and monitor clinical responses in clinical trials. The gluten challenge has provided substantial benefit in the advance of novel therapeutics as an adjuvant treatment to the gluten free diet. Generally, a strict gluten-free diet is highly burdensome to patients and can be limited in its efficacy. Alternative therapies-including gluten modification, modulation of intestinal permeability and immune response-could be central to the future treatment of celiac disease.
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      Siegel, M.; Bethune, M. T.; Gass, J.; Ehren, J.; Xia, J.; Johannsen, A.; Stuge, T. B.; Gray, G. M.; Lee, P. P.; Khosla, C. Chem. Biol. 2006, 13, 649 DOI: 10.1016/j.chembiol.2006.04.009
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      Rational Design of Combination Enzyme Therapy for Celiac Sprue
      Siegel, Matthew; Bethune, Michael T.; Gass, Jonathan; Ehren, Jennifer; Xia, Jiang; Johannsen, Alexandre; Stuge, Tor B.; Gray, Gary M.; Lee, Peter P.; Khosla, Chaitan
      Chemistry & Biology (Cambridge, MA, United States) (2006), 13 (6), 649-658CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      Celiac sprue (also known as celiac disease) is an inheritable, gluten-induced enteropathy of the upper small intestine with an estd. prevalence of 0.5%-1% in most parts of the world. The ubiquitous nature of food gluten, coupled with inadequate labeling regulations in most countries, constantly poses a threat of disease exacerbation and relapse for patients. Here, the authors demonstrate that a two-enzyme cocktail comprised of a glutamine-specific cysteine protease (EP-B2) that functions under gastric conditions and a PEP, which acts in concert with pancreatic proteases under duodenal conditions, is a particularly potent candidate for celiac sprue therapy. At a gluten:EP-B2:PEP wt. ratio of 75:3:1, grocery store gluten is fully detoxified within 10 min of simulated duodenal conditions, as judged by chromatog. anal., biopsy-derived T cell proliferation assays, and a com. antigluten antibody test.
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      Mitea, C.; Havenaar, R.; Drijfhout, J. W.; Edens, L.; Dekking, L.; Koning, F. Gut 2008, 57, 25 DOI: 10.1136/gut.2006.111609
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      Gordon, S. R.; Stanley, E. J.; Wolf, S.; Toland, A.; Wu, S. J.; Hadidi, D.; Mills, J. H.; Baker, D.; Pultz, I. S.; Siegel, J. B. J. Am. Chem. Soc. 2012, 134, 20513 DOI: 10.1021/ja3094795
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      Richter, F.; Leaver-Fay, A.; Khare, S. D.; Bjelic, S.; Baker, D. PLoS One 2011, 6, e19230 DOI: 10.1371/journal.pone.0019230
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      Mak, W. S.; Siegel, J. B. Curr. Opin. Struct. Biol. 2014, 27, 87 DOI: 10.1016/j.sbi.2014.05.010
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      Computational enzyme design: Transitioning from catalytic proteins to enzymes
      Mak, Wai Shun; Siegel, Justin B.
      Current Opinion in Structural Biology (2014), 27 (), 87-94CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
      A review. The widespread interest in enzymes stems from their ability to catalyze chem. reactions under mild and ecol. friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biol. catalysts capable of performing the desired chem. transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chem. and force-field based protein mol. modeling have led to the design of novel proteins capable of catalyzing chem. reactions not catalyzed by naturally occurring enzymes. Here, the authors discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward.
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      Shan, L.; Molberg, O.; Parrot, I.; Hausch, F.; Filiz, F.; Gray, G. M.; Sollid, L. M.; Khosla, C. Science 2002, 297, 2275 DOI: 10.1126/science.1074129
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      Shan, L. J. Proteome Res. 2005, 4, 1732 DOI: 10.1021/pr050173t
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      Identification and Analysis of Multivalent Proteolytically Resistant Peptides from Gluten: Implications for Celiac Sprue
      Shan, Lu; Qiao, Shuo-Wang; Arentz-Hansen, Helene; Molberg, Oeyvind; Gray, Gary M.; Sollid, Ludvig M.; Khosla, Chaitan
      Journal of Proteome Research (2005), 4 (5), 1732-1741CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Dietary gluten proteins from wheat, rye, and barely are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent mol. and structural analyses of representative gluten proteins, most notably α- and γ-gliadin proteins from wheat, have improved the authors' understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from α-gliadin under physiol. conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here, the authors report three lines of investigation in support of this hypothesis. First, biochem. and immunol. anal. of deletion mutants of α-2 gliadin confirmed that the DQ2 restricted T cell response to the α-2 gliadin are directed toward the epitopes clustered within the 33-mer. Second, proteolytic anal. of a representative γ-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degrdn., and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico anal. of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiol. generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.
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      Sollid, L. M.; Qiao, S. W.; Anderson, R. P.; Gianfrani, C.; Koning, F. Immunogenetics 2012, 64, 455 DOI: 10.1007/s00251-012-0599-z
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      Tye-Din, J. A.; Stewart, J. A.; Dromey, J. A.; Beissbarth, T.; van Heel, D. A.; Tatham, A.; Henderson, K.; Mannering, S. I.; Gianfrani, C.; Jewell, D. P.; Hill, A. V.; McCluskey, J.; Rossjohn, J.; Anderson, R. P. Sci. Transl. Med. 2010, 2, 41ra51 DOI: 10.1126/scitranslmed.3001012
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      Arentz-Hansen, H.; Korner, R.; Molberg, O.; Quarsten, H.; Vader, W.; Kooy, Y. M.; Lundin, K. E.; Koning, F.; Roepstorff, P.; Sollid, L. M.; McAdam, S. N. J. Exp. Med. 2000, 191, 603 DOI: 10.1084/jem.191.4.603
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      Chang, J. H.; Choi, M. G.; Yim, D. S.; Cho, Y. K.; Park, J. M.; Lee, I. S.; Kim, S. W.; Chung, I. S. Dig. Dis. Sci. 2009, 54, 578 DOI: 10.1007/s10620-008-0399-3
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      A novel placement method of the Bravo wireless pH monitoring capsule for measuring intragastric pH
      Chang Jae Hyuck; Choi Myung Gyu; Yim Dong-Seok; Cho Yu Kyung; Park Jae Myung; Lee In Seok; Kim Sang Woo; Chung In Sik
      Digestive diseases and sciences (2009), 54 (3), 578-85 ISSN:.
      PURPOSE: The delivery system of the Bravo capsule was designed for placement on the esophagus. We evaluated the feasibility of our novel placement method of the Bravo capsule using a clip to monitor intragastric pH and to compare the accuracy of the Bravo wireless system to the traditionally used Slimline catheter-Mark III Digitrapper pH monitoring system. METHODS: The Bravo capsule was placed by clip or conventional delivery system using suction on the gastric wall in 20 fasted subjects. A separate group of ten healthy volunteers underwent simultaneous intragastric pH monitoring for comparison of the two systems with meals. RESULTS: Early dislodgment rate of the capsules was lower when placed using clipping (20%) than using conventional delivery system (70%) within 48 h after placement. We observed prominent movement of one catheter in the stomach during the study. Post-test calibration drifts of the catheters at pH 7.01 were significantly greater than those of the Bravo capsules (P = 0.02). CONCLUSION: Our novel clipping method of the Bravo pH capsule placement provided accurate monitoring of intragastric pH with merits of tolerability, acid stability, and fixing position.
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      Lupo, A.; Roebuck, C.; Walsh, A.; Mozola, M.; Abouzied, M. J. AOAC Int. 2013, 96, 121 DOI: 10.5740/jaoacint.12-271
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      Moron, B.; Cebolla, A.; Manyani, H.; Alvarez-Maqueda, M.; Megias, M.; Thomas Mdel, C.; Lopez, M. C.; Sousa, C. Am. J. Clin. Nutr. 2008, 87, 405
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      26
      Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide
      Moron, Belen; Cebolla, Angel; Manyani, Hamid; Alvarez-Maqueda, Moises; Megias, Manuel; del Carmen Thomas, Maria; Lopez, Manuel Carlos; Sousa, Carolina
      American Journal of Clinical Nutrition (2008), 87 (2), 405-414CODEN: AJCNAC; ISSN:0002-9165. (American Society for Nutrition)
      Background: Celiac disease is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Immunotoxic gluten peptides that are recalcitrant to degrdn. of digestive enzymes appear to trigger celiac syndromes. A 33-mer peptide from α-2 gliadin has been identified as a principal contributor to gluten immunotoxicity. A gluten-free diet is the usual first therapy for celiac disease patients; therefore, the characterization and quantification of the toxic portion of the gluten in foodstuffs is crucial to avoid celiac damage. Objective: We aimed to develop immunol. assays as a novel food anal. tool for measuring cereal fractions that are immunotoxic to celiac disease patients. Design: The design focused on the prodn. of monoclonal antibodies against the gliadin 33-mer peptide and the development of enzyme-linked immunosorbent assays (ELISAs) and Western blot anal. with the use of novel antibodies. Results: A sandwich ELISA method showed a detection limit for wheat, barley, and rye of <1 ppm prolamine. However, the method required a sample that was ≥1 order of magnitude greater for the detection of low-toxic oats, and there was no signal with the safe cereals maize and rice. A competitive ELISA method was also developed for detection of the toxic peptide in hydrolyzed food, which had a detection limit of <0.5 ppm gliadin. Conclusions: Both ELISAs designed for use with the toxic gliadin 33-mer peptide suggested a high correlation between the presence of the peptide and the amt. of cereal that was toxic to celiac disease patients. The sensitivity was significantly higher than that of equiv. methods recognizing other gluten epitopes.
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      Bethune, M. T.; Strop, P.; Tang, Y.; Sollid, L. M.; Khosla, C. Chem. Biol. 2006, 13, 637 DOI: 10.1016/j.chembiol.2006.04.008
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      27
      Heterologous Expression, Purification, Refolding, and Structural-Functional Characterization of EP-B2, a Self-Activating Barley Cysteine Endoprotease
      Bethune, Michael T.; Strop, Pavel; Tang, Yinyan; Sollid, Ludvig M.; Khosla, Chaitan
      Chemistry & Biology (Cambridge, MA, United States) (2006), 13 (6), 637-647CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/L) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purifn. and refolding. The zymogen was rapidly auto-activated to its mature form under acidic conditions at a rate independent of proEP-B2 concn., suggesting a cis mechanism of auto-activation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, α2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 Å resoln. and provided at. insights into the obsd. subsite specificity of the endoproteases. Our findings suggest that orally administered proEP-B2 may be esp. well suited for treatment of celiac sprue.
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      Ehren, J.; Govindarajan, S.; Moron, B.; Minshull, J.; Khosla, C. Protein Eng., Des. Sel. 2008, 21, 699 DOI: 10.1093/protein/gzn050
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      28
      Protein engineering of improved prolyl endopeptidases for celiac sprue therapy
      Ehren, Jennifer; Govindarajan, Sridhar; Moron, Belen; Minshull, Jeremy; Khosla, Chaitan
      Protein Engineering, Design & Selection (2008), 21 (12), 699-707CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
      Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concn.) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clin. relevance. Through two rounds of iterative mutagenesis and anal., variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, esp. in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and anal. cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacol. significant improvements in gluten detoxification activity.
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      Gass, J.; Bethune, M. T.; Siegel, M.; Spencer, A.; Khosla, C. Gastroenterology 2007, 133, 472 DOI: 10.1053/j.gastro.2007.05.028
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      Petersen, J.; Montserrat, V.; Mujico, J. R.; Loh, K. L.; Beringer, D. X.; van Lummel, M.; Thompson, A.; Mearin, M. L.; Schweizer, J.; Kooy-Winkelaar, Y.; van Bergen, J.; Drijfhout, J. W.; Kan, W. T.; La Gruta, N. L.; Anderson, R. P.; Reid, H. H.; Koning, F.; Rossjohn, J. Nat. Struct. Mol. Biol. 2014, 21, 480 DOI: 10.1038/nsmb.2817
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      T-cell receptor recognition of HLA-DQ2-gliadin complexes associated with celiac disease
      Petersen, Jan; Montserrat, Veronica; Mujico, Jorge R.; Loh, Khai Lee; Beringer, Dennis X.; van Lummel, Menno; Thompson, Allan; Mearin, M. Luisa; Schweizer, Joachim; Kooy-Winkelaar, Yvonne; van Bergen, Jeroen; Drijfhout, Jan W.; Kan, Wan-Ting; La Gruta, Nicole L.; Anderson, Robert P.; Reid, Hugh H.; Koning, Frits; Rossjohn, Jamie
      Nature Structural & Molecular Biology (2014), 21 (5), 480-488CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      Celiac disease is a T cell-mediated disease induced by dietary gluten, a component of which is gliadin. 95% of individuals with celiac disease carry the HLA (human leukocyte antigen)-DQ2 locus. Here we detd. the T-cell receptor (TCR) usage and fine specificity of patient-derived T-cell clones specific for two epitopes from wheat gliadin, DQ2.5-glia-α1a and DQ2.5-glia-α2. We detd. the ternary structures of four distinct biased TCRs specific for those epitopes. All three TCRs specific for DQ2.5-glia-α2 docked centrally above HLA-DQ2, which together with mutagenesis and affinity measurements provided a basis for the biased TCR usage. A non-germline encoded arginine residue within the CDR3β loop acted as the lynchpin within this common docking footprint. Although the TCRs specific for DQ2.5-glia-α1a and DQ2.5-glia-α2 docked similarly, their interactions with the resp. gliadin determinants differed markedly, thereby providing a basis for epitope specificity.
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      Bethune, M. T.; Khosla, C. Methods Enzymol. 2012, 502, 241 DOI: 10.1016/B978-0-12-416039-2.00013-6
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      Camarca, A.; Anderson, R. P.; Mamone, G.; Fierro, O.; Facchiano, A.; Costantini, S.; Zanzi, D.; Sidney, J.; Auricchio, S.; Sette, A.; Troncone, R.; Gianfrani, C. J. Immunol. 2009, 182, 4158 DOI: 10.4049/jimmunol.0803181
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      Pera, P.; Bucca, C.; Borro, P.; Bernocco, C.; De Lillo, A.; Carossa, S. J. Dent. Res. 2002, 81, 179 DOI: 10.1177/154405910208100307
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      Influence of mastication on gastric emptying
      Pera P; Bucca C; Borro P; Bernocco C; De Lillo A; Carossa S
      Journal of dental research (2002), 81 (3), 179-81 ISSN:0022-0345.
      The role of mastication on digestion efficiency remains to be demonstrated. This study investigates whether masticatory function influences gastric emptying rate. Twelve normal volunteers were studied on two occasions after ingestion of the same test meal containing ham cubes, crackers, and egg (mixed with 13C-octanoic acid), chewed, in random order, either with 50 masticatory cycles or with 25 cycles, swallowing ham cubes whole. Lag phase (Tlag) and gastric half-emptying time (T1/2) were measured by means of the 13C-octanoic acid breath test. Trituration performance was assessed by the sieve test, and was expressed as the percentage of ham particles < or = 1 mm after 50 masticatory cycles. Tlag and T1/2 were significantly shorter when the meal was chewed with 50 cycles than with 25 cycles (Tlag 25.9+/-3.8 vs. 36.4+/-4.1 min, p=0.017; T1/2 49.1+/-5.7 vs. 62.5+/-6 min, p=0.009). Trituration performance was inversely related to both Tlag (r=0.621, p=0.031) and T1/2 (r=0.699, p=0.012). Comminution of food influences significantly gastric emptying rates.
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      Siegel, M.; Garber, M. E.; Spencer, A. G.; Botwick, W.; Kumar, P.; Williams, R. N.; Kozuka, K.; Shreeniwas, R.; Pratha, V.; Adelman, D. C. Dig. Dis. Sci. 2012, 57, 440 DOI: 10.1007/s10620-011-1906-5
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      Safety, Tolerability, and Activity of ALV003: Results from Two Phase 1 Single, Escalating-Dose Clinical Trials
      Siegel, Matthew; Garber, Mitchell E.; Spencer, Andrew G.; Botwick, Wendy; Kumar, Pawan; Williams, Robert N.; Kozuka, Kenji; Shreeniwas, Revati; Pratha, Vijaya; Adelman, Daniel C.
      Digestive Diseases and Sciences (2012), 57 (2), 440-450CODEN: DDSCDJ; ISSN:0163-2116. (Springer)
      Background. Celiac disease is the most common hereditary autoimmune disease in humans. The only treatment option for non-refractory celiac disease patients is adherence to a strict life-long gluten-free diet, which often fails to normalize small bowel histol. ALV003 is a mixt. of two proteases that degrades gluten and is in clin. development as an oral therapy for patients with celiac disease. Aims. The safety, tolerability, and activity of ALV003 were assessed in two phase 1 clin. trials. Methods. In study 1 (N = 28) the study drug was administered in the fasted state; in study 2 (N = 53) the study drug was administered together with a gluten-contg. meal. Both studies were single-dose, single-blind, placebo-controlled, cross-over trials. ALV003 was dosed at escalating dose levels by cohort (100, 300, 900, and 1,800 mg) and gastric samples were aspirated using a nasogastric tube. Adverse events, serum drug levels, and anti-drug antibody titers were measured. Gastric samples were assessed for ALV003 enzymic activity over time (gastric pharmacokinetics) and gluten degrdn. (gastric pharmacodynamics). Results. All doses were well tolerated, and no serious adverse events or allergic reactions were obsd. Gastric aspirates collected 30 min following a meal showed that 100 and 300 mg ALV003 degraded 75 ± 10% (N = 8) and 88 ± 5% (N = 8), resp., of one gram of wheat bread gluten. Conclusions. ALV003 is an orally active protease that appears to be stable in the fed stomach and degrades dietary gluten in this compartment. Single doses of oral ALV003 were not assocd. with serious adverse reactions.
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      Picariello, G.; Mamone, G.; Cutignano, A.; Fontana, A.; Zurlo, L.; Addeo, F.; Ferranti, P. J. Agric. Food Chem. 2015, 63, 3579 DOI: 10.1021/acs.jafc.5b00631
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      McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. J. Appl. Crystallogr. 2007, 40, 658 DOI: 10.1107/S0021889807021206
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      Phaser crystallographic software
      McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Winn, Martyn D.; Storoni, Laurent C.; Read, Randy J.
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