Noninflammatory gluten peptide analogs as biomarkers for celiac sprue

Abstract

New tools are needed for managing celiac sprue, a lifelong immune disease of the small intestine. Ongoing drug trials are also prompting a search for noninvasive biomarkers of gluten-induced intestinal change. We have synthesized and characterized noninflammatory gluten peptide analogs in which key Gln residues are replaced by Asn or His. Like their proinflammatory counterparts, these biomarkers are resistant to gastrointestinal proteases, susceptible to glutenases, and permeable across enterocyte barriers. Unlike gluten peptides, however, they are not appreciably recognized by transglutaminase, HLA-DQ2, or disease-specific T cells. In vitro and animal studies show that the biomarkers can detect intestinal permeability changes as well as glutenase-catalyzed gastric detoxification of gluten. Accordingly, controlled clinical studies are warranted to evaluate the use of these peptides as probes for abnormal intestinal permeability in celiac patients and for glutenase efficacy in clinical trials and practice.

Figure 1. Synthetic Gluten and Biomarker Peptide Sequences

Sequences are shown for the native 33-mer (designated QQQ-33-mer) derived from α2-gliadin, the synthetically deamidated 33-mer (EEE-33-mer), the biomarkers NNN-33-mer and HHH-33-mer, and a noninflammatory control peptide of unrelated sequence derived from myoglobin. Bonds that are scissile to EP-B2-mediated cleavage are designated by arrowheads. Glutamine residues that are selectively deamidated by TG2 or synthetically replaced in the biomarker peptides are in bold. Leucine residues that are isotope labeled for in vivo experiments are underlined.

Figure 2. The 33-mer Gluten Peptide and Gluten Peptide-Based Biomarkers Are Similarly Resistant to Cleavage by Pepsin and Susceptible to Cleavage by the Glutenase EP-B2

(A–F) Reverse-phase HPLC traces for QQQ-33-mer (A and D), NNN-33-mer (B and E), and HHH-33-mer (300 μM) (C and F) after simulated gastric digestion for specified durations with either pepsin alone (A–C) or pepsin and 120 μg/mL EP-B2 (D–F). The TAME internal standard (T), intact peptide (peak 5), minimally processed peptide lacking only the N-terminal LQ (peak 4), and other major digestion products are indicated for each peptide trace overlay. (G–J) Integrated area-under-the-curve analysis for intact peptides QQQ-33-mer (G), NNN-33-mer (H), HHH-33-mer (I), and myoglobin peptide (J) showing dose and time dependency of EP-B2-mediated digestion. Each peptide (300 μM) was digested in vitro with pepsin supplemented with specified concentrations of EP-B2, and reaction products were analyzed by HPLC. The area under the curve for each intact peptide peak (and, where applicable, the minimally processed -LQ peptide peak) was calculated and normalized to that for the internal standard. Values are expressed as the percentage of intact peptide remaining after a given digestion duration relative to the initial peak area. (K) LC-MS identification of major digestion products from simulated gastric digests with pepsin ± EP-B2. HPLC peak number corresponds to the peak numbers in (A)–(F). Data shown are representative of three independent experiments.

Figure 3. The 33-mer Gluten Peptide and Gluten Peptide-Based Biomarkers Are Similarly Resistant to Cleavage by Pancreatic Enzymes and Susceptible to Cleavage by Prolyl Endopeptidase

(A) Integrated area-under-the-curve analysis for intact QQQ-33-mer, NNN-33-mer, and HHH-33-mer after simulated intestinal digestion ± supplementation with PEP from FM PEP. Following treatment with pepsin, each peptide (300 μM) was digested in vitro with pancreatic proteases (TCEC) and rat intestinal BBM ± 1.2 U/mL FM PEP. Reaction products were analyzed by reverse-phase HPLC. The area under the curve for each intact peptide peak was calculated and normalized to that for the internal standard. Values are expressed as the percentage of intact peptide remaining after a given duration relative to the initial peak area. (B) Cleavage map derived from LC-MS/MS analysis of major digestion products following simulated gastrointestinal digests. Blue arrowheads designate major cleavage sites resulting from EP-B2 supplementation. Red arrowheads designate major cleavage sites resulting from FM PEP supplementation. Underlined, numbered sequences designate major products of digestion with EP-B2. (C–E) HPLC traces for QQQ-33-mer (C), NNN-33-mer (D), and HHH-33-mer (E) (300 μM) after simulated gastric digestion with pepsin + 120 μg/mL EP-B2 for 60 min followed by treatment with TCEC + BBM ± 1.2 U/mL FM PEP for 10 or 60 min. T, TAME internal standard. HPLC peak numbers corresponds to the numbered sequences in B. Data shown are representative of two independent experiments.

Figure 4. Biomarkers Are Noninflammatory in the Context of Celiac Sprue

(A) Specific activity of TG2 (5 μM) in the presence of 100 μM gluten peptide QQQ-33-mer, biomarkers NNN-33-mer or HHH-33-mer, control myoglobin peptide, or no peptide. Means ± SD for triplicate assays are shown. Data are representative of three independent experiments. Statistical comparisons were performed with respect to samples containing QQQ-33-mer. ***p < 0.001. (B) Ratio of DQ2-bound to unbound fluorescein-conjugated (f-)peptides following incubation of thrombin-cleaved DQ2-αI-gliadin peptide complexes (9.4 μM) with 0.185 μM f-QQQ-33-mer, f-EEE-33-mer, f-NNN-33-mer, -f-HHH-33-mer, or f-myoglobin peptide in a citrate-PBS buffer (pH 5.5) for 45 hr at 37°C. Means ± SD for triplicate assays are shown. Data are representative of three independent experiments. Statistical comparisons were performed with respect to samples containing f-EEE-33-mer. **p < 0.01. (C) Activation of celiac patient-derived intestinal T cell lines (TCL) and T cell clones (TCC) in response to incubation with DQ2-homozygous antigen-presenting cells preloaded with varied concentrations of ±TG2-treated QQQ-33-mer, NNN-33-mer, or HHH-33-mer. Positive (2000 nM synthetically deamidated EEE-33-mer) and negative (no peptide) controls are shown for comparison. Activation is measured in terms of counts per minute (cpm) of [³H]-thymidine incorporated into harvested DNA of proliferating T cells. Means ± SD for triplicate wells are shown.

Figure 5. Glutenase-Mediated Biomarker Metabolism Parallels Gluten Digestion In Vivo

Three groups of rats (n = 4 animals/group) were administered a gluten-containing meal supplemented with 0, 10, or 40 mg EP-B2 glutenase (for groups 1, 2, or 3, respectively), 19.7 mg [¹³C₃]-HHH-33-mer biomarker, 3.3 mg [D₃]-myoglobin peptide control, and 10 mg vancomycin dosing standard. Gastric contents were collected after 90 min and analyzed by HPLC, competitive ELISA, and 3Q LC-MS/MS. (A) Representative HPLC traces of gastric contents from one animal per group. Internal standards for dosing (V) and HPLC analysis (T) correspond roughly to cut-offs between regions of nontoxic metabolites, partially digested peptides, and proteins and peptides retaining immunogenicity (Gass et al., 2007). Asterisk indicates a nongluten peak that was omitted from area-under-the-curve analysis. (B) Area-under-the-curve analysis quantifying the data from (A). The area under the curve for HPLC trace regions corresponding to nontoxic metabolites (2.7–5.6 min), partially digested peptides (6.3–15.1 min), and immunogenic proteins and peptides (16.0–30.0 min) was normalized to the area under the curve for the vancomycin and TAME internal standards. Means ± SD for each animal group are shown. Data are representative of four similar experiments. Statistical comparisons were performed with respect to group 1 (no glutenase). **p < 0.01. (C) Competitive ELISA analysis quantifying the concentration of peptides containing the gluten peptide sequence QPQLPY in gastric contents. Samples from each animal were tested in triplicate, with a typical error of 5%–10% of the sample mean. Means ± SD for each animal group are shown. Data are representative of four similar experiments. Statistical comparisons were performed with respect to group 1 (no glutenase). **p < 0.01. (D) 3Q LC-MS/MS analysis quantifying the concentration of QQQ-33-mer (derived from ingested gluten), [¹³C₃]-HHH-33-mer biomarker, and [D₃]-myoglobin peptide in gastric contents. Samples from each animal were tested in triplicate, with a typical error of <10% of the sample mean for group 1. Means ± SD for each animal group are shown. Statistical comparisons were performed with respect to group 1 (no glutenase). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. Biomarker Transepithelial Transport Parallels Gluten Peptide Transport In Vitro and In Vivo

(A) Experimental design for in vitro studies. Transwell supports bearing mature T84 epithelial cell monolayers were preincubated with media alone or with 600 U/mL IFN-γ in the basolateral chamber. After 48 hr, 20 μM Cy5-labeled QQQ-33-mer, NNN-33-mer, or HHH-33-mer was added to the apical chamber, and samples from the apical and basolateral chambers were sampled over time to determine the stability and apical-to-basolateral flux of each intact peptide. (B) Apical-to-basolateral flux of each Cy5-labeled peptide under basal (0 U/mL IFN-γ) and simulated inflammatory (600 U/mL IFN-γ) conditions. Means ± SD for triplicate assays are shown. Data are representative of two similar experiments. Statistical comparisons were performed with respect to QQQ-33-mer; no significant differences were observed. (C–E) Reverse-phase HPLC traces from LC-MS analysis of samples taken from the apical and basolateral chambers at 0 and 10 hr. Elution of Cy5-QQQ-33-mer (C), Cy5-NNN-33-mer (D), and Cy5-HHH-33-mer (E) were monitored by their absorbance at 640 nm. Intact Cy5-peptides elute as the major peak between 9 and 10 min. The peak eluting in each trace at 8 min is Cy5-LQ, signifying limited processing of the N terminus by T84 cells. (F) Area-under-the-curve analysis quantifying the data from C–E. (G) 3Q LC-MS/MS analysis quantifying the concentration of [¹³C₃]-HHH-33-mer in the peripheral plasma of a gluten-sensitive rhesus macaque (FH45) following intragastric biomarker administration. Means ± SD for triplicate assays are shown. Statistical comparisons were performed with respect to the 0 min time point (prior to biomarker administration). ****p < 0.0001.