Enhanced Peptide-Binding Capacities of Small Intestinal Brush Border Membranes in Celiac Disease

Abstract In pathogenesis of celiac disease, the significance of prolamin peptide interactions with enterocytes is controversial. Changes in cellular metabolism induced by gliadin peptides, as well as uptake and presentation by enterocytes, are discussed. We analyzed peptide binding to enterocytic membranes as a potential key event. Binding capacities of brush border membranes isolated from small intestinal biopsies of untreated (n= 49) and treated celiac patients on a gluten-free diet (n= 30), as well as control subjects (n= 43), were measured with a dot blot chemiluminescence assay. Synthetic gliadin peptides comprising amino acid position 8–19 (G XIV) and 30–41 (G XI) of α-gliadins, a peptic-tryptic digest of gliadin (PT-GLI), and a synthetic zein peptide were used. Comparing treated celiac patients with controls, we observed significantly enhanced membrane-binding of PT-GLI [mean 122.4 densitometric units/μg (95% confidence interval 116.0–128.9) vs 108.9 (102.1–115.7)] and of zein peptide [50.2 (38.4–61.9) vs 28.8 (13.4–44.2)], but only slightly increased binding of the synthetic gliadin peptides G XIV [65.5 (60.6–70.5) vs 62.4 (56.3–68.5) and G XI [75.2 (69.8–80.6) vs 65.9 (55.2–76.5)]. Independent of patient group, membrane-binding capacities for celiac-active gliadin peptides exceeded those of the zein peptide. Thus, interaction of gliadin peptides with the apical enterocytic membrane was not found exclusively in celiac disease. Furthermore, increased binding capacities in treated celiac disease were not confined to celiac-active peptides. Quantitative differences in gliadin peptide binding as a primary characteristic in celiac disease might contribute to pathogenetic effects exerted on small intestinal epithelial cells. Main The complex interplay of environmental and inherent factors in the pathogenesis of celiac disease is far from being understood. Celiac disease is induced by ingestion of prolamins and is characterized by small intestinal villous atrophy, crypt cell hyperplasia, and lamina propria infiltration by lymphocytes (1, 2). It has been proposed that the presentation of prolamin peptides by HLA class II molecules to T cells initiates immunopathogenesis (2, 3). There is increasing evidence that tissue transglutaminase plays a role in celiac disease. Enzymatic deamidation of gliadin peptides unmasks an epitope that binds to HLA-DQ2 and is recognized by gut-derived T cells (4). Furthermore, new antigenic complexes are created by cross-linking of gliadin with tissue transglutaminase (5). Although celiac disease is viewed mainly as a T cell-mediated disorder, interactions between prolamin peptides and small intestinal epithelial cells might be involved in several ways (6). First, apical binding and uptake of prolamin fragments might show direct effects on enterocytes and lead to cell damage. HLA-DR overexpression (7), reduction of cell height (8), inhibition of biosynthesis of BBM hydrolases (9), and impaired cellular metabolism (10) have been demonstrated as effects of gliadin peptides on enterocytes, mediated in part by other cell types. Second, uptake, transcellular transport, and basolateral release of prolamin peptides by enterocytes might enable transglutaminase-mediated modification in the subepithelial region, followed by antigen presentation and T cell stimulation (4, 11). Third, presentation of modified peptides by enterocytes might cause T cell activation directly (12–14). In each case, initial binding to the apical BBM is supposed to be a prerequisite to the subsequent steps. Conflicting results have been reported concerning gliadin peptide binding to enterocytes in celiac disease (15, 16). The aim of our study was therefore to clarify whether BBM-binding capacity for gliadin peptides is altered in celiac disease. Furthermore, we compared the BBM of patients with active disease with that of patients with treated disease to detect whether membrane-binding alterations in celiac disease are primary or secondary. METHODS Patients. Small intestinal biopsies were obtained for routine diagnostic procedures from the following three patient groups:1) CON: 25 females and 18 males (0.4–14.3 y, median 3.1 y) with normal mucosa on a diet of gluten-containing food (diarrhea, failure to thrive);2) CD/A: 25 females and 24 males (0.3–18.3 y, median 4.1 y) with untreated active celiac disease, showing severe mucosal damage, according to Marsh (1), and 3) CD/R: 20 females and 10 males (2.8–69.2 y, median 46.4 y) with celiac disease in remission, showing intact intestinal mucosa on a gluten-free diet for at least 6 months. The protocol of the study was approved by the local ethics committee. BBM. Small intestinal biopsies (wet weight 3–20 mg) were homogenized with a glass/Teflon homogenizer in 500 mM mannitol, 10 mM HEPES, pH 7.5. CaCl2 was added to reach a final concentration of 10 mM. After an incubation period of 15 min at 4°C, the homogenate was centrifuged (3,000 ×g, 15 min, 4°C) to separate aggregated membranes. After further centrifugation of the supernatant (30,000 ×g, 30 min, 4°C), the pellet of BBM vesicles was resuspended in 100 mM mannitol, 10 mM Tris, pH 7.4. For investigation of membrane morphology by electron microscopy, membrane vesicles were fixed with glutaraldehyde as previously described (17). The purity of BBM preparations was assessed by measuring the BBM enzymes sucrase (18) and alkaline phosphatase (test kit from Sigma Chemical Co., Deisenhofen, Germany). Enzyme units were defined as the amount of enzyme transforming 1 μmol of substrate/min under the experimental conditions. Specific enzyme activities were expressed as milliunits of enzyme/milligram of protein. Protein content was determined with the DC protein assay (Bio-Rad, Munich, Germany). PT-GLI. A crude mixture of gliadin peptides, free of contaminating proteases, was obtained by peptic-tryptic digestion of gliadin and was biotinylated with biotinamidocaproate-N-hydroxysulfosuccinimide ester (Sigma Chemical Co.), as described elsewhere (19). Peptide synthesis. Synthesis of gliadin peptides G XI and G XIV and zein peptide Z I was performed automatically by a solid phase peptide synthesizer (model 431 A, Applied Biosystems, Foster City, CA) using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry on a small scale (1 mmol/Fmoc amino acid). Amino acid side chain protection during synthesis was as follow: Ser (t-butyl), Asn (trityl), and Gln (trityl). Peptide G XI consisted of 12 amino acids at position 30–41 of α-gliadins (amino acid sequence: FPGQQQPFPPQQ) and peptide G XIV consisted of 12 amino acids at position 8–19 (LQPQNPSQQQPQ) (20). Zein peptide Z I (= control) was 14 amino acids long and covered the sequence of nontoxic zein SF4 at position 8–21 (sequence: LAPSAIIPQFLPPV). Crude peptides were checked for amino acid sequences with a pulsed liquid sequencer (model 471 A; Applied Biosystems). Biotinylation of synthetic peptides. Synthesized crude peptides (10.0 mg) wer (...truncated)