Cross linking to tissue transglutaminase and collagen favours gliadin toxicity in coeliac disease.

BACKGROUND AND AIMS: Intestinal inflammation in coeliac disease is driven by the gluten fraction of wheat proteins. Deamidation or cross linking of gluten peptides by tissue transglutaminase (tTG), the coeliac disease autoantigen, creates potent T cell stimulatory peptides. Therefore, our aim was to identify the reaction patterns of gluten peptides, intestinal extracellular matrix proteins, and tTG. METHODS: tTG activity was analysed by incorporation of monodansyl cadaverine into gliadins. Fluorescence labelled tTG reactive short gliadin peptides were used to demonstrate their deamidation and explore their cross linking patterns with tTG itself or extracellular matrix proteins. Patient sera and controls were checked for autoantibodies to matrix proteins. RESULTS: Gliadins alpha1-alpha11, gamma1-gamma6, omega1-omega3, and omega5 were substrates for tTG. tTG catalysed the cross linking of gliadin peptides with interstitial collagen types I, III, and VI. Coeliac patients showed increased antibody titres against the collagens I, III, V, and VI. CONCLUSIONS: tTG formed high molecular weight complexes with all tested gliadins. As all tested gliadins were substrates for tTG, the tTG catalysed modifications were not restricted to single gliadin types and epitopes. Furthermore, haptenisation and long term immobilisation of gliadin peptides by tTG catalysed binding to abundant extracellular matrix proteins could be instrumental in the perpetuation of intestinal inflammation and some associated autoimmune diseases in coeliac disease.

Enhanced peptide-binding capacities of small intestinal brush border membranes in celiac disease.

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 alpha-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/microg (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.

Comparative investigations of partial amino acid sequences of prolamins and glutelins from cereals. VIII. Amino acid sequences of glutelin peptides.

The peptide fractions isolated from chymotryptic hydrolysates of wheat, rye and barley glutelins were separated by high-performance liquid chromatography on octadecyl silica gel. The peptides obtained were analysed for their amino acid composition and some also for their amino acid sequence. Characteristic sequences of peptides from wheat glutelin can be grouped into three types. The first type contains a high amount of Gly and frequently Tyr in the N-terminal positions. A typical partial sequence, which occurs repeated in two peptides, is QGQQPGQGQ. Sequences of this type are found in high-molecular-weight subunits. The second type is characterized by the sequence SQn (n = 3-5) followed by a hydrophobic tripeptide e.g., PPF, PVL; the most frequent sequence is SQQQQPPF. Low-molecular-weight subunits contain sequences of this type. The third type, which has partial sequences such as QQPQQPFP, corresponds to typical peptides from prolamin. Most sequences of peptides from rye and barley glutelins can be divided into two groups. The predominant type shows prolamin-like sequences, e.g., PQQPXPQQ with X being F or I. The second type is similar to glycine-rich peptides from wheat glutelin, except that repeating sequences are less frequent.

High-performance liquid chromatography of reduced glutenin: amino acid composition of fractions and components.

Reduced glutenin is separated by gel permeation high-performance liquid chromatography into three major and five minor fractions, which significantly differ in their amino acid compositions. By reversed-phase high performance liquid chromatography, about 20 glutenin components are obtained. These can be classified into three groups according to their amino acid compositions: a hydrophilic group with relatively high values of Glx and Phe, a more hydrophobic group with a high content of Gly, and a strongly hydrophobic group with higher values of Val and Leu. Groups 1, 2 and 3 contain middle-, high- and low-molecular-weight (MMW-, HMW-, LMW-) subunits respectively.

[Comparative study of partial amino acid sequences of prolamins and glutelins from various cereals. VII. Amino acid sequences of prolamin peptides]. / Vergleichende Untersuchungen über partielle Aminosäuresequenzen von Prolaminen und Glutelinen verschiedener Getreidearten. VII. Aminosäuresequenzen von Prolaminpeptiden.

The peptide fractions isolated from chymotryptic hydrolysates of wheat, rye and barley prolamines [this journal (1984) 178:173] were separated into pure peptides by high-performance liquid chromatography on octadecyl silica gel. The peptides which were most abundant were analyzed for amino acid composition and in part for amino acid sequence. Besides peptides which are typical for only one of the cereals investigated, peptides of similar composition were found in all three prolamines. These contain repeating sequences and are built up of mainly Gln (Q), Pro (P) and hydrophobic amino acids (X) such as Phe, Tyr, Ile, Val and Leu. One of the most frequent partial sequences is QQPQQPXP.

[Comparative studies of partial amino acid sequences of prolamins and glutelins from various cereals. VI. Anion exchange chromatography of peptide fractions obtained by cation exchange chromatography]. / Vergleichende Untersuchungen über partielle Aminosäuresequenzen von Prolaminen und Glutelinen verschiedener Getreidearten. VI. Anionenaustauschchromatographie der durch Kationenaustauschchromatographie erhaltenen Peptidfraktionen.

The main peptide fractions obtained from prolamins and glutelins of wheat, rye, barley, and corn by chymotryptic hydrolysis, gel filtration and cation exchange chromatography [1, 2] were further separated by anion exchange chromatography. The amino-acid compositions of the peptides obtained from wheat, rye and barley prolamins are closely related. Typical compositions are Glx greater than 44, Pro greater than 28, and Phe greater than 7 mol-%. The peptide fractions from corn prolamine are different (Glx greater than 20, Leu greater than 20, Ala greater than 12, Pro greater than 10 mol-%). The peptide fractions from wheat, rye and barley glutelins are less similar. Typical compositions are Glx greater than 30, Pro greater than 15, and Gly greater than 8 mol-%. Most of the fractions from corn glutelin contain many amino-acids with Glx, Leu, and Ala predominating. Wheat differs significantly from the other cereals because of the unique composition of some high molecular weight peptide fractions from glutelin, which are rich in Glx (greater than 49) and Gly (greater than 19 mol-%).

[Tryptophan content of cereal proteins]. / Tryptophangehalt von Getreideproteinen.

The tryptophan contents of the albumin, globulin, prolamine and glutelin fractions from wheat, rye, barley, oat, sorghum, rice and maize were estimated by HPLC after alkaline hydrolysis. Complete amino acid compositions, including the amide contents, are given for all these protein fractions.

[Comparative investigations of partial amino acid sequences of prolamines and glutelins from cereals. II. Fractionation of glutelins (author's transl)]. / Vergleichende Untersuchungen über partielle Aminosäuresequenzen von Prolaminen und Glutelinen verschiedener Getreidearten. II. Fraktionierung der Gluteline.

Glutelins of wheat, rye, barley, oats, rice, sorghum and maize, which remain as residual proteins after extraction of defatted flours with water, salt solution and aqueous ethanol, were successively extracted with dilute acetic acid. Protein distribution and amino acid composition differ greatly with wheat, rye, barley, oats and rice and depend on the number of extraction steps and the concentration of acid used. The glutelins of sorghum and maize are only slightly soluble in acetic acid. In the case of barley glutelin fractions with very different compositions were obtained. The solubility of wheat and maize glutelins was examined in disulfide reducing and surface active agents. Wheat glutelin but not maize glutelin is soluble in HgCl2- and 2-mercaptoethanol solutions. A mixture of 2-mercaptoethanol and sodium dodecylsulfate is an excellent solvent for both glutelins.