HYDROPHOBIC INTERACTIONS BETWEEN GLIADIN AND PROTEINS AND CELIAC DISEASE (1)
Celiac disease(CD) is characterized by damage of the small intestinal mucosa developed by the presence of the gliadin fraction of wheat gluten. CD is most probably a T-cell mediated immunologic disease, and CD is caused by a direct toxic effect on the intestinal mucosa. Among gliadms intrinsic properties are protease resistance, repeated amino acid domains and hydrophobicity.
Gluten was prepared from commercial wheat flour. Gliadins were obtained by 70% ethanol extraction from our laboratory prepared and commercial gluten. Extracts were centrifuged at 4000 xg, 20 min, at 4°C; and the supernatant was dialyzed against 0.01 N acetic acid before freeze-drying. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 17% polyacrylamide gels.
Wheat Germ Agglutinin was not detected in commercial gluten or in our laboratory preparation. The two gliadin preparations have a similar pattern, with polypeptides sizes in two ranges; around 15 kDa corresponding to a-gliadins and between 30-55 kDa to y-gliadins.
Digested gliadins interacted weakly with BSA and strongly with both light and heavy IgG chains but not with IgM. Digested gliadins interacted also with the Fc fraction of IgG. The binding of Gd-IgG heavy chain, Gd-IgG light chain and IgG Fc fraction were not inhibited by carbohydrates.
The IgG glycosidic moiety was recognized by lectins both in unboiled or boiled membranes. In contrast, gliadin digest interacted only with the unboiled IgG, demonstrating no lectin-like interaction with the IgG carbohydrate structure. Anti-IgG reacted with human IgG as an antigen even in the denatured conformation. The majority of the CD patients presented circulating IgA antibodies against a 55 kDa aggregate of digested gliadins. This 55 kDa aggregate was also the most common antigen recognized by IgG antibodies from CD patients as well as from healthy individuals.
Approximately 86% of the polypeptides were found in the unbound fraction (FI) when the supernatant of the 1 M ammonium sulfate precipitation of gliadin digest (Fs) was applied to the T-Gel (g-mercaptoethanol coupled to divinylsulphone-activated agarose) column. The main bound fraction (FII) eluted with 0.08 M (NH4)2SO4 was composed of high molecular weight aggregates and corresponds to 9.5% of the Fs loaded. The remaining protein (FIII) was eluted with water. The bulk of the material was under 25 kDa by SDS-PAGE for fresh-resuspended freeze-dried gliadin digest (Gd). Aggregation of Gd was strongly increased by high concentration of NaCl or by (NH4)2SO4.
Gd addition led to a 60% reduction in the slope of CPA fluorescence intensity against BSA concentration. Exposure of BSA to different Gd fractions caused different changes in the fluorescence of CPA. FII fraction was the most hydrophobic fraction, and gave the larger reduction in the fluorescence intensity of CPA bound to BSA.
Although IgA antigliadin antibodies are specific for CD, both celiac and healthy individuals present IgG antigliadin antibodies. Gliadin digest-IgG interaction is not completely immunological but hydrophobic, because of the gliadin binding to the IgG Fc fraction.