Gliadin Preparations Research Articles

Heterogeneous reactivity patterns of HLA-DQ-restricted, small intestinal T-cell clones from patients with celiac disease

BACKGROUND & AIMS: T-cell immune reactions toward wheat gliadins seem important in the pathogenesis of celiac disease. We have previously shown that gliadin-specific T-cell clones (TCCs) from the small intestinal mucosa of patients with celiac disease are predominantly restricted by the celiac disease-associated HLA-DQ2 and HLA-DQ8 molecules, suggesting a link between the HLA association and immunopathogenesis. The aim of the present study was to investigate the nature of the stimulating gliadin antigens.METHODS: Three different pools of gliadins and one purified alpha-type and two purified gamma- type gliadin preparations were prepared by ion exchange chromatography and tested for recognition by a panel of small intestinal gliadin- specific TCCs.RESULTS: Evidence suggested that enzymatic digestion and heating of the gliadins influenced TCC stimulation. Most of the TCCs recognized all three gliadin pools, but some had distinct reactivity patterns. Thirteen of 21 TCCs responded to one or more of the three purified gliadins discerning highly discriminative patterns.CONCLUSIONS: Small intestinal, gliadin-specific T cells from patients with celiac disease show diverse reactivity patterns. Stimulation of large numbers of different gliadin-specific T cells in the small intestinal mucosa of patients with celiac disease may occur; this may be an important immunopathogenic step in the disease.(Gastroenterology 1997 Mar;112(3):752-9)

Hydrophobic interactions between gliadin and proteins and celiac disease.

Gliadin-protein interaction and its relationship to the pathogenesis hypotheses of celiac disease was investigated. Wheat germ agglutinin was not immunodetected in gliadin preparations. Peptic-tryptic gliadin digest was used to study the gliadin-protein interactions by crossed immunoelectrophoresis and affinity blotting. Biotinylated gliadin digest interacted with IgG and bovine serum albumin but not with several glycoproteins. Since albumin and IgG light chains are not glycosylated, this interaction is not lectin-like, neither completely immunological because of recognition of the IgG Fc fraction. Immobilized and boiled IgG was not recognized by gliadin digest as a lectin. Gliadin digest fractions from T-gel chromatography reduced the fluorescence intensity of cis-parinaric acid bound to albumin. The gliadin-protein interaction is not lectin-like or completely immunological but hydrophobic. Hydrophobicity of gliadins may contribute to the pathogenic events that result in celiac disease.

Protein–Lipid Interactions in Wheat Gluten: Reassessment of the Occurrence of Lipid-Mediated Aggregation of Protein in the Gliadin Fraction

Gliadin preparations, isolated by AUC extraction and pH precipitation from undefatted- and water-saturated butan-1-ol (WSB) defatted cv. Neepawa gluten, were chromatographed on Sephacryl S-200, resulting in separation into five peaks, one at the void volume (Vo) and the other four included by the resin. Defatting resulted in a diminishedVopeak, but the other peaks were unaffected. Electrophoretic analysis (SDS–PAGE) showed that theVofraction contained both high and lowMrsubunits of glutenin but very little gliadin except for traces of ω-gliadins. Re-chromatography of the Sephacryl S-200Vofraction from undefatted gliadin and chromatography of whole undefatted gliadin on a larger pore size Sephacryl S-400 column resulted in the separation of two apparently UV absorbing fractions, one at theVo, and an included fraction that was eluted over a broad area of the chromatogram following theVopeak. In the case of whole undefatted gliadin, several overlapping peaks corresponding to lowerMrproteins were also observed. The Sephacryl S-400Vopeak from both the gliadin preparation and the re-chromatographed Sephacryl S-200Vofraction were removed by defatting, and no protein was detected in those Sephacryl S-400Vofractions. The components included by Sephacryl S-400, which were eluted over a broad range in the chromatogram, contained high and lowMrsubunits of glutenin, and the later peaks in the gliadin chromatograms contained gliadin polypeptides and lowerMrproteins. No evidence was obtained for the occurrence of lipid-mediated aggregation of gluten proteins in gliadin isolated by the pH precipitation procedure. The material that can be removed by defatting with WSB (presumably lipid) may be present in the form of large vesicles, which are detected as an apparentVopeak because of light scattering.

Characterization of gliadin-galactomannan incubation mixtures by analytical ultracentrifugation—Part I. Sedimentation velocity

The aim of this work is to examine the possible interaction and extent thereof of the polysaccharide galactomannan (GAL) with the cereal protein gliadin (GLI) and a peptic-tryptic degraded gliadin (PT-GLI) by analytical ultracentrifugation. The work is part of a series of investigations into the field of coeliac disease (gluten-induced enteropathy) as gliadins are known to be toxic for patients with this disease. The molecular integrity of the GAL and GLI preparations was first checked by sedimentation velocity and sedimentation equilibrium. Sedimentation velocity showed single boundaries indicating homogeneity and low-speed sedimentation equilibrium gave plausible apparent weight average molar masses of 180,000 g/ mol for GAL and 20,000 g/mol for GLI. PT-GLI, GLI and GAL in phosphate buffer (pH 6.5) and the incubated mixtures (stirred for 3 h at 37 °C; PT-GLI: GAL = 3.53:1, wt.wt.; GLI:GAL = 0.23 and 0.55:1, wt.wt.) were then investigated by sedimentation velocity at a temperature of 20 °C. The plots of 1 s 20 vs. c of GAL, PT-GLI-GAL and GLI-GAL mixtures after incubation show a significantly different shape suggesting the presence of interactions. According to the equation 1 s 20 = 1 s o 20 (1 + k sc) , values for { s o 20, k s } of {(4.02 ± (490.9 ± 28.9) ml/ g, {(5.92 ± 0.24) S, (1152 ± 44) ml/g} and {(5.38 ± 0.19) S, (1141 ± 38) ml/g} for GAL and PT-GLI-GAL and GLI-GAL mixtures, respectively, were obtained. The concentration of GAL ranged from 0.75–3.0 mg/ml for GAL alone and from 0.34–1.50 mg/ml in the incubated mixtures. This apparent indication for a weak non-covalent protein-polysaccharide interaction was further supported by UV absorption spectrometry and gel filtration.

Comparative Studies of Different Gliadin Preparations in Detecting Antigliadin Antibodies

SummaryAntigliadin antibodies (AGA) have been used as indicators of celiac disease. The presence of these antibodies in other gastrointestinal and liver disorders and even in normal healthy controls casts a shadow on the diagnostic significance of AGA. We examined 91 normal controls of varying ages and 97 patients with various gastrointestinal and liver disorders. Forty‐eight of 97 nonceliac patients were positive for AGA, and diagnosis‐specific incidences ranged as high as 75% in patients with small bowel disease. In addition, the levels of AGA were dependent upon age as their presence increased from 12% in children with a mean age of 10, to 35–40% in normals within the 60–70 age group. The lack of celiac disease specificity of AGA was not due to either the source of gliadin nor to the sensitivities of the test methods. Both the enzyme‐linked immunosorbent assay (ELISA) and the immunofluorescence methods gave comparable results. ELISA was more sensitive than immunofluorescence. These results thus strongly suggest that AGA are not markers of celiac disease and increase with age in normals.

Reappraisal of the 'lectin hypothesis' in the aetiopathogenesis of coeliac disease.

The agglutinating properties of a crude gluten digest, purified gliadin fractions and established plant lectins were investigated using mammalian erythrocytes, rat enterocytes and normal and coeliac human enterocytes as the target systems. Gliadin preparations failed to cause agglutination of any of the cells tested, whereas established pure plant lectins were active cell agglutinins. These studies indicate that gliadin peptides do not interact with intestinal cells in a polyvalent, lectin-like manner and as such cannot be regarded as true lectins. Mucosal damage in coeliac disease is unlikely therefore to be related to lectin-like activity of gliadin.

Fractionation of water-insoluble protein using sephacryl S-200 in formamide.

The ability of Sephacryl S-200 to fractionate water-insoluble protein by gel permeation chromatography was investigated. Elution of protein standards from colums of Sephacryl S-200 equilibrated with formamide, an excellent solvent for both hydrophobic and hydrophilic proteins, was molecular weight dependent. Advantages of chromatography of water-insoluble proteins using Sephacryl S-200 include rapidity, recovery of protein free of detergent or solvent, safety, ability to purify large amounts of protein, and ability to separate proteins as large as 100,000 daltons. Separation of water-insoluble polypeptides in a crude preparation of gliadin in formamide demonstrated the practicality of the method.

Characterisation of three wheat proteins found in chloroform‐methanol extracts of flour

AbstractThree wheat proteins, CM1, CM2 and CM3 constituting in total more than 1% of flour proteins, have been isolated from chloroform—methanol (2:1 v/v) extracts of flour. CM1 and 2 have single chains with m.w. of 13 000 and isoleucine as the C‐terminal amino acid. They appear to be closely related even in amino acid sequence as judged from fingerprints. All three proteins are soluble in chloroform—methanol after freeing from lipid, but only moderately so in water. CM3 becomes insoluble after reduction of SS bonds due to aggregation of the reduced poly‐peptide chains by secondary forces. It differs appreciably from CM1 and 2 in sequence and has C‐terminal leucine and a m.w. of 15 000.The mobilities of CM2 and 3 at pH 3.2 are very close to ALB 13A and ALB 13B, respectively, and the albumins found in electrophoretic patterns of crude gliadin preparations do in fact represent mixtures of both classes.

Preparation of gliadin by urea extraction

AbstractA method in which, after a preliminary water extraction, all the gluten proteins in flour may be extracted by 2M urea is described. The protein so extracted is mainly gliadin, relatively free from high‐molecuiarweight glutenin. Gel filtration with Bio Gel P‐150 polyacrylamide beads and electrophoresis in starch gel of varying porosity shows that the molecular weight distribution is relatively narrow. It is suggested that, under some conditions, an unidentified compound of low molecular weight is responsible for the intermolecular crosslinking of gliadin molecules to give high‐molecular‐weight glutenin.

Preparation of Gliadin and Zein.

SummaryMethods are described for the preparation of gliadin from wheat and of zein from corn (maize). The products are of a grade suitable for many types of nutrition investigations.

A COMPARISON OF GLUTENIN AND GLIADIN PREPARED FROM ONE FLOUR BY VARIOUS METHODS

Gliadin and glutenin were prepared from gluten of hard red spring wheat flour by five different methods and analyzed by the Van Slyke procedure. The gliadin preparations gave very similar results, indicating that differences in manipulation have little effect on this protein. The glutenin preparations showed very great differences in nitrogen distribution, the greatest being in ammonia nitrogen and in basic nitrogen. There is considerable evidence that these two nitrogen fractions tend to be inversely proportional. This is particularly applicable to preparations made by methods involving use of alkaline solutions. Glutenin isolated by use of Blish and Sandstedt's acetic acid method, using 0.007 N acid appeared to be purer than the other preparations and contained the highest percentage of ammonia nitrogen. This protein, when dissolved in 0.025 N sodium hydroxide solution and allowed to stand for one week, lost 4.8% of its nitrogen in the form of ammonia, but the basic fraction was unchanged. It seems likely that any method for isolating glutenin involving use of alkaline solutions would involve loss of nitrogen in the course of the preparation and the nitrogen distribution obtained by the Van Slyke analysis would therefore be in error. The use of very dilute acetic acid is recommended but exposure to even dilute solutions of the strong alkalies should be rigorously avoided in the preparation of glutenin.