Structural conformation and self-assembly process of p31-43 gliadin peptide in aqueous solution. Implications for celiac disease.

Celiac disease (CeD) is a highly prevalent chronic immune-mediated enteropathy developed in genetically predisposed individuals after ingestion of a group of wheat proteins (called gliadins and glutenins). The 13mer α-gliadin peptide, p31-43, induces proinflammatory responses, observed by in vitro assays and animal models, that may contribute to innate immune mechanisms of CeD pathogenesis. Since a cellular receptor for p31-43 has not been identified, this raises the question of whether this peptide could mediate different biological effects. In this work, we aimed to characterize the p31-43 secondary structure by different biophysical and in silico techniques. By dynamic light scattering and using an oligomer/fibril-sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, atomic force microscopy analysis showed p31-43 oligomers with different height distribution. Also, peptide concentration had a very strong influence on peptide self-organization process. Oligomers gradually increased their size at lower concentration. Whereas, at higher ones, oligomers increased their complexity, forming branched structures. By CD, we observed that p31-43 self-organized in a polyproline II conformation in equilibrium with ß-sheets-like structures, whose pH remained stable in the range of 3-8. In addition, these findings were supported by molecular dynamics simulation. The formation of p31-43 nanostructures with increased ß-sheet structure may help to explain the molecular etiopathogenesis in the induction of proinflammatory effects and subsequent damage at the intestinal mucosa in CeD.

Genetic diversity and relationships among accessions of five crested wheatgrass species (Poaceae: Agropyron) based on gliadin analysis.

Agropyron Gaertn. is the most important genus in Triticeae (Poaceae), which includes many forage grasses with high economic value. The genetic diversity and relationships of 36 accessions from five crested wheatgrass species were analyzed by gliadin markers. A total of 54 product bands were detected after acid polyacrylamide gel electrophoresis (A-PAGE), of which 100% were polymorphic. The genetic similarity coefficient based on Nei-Li's method ranged from 0.065 to 0.755 with an average of 0.451. The Shannon diversity information index showed that there was a high level of genetic diversity among the accessions. An unweighted pair group method with arithmetic average (UPGMA) dendrogram was constructed based on the Nei-Li's genetic similarity coefficients, which showed the phylogenetic relationships among accessions of different species. Analysis of molecular variance (AMOVA) showed that the proportion of variance explained by inter- and intraspecific variance was 9.34 and 90.66%, respectively, which revealed that the genetic variations within species were higher than the variations among species. Based on pairwise genetic distances (ΦST) among species, the cluster analysis indicated that A. mongolicum had a low-affinity relationship with other species, while A. fragile showed a close relationship with A. cristatum ssp pectinatum. Finally, the implications of the results for the taxonomy of Agropyron were discussed.

Isolation and phylogenetic analysis of novel γ-gliadin genes in genus Dasypyrum.

As the most ancient member of the wheat gluten family, the γ-gliadin genes are suitable for phylogenetic analysis among wheat and related species. Species in the grass genus Dasypyrum have been widely used for wheat cross breeding. However, the genomic relationships among Dasypyrum species have been little studied. We isolated 22 novel γ-gliadin gene sequences, among which 10 are putatively functional. The open reading frame lengths of these sequences range from 642 to 933 bp, and these putative proteins consist of five domains. Phylogenetic analyses showed that all Dasypyrum γ-gliadin gene sequences clustered in a large group; D. villosum and tetraploid D. breviaristatum γ-gliadin gene sequences clustered in a subgroup, while diploid D. breviaristatum γ-gliadin gene sequences clustered at the edge of the subgroup. All of the Dasypyrum γ-gliadin gene sequences were absent in three major T cell-stimulatory epitopes binding to HLA-DQ2/8 in celiac disease patients. Based on the phylogenetic analyses, we suggest that D. villosum and tetraploid D. breviaristatum evolved in parallel from a diploid ancestor D. breviaristatum.

Molecular cytogenetic characterization of a new leaf rolling triticale.

Leaf rolling occurs in some cereal genotypes in response to drought. We identified and made a phenotypic, cytological and physiological analysis of a leaf-rolling genotype (CMH83) of hexaploid triticale (X Triticosecale Wittmack) that exhibited reduced plant height, rolled and narrow leaves. Gliadin electrophoresis of seed protein showed that CMH83 was genetically stable. Sequential Giemsa-C-banding and genomic in situ hybridization showed that CMH83 contains 12 rye chromosomes; two pairs of these chromosomes have reduced telomeric heterochromatin bands. Tests of relative water content and water loss rate of leaves of CMH83 compared with those of wheat cultivars indicated that rapid water loss after drought stress in CMH83 is associated with the leaf rolling phenotypes. Leaf rolling in CMH83 was a dominant trait in our inheritance studies. Triticale line CMH83 could be used to study drought resistance mechanisms in triticale.

Linkage relationships between prolamin genes located on chromosome 1Hch in Hordeum chilense.

The endosperm storage proteins of Hordeum chilense Roem. et Schult., a species used in the synthesis of the amphiploid tritordeum (x Tritordeum Ascherson et Graebner), have a great effect on the gluten strength of this amphiploid. We have analysed electrophoretically the heredity of these proteins, which are synthesised by genes located on chromosome 1H(ch), and detected up to five loci in a cross between two lines of H. chilense. These loci present a certain homology with loci synthesising the same proteins in wheat. The genetic distances between these loci were calculated.