Dynamic Evolution of α-Gliadin Prolamin Gene Family in Homeologous Genomes of Hexaploid Wheat.

Wheat Gli-2 loci encode complex groups of α-gliadin prolamins that are important for breadmaking, but also major triggers of celiac disease (CD). Elucidation of α-gliadin evolution provides knowledge to produce wheat with better end-use properties and reduced immunogenic potential. The Gli-2 loci contain a large number of tandemly duplicated genes and highly repetitive DNA, making sequence assembly of their genomic regions challenging. Here, we constructed high-quality sequences spanning the three wheat homeologous α-gliadin loci by aligning PacBio-based sequence contigs with BioNano genome maps. A total of 47 α-gliadin genes were identified with only 26 encoding intact full-length protein products. Analyses of α-gliadin loci and phylogenetic tree reconstruction indicate significant duplications of α-gliadin genes in the last ~2.5 million years after the divergence of the A, B and D genomes, supporting its rapid lineage-independent expansion in different Triticeae genomes. We showed that dramatic divergence in expression of α-gliadin genes could not be attributed to sequence variations in the promoter regions. The study also provided insights into the evolution of CD epitopes and identified a single indel event in the hexaploid wheat D genome that likely resulted in the generation of the highly toxic 33-mer CD epitope.

Biochemical and functional properties of wheat gliadins: a review.

Gliadins account for 40-50% of the total storage proteins of wheat and are classified into four subcategories, α-, ß-, γ-, and ω-gliadins. They have also been classified as ω5-, ω1, 2-, α/ß-, and γ-gliadins on the basis of their primary structure and molecular weight. Cysteine residues of gliadins mainly form intramolecular disulfide bonds, although α-gliadins with odd numbers of cysteine residues have also been reported. Gliadins are generally regarded to possess globular protein structure, though recent studies report that the α/ß-gliadins have compact globular structures and γ- and ω-gliadins have extended rod-like structures. Newer techniques such as Mass Spectrometry with the development of matrix-assisted laser desorption/ionization (MALDI) in combination with time-of-flight mass spectrometry (TOFMS) have been employed to determine the molecular weight of purified ω- gliadins and to carry out the direct analysis of bread and durum wheat gliadins. Few gliadin alleles and components, such as Gli-B1b, Gli-B2c and Gli-A2b in bread wheat cultivars, γ-45 in pasta, γ-gliadins in cookies, lower gliadin content for chapatti and alteration in Gli 2 loci in tortillas have been reported to improve the product quality, respectively. Further studies are needed in order to elucidate the precise role of gliadin subgroups in dough strength and product quality.

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.

Expansion of the gamma-gliadin gene family in Aegilops and Triticum.

BACKGROUND: The gamma-gliadins are considered to be the oldest of the gliadin family of storage proteins in Aegilops/Triticum. However, the expansion of this multigene family has not been studied in an evolutionary perspective. RESULTS: We have cloned 59 gamma-gliadin genes from Aegilops and Triticum species (Aegilops caudata L., Aegilops comosa Sm. in Sibth. & Sm., Aegilops mutica Boiss., Aegilops speltoides Tausch, Aegilops tauschii Coss., Aegilops umbellulata Zhuk., Aegilops uniaristata Vis., and Triticum monococcum L.) representing eight different genomes: Am, B/S, C, D, M, N, T and U. Overall, 15% of the sequences contained internal stop codons resulting in pseudogenes, but this percentage was variable among genomes, up to over 50% in Ae. umbellulata. The most common length of the deduced protein, including the signal peptide, was 302 amino acids, but the length varied from 215 to 362 amino acids, both obtained from Ae. speltoides. Most genes encoded proteins with eight cysteines. However, all Aegilops species had genes that encoded a gamma-gliadin protein of 302 amino acids with an additional cysteine. These conserved nine-cysteine gamma-gliadins may perform a specific function, possibly as chain terminators in gluten network formation in protein bodies during endosperm development. A phylogenetic analysis of gamma-gliadins derived from Aegilops and Triticum species and the related genera Lophopyrum, Crithopsis, and Dasypyrum showed six groups of genes. Most Aegilops species contained gamma-gliadin genes from several of these groups, which also included sequences from the genera Lophopyrum, Crithopsis, and Dasypyrum. Hordein and secalin sequences formed separate groups. CONCLUSIONS: We present a model for the evolution of the gamma-gliadins from which we deduce that the most recent common ancestor (MRCA) of Aegilops/Triticum-Dasypyrum-Lophopyrum-Crithopsis already had four groups of gamma-gliadin sequences, presumably the result of two rounds of duplication of the locus.

The wheat omega-gliadin genes: structure and EST analysis.

A survey and analysis is made of all available omega-gliadin DNA sequences including omega-gliadin genes within a large genomic clone, previously reported gene sequences, and ESTs identified from the large wheat EST collection. A contiguous portion of the Gli-B3 locus is shown to contain two apparently active omega-gliadin genes, two pseudogenes, and four fragments of the 3' portion of omega-gliadin sequences. Comparison of omega-gliadin sequences allows a phylogenetic picture of their relationships and genomes of origin. Results show three groupings of omega-gliadin active gene sequences assigned to each of the three hexaploid wheat genomes, and a fourth group thus far consisting of pseudogenes assigned to the A-genome. Analysis of omega-gliadin ESTs allows reconstruction of two full-length model sequences encoding the AREL- and ARQL-type proteins from the Gli-A3 and Gli-D3 loci, respectively. There is no DNA evidence of multiple active genes from these two loci. In contrast, ESTs allow identification of at least three to four distinct active genes at the Gli-B3 locus of some cultivars. Additional results include more information on the position of cysteines in some omega-gliadin genes and discussion of problems in studying the omega-gliadin gene family.

Characterization of alpha-gliadin genes from diploid wheats and the comparative analysis with those from polyploid wheats.

To carry out the comparative analysis of alpha-gliadin genes on A genomes of diploid and polyploid wheats, 8 full-length alpha-gliadin genes, including 3 functional genes and 5 pseudogenes, were obtained from diploid wheats, among which 2, 2 and 4 alpha-gliadin genes were isolated from T. urartu, T. monococcum and T. boeoticum, respectively. The results indicated that higher number of alpha-gliadin pseudogenes have been present in diploid wheats before the formation of polyploid wheats. Amino acid sequence comparative analysis among 26 alpha-gliadin genes, including 16 functional genes and 10 pseudogenes, from diploid and polyploid wheats was conducted. The results indicated that all alpha-gliadins contained four coeliac toxic peptide sequences (i.e., PSQQ, QQQP, QQPY and QPYP). The polyglutamine domains are highly variable, and the second polyglutamine stretch is usually disrupted by the lysine or arginine residue at the fourth position. The unique domain I is the most conserved domain. There are 4 and 2 conserved cysteine residues in the unique domains I and II, respectively. Comparative analysis indicated that the functional alpha-gliadin genes from A genome are highly conserved, whereas the identity of pseudogenes in diploid wheats are higher than those in hexaploid wheats. Phylogenetic analysis indicated that all the analyzed functional alpha-gliadin genes could be clustered into two major groups, among which one group could be further divided into 5 subgroups. The origin of alpha-gliadin pseudogene and functional genes were also discussed.

[Biochemical and molecular characterization of gliadins].

Gliadins account for about 40-50% of the total proteins in wheat seeds and play an important role on the nutritional and processing quality of flour. Usually, gliadins could be divided into alpha- (alpha/beta-), gamma- and omega-groups, whereas the low-molecular-weigh (LMW) gliadins were novel seed storage proteins. The low-molecular-weight glutenin subunits (LMW-GSs) were also designated as gliadins in a few literatures. The genes encoding gliadins were mainly located on the short arms of group 6 and group 1 chromosomes, and not evenly distributed. Repetitive sequences covered most of un-coding regions, which attributed greatly to the evolution of wheat genome. Primary structure of each gliadin has been divided into several domains, and the long repetitive domains consisted of peptide motifs. Conserved cysteine residues mainly formed intramolecular disulphide bonds. The rare potential intermolecular disulphide bonds and the long repetitive domains played an important role in the wheat flour quality. There was a general idea that gliadin genes, even prolamin genes, have a common origin and subsequent divergence lead to the gene polymorphism. The gamma-gliadins have been considered to be the most ancient of the wheat prolamin family. Several elements in the 5'-flanking (e.g. CAAT and TATA box) and the 3'-flanking sequences had been detected, which had been shown necessary for the proper expression of gliadins.

[Isolation and sequence analysis of a omega-gliadin homologous gene from wheat].

The DNA sequence of a full-length Triticum astivum CV. Jinan 177omega-gliadin homologous gene (omega1236) containing partial 5' and 3' flanking sequences with no intron was cloned by genomic PCR-based technology. The omega1236 sequence possibly encode a putative 47.2 kDa protein except for eight stop codons at amino acid residue positions 87, 117, 125, 157, 198, 313, 357 and 365 respectively. All the eight stop codons were caused by base transition. Sequence analysis revealed that omega1236 had 98% homology to a omega-gliadin gene of wheat (AB059812). Like all other gliadin gene families characterized in cereals, this gene possessed all the features in other plant reported previously. Phylogenetic analysis of the completely sequenced gene as well as those omega-genes in wheat, omega-secalin and C-horden genes in rye and barley, and alpha-, beta- and gamma-gliadin genes in wheat indicated that the omega1236 was more closely related to omega-gliadin gene family, much less homology to alpha- , beta- and gamma-gliadin gene families. Short peptide was produced in the culture of transformed E. coli induced by IPTG in early 2 h. It indicated that stop codon would be in omega1236. The result is consistent with that of the sequenced gene. The present paper could accumulate data useful for both omega-gliadin gene cloning by PCR and the study on structures and functions of these genes.

High microbial production and characterization of strictly periodic polymers modelled on the repetitive domain of wheat gliadins.

Primary structures of wheat prolamins contain repetitive domains involved in the mechanical properties of gluten. In order to experience the ability of recombinant strictly periodic polypeptides, modelled on a consensus sequence of wheat gliadins (PQQPY)(8) and (PQQPY)(17) (SPR8 and SPR17 polypeptides, respectively), to be formulated in film solutions, their heterologous expression conditions, in batch culture and low cell densities, were optimized to match the high requirements of this process. A convenient and general purification procedure was also devised. Moreover, FTIR-ATR characterizations indicated that these periodic polypeptides prepared as hydrated doughy state and dried have the tendency to form a protein network through intermolecular beta-sheets, strongly maintained by hydrogen bonds. Accordingly, these recombinant polypeptides are assumed to be a suitable candidate for potential application.

[Allergenicity of wheat flour]. / Allergénicité de la farine de blé.

Food allergy to wheat flour is a pathology that is found less frequently than coeliac disease or respiratory allergy to flour; it seems however to be a constant argument. Our study used a panel of 28 patients diagnosed with food allergy to wheat flour. Our objective was to characterise the reactivity of type IgE and IgG antibodies of these patients with regard to the different classes of proteins of wheat flour so as to establish an antigenic profile of the allergens of wheat in the framework of food allergy to flour. Our results show the implication of different classes of wheat proteins and notably the major reserve proteins (gliadins and glutens) in food allergy.

Determination of disulfide bonds in gamma-46 gliadin.

The disulfide bonds in gamma-46 gliadin were identified: Cys173--Cys192, Cys212--Cys291, Cys165--Cys199 (or Cys200), Cys283--Cys200 (or Cys199). The disulfide-containing peptides were obtained by limited hydrolysis of the intact protein with chymotrypsin at an enzyme/substrate ratio of 1:1000 at 20 degrees C for 22 h with subsequent digestion of disulfide-containing fragments with trypsin and chymotrypsin. The locations of disulfide bonds were determined by sequencing disulfide-containing fractions and constituent peptides and comparison of the obtained sequences with the partial amino acid sequence of gamma-46 gliadin determined earlier.

Specificity of antigliadin antibody in celiac disease.

Celiac disease is activated in genetically susceptible individuals by the dietary ingestion of wheat gluten and similar proteins in other grains. Gliadins are a complex mixture of proteins that contain at least 40 different components in a single variety of wheat. We have purified the four major electrophoretic fractions of wheat gliadin and examined the specificity of antigliadin antibody for those fractions by radioimmunoassay in 30 patients with celiac disease and 30 matched controls. All patients had been on a gluten-free diet for more than 18 mo and were clinically asymptomatic at the time of study. Seventeen of 30 patients had increased antibody levels to one or more of the gliadin fractions. Twelve of 17 patients had elevated antibody to A or 6D alpha-gliadin, 9 of 17 to beta-gliadin, 10 of 17 to gamma-gliadin, and 8 of 17 to omega-gliadin. Of the 17 subjects, 5 had increased antigliadin antibody levels to one gliadin fraction only, whereas 12 had increased levels to two or more fractions. Of the 17 patients with increased antibody titers, 16 had the G2m(n) immunoglobulin heavy chain allotype marker and 14 had the serologic HLA specificities-B8 or-DR3, or both. Definition of the wheat gliadin fractions and specific gliadin peptides that can activate celiac disease remains an open question. These data indicate that antigliadin antibody in the serum of asymptomatic patients with celiac disease who are maintained on a gluten-free diet can be directed against one or a multiple of gliadin fractions.

A solid-phase radioimmunoassay for measurement of circulating antibody titres to wheat gliadin and its subfractions in patients with adult coeliac disease.

A solid-phase radioimmunoassay for the measurement of circulating antibody titres to wheat gliadin is described. Using this assay, we have measured antibody titres to unfractionated gliadin in normal healthy controls, in coeliac patients on a gluten-free or a normal diet, and in patients with ulcerative colitis and Crohn's disease. High titres of antibodies to unfractionated gliadin were observed only in the patients with untreated coeliac disease. Antibody titres to alpha, beta, gamma and omega gliadin subfractions were measured in patients with untreated coeliac disease and compared with titres in normal controls. Patients with untreated coeliac disease had higher antibody titres to the gliadin subfractions. No specific pattern of circulating antibody titres to gliadin subfractions was observed in the untreated coeliac patients which would provide a diagnostic profile. These results suggest shared antigenicity between the gliadin subfractions.