Two-Dimensional Gel Mapping and N-Terminal Sequencing of LMW-Glutenin Subunits

Low-molecular-weight glutenin subunits from the wheat (Triticumaestivum L.) cultivar ‘Chinese Spring’ were mappedusing two dimensional electrophoresis (isoelectric focusingversus SDS-polyacrylamide gel electrophoresis). Protein spotswere electroblotted on to polyvinylidene difluoride membraneand characterized by N-terminal amino acid sequencing. Mostof the proteins that had accessible N-termini gave sequencesin agreement with known sequences for low-molecular-weight glutenins,-type gliadins, and -type gliadins. Two variants which havenot been seen in direct sequencing before were found. The gluteninstarting material was also reduced, alkylated, and partiallyfractionated by reverse-phase high performance liquid chromatography.Sequencing of these fractions, each of which contained a mixtureof proteins, confirmed the presence of the glutenin variantsfound in the spot sequencing. Key words: Glutenin, electroblotting, amino acid sequences     

In vitro assessment of acetic-acid-soluble proteins (glutenin) toxicity in celiac disease

Acetic-acid-soluble storage proteins from gluten of the bread wheat cv. Sprint 3 were fractionated by adsorption chromatography on 2000 Å controlled-pore glass (CPG) beads, and glutenin polymers with molecular mass higher than 10⁷ Da and free from monomeric gliadins were recovered. The glutenin polymers were found to consist of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits. Peptic-tryptic (PT) digests of glutenins were examined for their agglutination activity on human myelogenous leukemia K 562(S) cells, agglutination being strongly correlated with toxicity for the celiac intestine. The peptide fraction at a concentration of 1 g/L of culture medium was able to agglutinate 30% of K 562(S) cells, suggesting a moderate toxic effect. This toxicity may be accounted for by homologies in amino acid sequences between glutenin subunits and α/β-and γ-gliadins. © 1997 John Wiley & Sons, Inc.     

Characterization of low-molecular-weight glutenin genes in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

This paper reports the characterization of the low-molecular-weight (LMW) glutenin gene family of Aegilops tauschii (syn. Triticum tauschii), the D-genome donor of hexaploid wheat. By analysis of bacterial artificial chromosome (BAC) clones positive for hybridization with an LMW glutenin probe, seven unique LMW glutenin genes were identified. These genes were sequenced, including their untranslated 3 and 5 flanking regions. The deduced amino acid sequences of the genes revealed four putative active genes and three pseudogenes. All these genes had a very high level of similarity to LMW glutenins characterized in hexaploid wheat. The predicted molecular weights of the mature proteins were between 32.2 kDa and 39.6 kDa, and the predicted isoelectric points of the proteins were between 7.53 and 8.06. All the deduced proteins were of the LMW-m type. The organization of the seven LMW glutenin genes appears to be interspersed over at least several hundred kilo base pairs, as indicated by the presence of only one gene or pseudogene per BAC clone. Southern blot analysis of genomic DNA of Ae. tauschii and the BAC clones containing the seven LMW glutenin genes indicated that the BAC clones contained all LMW glutenin-hybridizing bands present in the genome. Two-dimensional gel electrophoresis of an LMW glutenin extract from Ae. tauschii was conducted and showed the presence of at least 11 distinct proteins. Further analysis indicated that some of the observed proteins were modified gliadins. These results suggest that the actual number of typical LMW glutenins may in fact be much lower than previously thought, with a number of modified gliadins also being present in the polymeric fraction.     

Gliadins polymerized with cysteine: effects on the physical and water barrier properties of derived films

To study the effects of disulfide bonds on certain functional properties of films made from the wheat gluten proteins gliadin and glutenin, cysteine was used to promote the formation of interchain disulfide bridges between gliadins in 70% ethanolic solution. Disulfide-mediated polymerization of gliadins was confirmed by means of SDS-PAGE analysis. After chemical treatment of gliadins, films were solution cast and the effects of both glycerol (used as a plasticizer) and relative humidity were studied on water vapor permeability, moisture sorption isotherms at 23 degrees C, and the optical properties of the films. The results were compared with those obtained from analogous films made from untreated glutenin macromolecules. Cysteine-mediated polymerization of gliadins improved the water vapor resistance of films achieving values close to those obtained for glutenin films. Development of intra- and interchain disulfide bonds did not change the moisture sorption capacity of the films but transparency was slightly diminished.     

Biochemical and molecular characterization of gliadins

Gliadins account for about 40–50% of the total proteins in wheat seeds and play an important role in the nutritional and processing quality of flour. Usually, gliadins can be divided into α-(α/β), γ-, and ω-groups, whereas the low-molecular-weight (LMW) gliadins are novel seed storage proteins. The low-molecular-weight glutenin subunits (LMW-GSs) are also designated as gliadins in a few publications. The genes encoding gliadins are mainly located on the short arms of group 6 and group 1 chromosomes, and not evenly distributed. Repetitive sequences cover most of the uncoding regions, which attributed greatly to the evolution of wheat genome. The primary structure of each gliadin is divided into several domains, and the long repetitive domains consist of peptide motifs. Conserved cysteine residues mainly form intramolecular disulfide bonds. The rare potential intermolecular disulfide bonds and the long repetitive domains play an important role in the quality of wheat flour. There is a general idea that gliadin genes, even prolamin genes, have a common origin and subsequent divergence leads to gene polymorphism. The γ-gliadins are 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 have been detected, which has been shown to be necessary for the proper expression of gliadins. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 796–807. The text was submitted by the authors in English.     

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.     

Characterization of gluten proteins and celiac disease-related immunogenic epitopes in the <Emphasis Type="Italic">Triticeae</Emphasis>: cereal domestication and breeding contributed to decrease the content of gliadins and gluten

Wheat proteins are important for the physico-chemical properties of bread-dough and contribute to the protein intake in the human diet. In certain individuals, an immunological reactivity of the gluten protein family is strongly implicated in the etiology of celiac disease (CD) and non-celiac wheat sensitivity (NCWS). There is evidence that gluten-related disorders have increased in frequency in recent years. Gluten proteins were characterized and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) while the occurrence of CD immunogenic epitopes was searched in the gliadin sequences of Triticeae within the NCBI database. We have observed a tendency toward low content of gliadins in cultivated species compared to that of the wild ancestors in all Triticeae members. Regarding the glutenin subunits, there was no clear trend, but levels tended to be higher in cultivated species. Thousand-kernel weight is higher for domesticated and cultivated species. Quantification of DQ2- and DQ8-restricted epitopes in gliadin sequences showed a great variability in the number of CD epitopes per species and genome. A higher frequency of immunnogenic epitopes was found to be associated with genomes of the DD, BBAADD, and RR type. Durum wheats tend to have a lower content of gluten and CD immunogenic epitopes. Cultivated barley could be an alternative cereal with low immunogenic epitopes and low gluten. The results reported in this study suggest that domestication and breeding have contributed to a decrease in the content of gliadins and total gluten in the Triticeae species over time.     

Accumulation of Gliadin and Glutenin Polypeptides during Development of Normal and Sulphur-deficient Wheat Seed: Analysis using Specific Monoclonal Antibodies

A panel of monoclonal antibodies with various specificitiesfor wheat (Triticum aestivum L.) gluten polypeptides has beenused to analyse the accumulation of these polypeptides in theendosperm of developing wheat seeds grown under normal and sulphur-deficientconditions. Immunoblots of polypeptides fractionated by SDS-PAGEallowed a qualitative analysis of gliadin and HMW glutenin accumulationfor high- and low-sulphur seeds 8 d to 30 d after anthesis (d.a.a.).In addition, quantitative analysis of the deposition of variousgluten polypeptides was performed, with a solid-phase radioimmunoassayon extracts of seeds harvested 4–36 d.a.a. The initialaccumulation of HMW glutenin subunits was detectable at an earlierstage of development than that of gliadins for both normal andsulphur-deficient seeds. The initiation of detectable gliadinaccumulation was asynchronous with an order of alpha-gliadins,beta-, gamma- and some omega-gliadins and finally the remainingomega-gliadins. In sulphur deficiency, all gliadins reacheda constant proportion of the dry weight of the endosperm earlierthan in normal wheat, while a more marked increase in the proportionof HMW glutenin occurred late in grain development. The proteinblot studies also identified a putative omega-gliadin polypeptidewhich was detectable late in seed development and only in sulphur-deficientseeds. Key words: Wheat, seed maturation, immunoassay     

Compositional Difference of Wheat Flour Glutens in Relation to Their Aggregation Behaviors

Comparison of the aggregation behavior of gluten was made by following the turbidity change of gluten suspension with four kinds of glutens prepared from flours different in flour quality. The four kinds of glutens showed different behaviors suggesting a relation to the flour quality. From the observation of the aggregation behavior of separated glutensis and gliadins and also of reconstituted glutens, it was concluded that the aggregation behavior of gluten is determined mainly by the nature of glutenin it contains. To investigate the difference of compositions of glutenins, component polypeptides of glutenin were fractionated by gel filtration into three fractions FI, FII and FIII after reduction and cyanoethylation of glutenin The glutenin from a strong flour was rich in FII, while the glutenin from a weak flour in FI. The observation of the aggregation behaviors of FI, FII and FIII suggested that the aggregation behavior of glutenin depends on the contents of these fractions.     

A second ‘overexpression’ allele at the <Emphasis Type="Italic">Glu-B1</Emphasis> high-molecular-weight glutenin locus of wheat: sequence characterisation and functional effects

Bread is one of the major constituents of the human diet and wheat (Triticum aestivum L.) is the most important cereal for bread making. The gluten proteins (glutenins and gliadins) are recognised as important components affecting the processing quality of wheat flour. In this research, we investigated a particular glutenin subunit allele in an Australian cultivar, H45. Based on protein and DNA assays, the Glu-B1 allele of H45 seems to be Glu-B1al, an allele that includes a functional duplication of a gene encoding an x-type high-molecular-weight glutenin subunit, and is thought to increase dough strength through overexpression of that subunit. Yet H45 does not have the dough properties that would be expected if it carries the Glu-B1al allele. After confirming that H45 overexpresses Bx subunits and that it has relatively low un-extractable polymeric protein (an indicator of weak dough), we cloned and sequenced two Bx genes from H45. The sequences of the two genes differ from each other, and they each differ by four single-nucleotide polymorphisms (SNPs) from the sequence that has been reported for the Glu-B1al x-type glutenin genes of the Canadian wheat cultivar Glenlea. One of the SNPs leads to an extra cysteine residue in one of the subunits. The presence of this additional cysteine may explain the dough properties of H45 through effects on cross-linkage within or between glutenin subunits. We propose that the Glu-B1 allele of H45 be designated Glu-B1br, and we present evidence that Glu-B1br is co-inherited with low un-extractable polymeric protein.     

The wheat γ-gliadin genes: characterization of ten new sequences and further understanding of γ-gliadin gene family structure

Ten new wheat γ-gliadin gene sequences are reported and an analysis of γ-gliadin gene family structure is carried out using all known γ-gliadin sequences. The new sequences comprise four genomic clones with significantly more flanking DNA than previously reported, and six cDNA clones from a wheat endosperm EST project. Analysis of extended flanking DNA from the genomic clones indicates the limits of conservation of γ-gliadin DNA sequence that are similar to those previously found with other gliadin and glutenin genes and that are theorized to define the DNA sequence necessary for gene control. Most of the flanking DNA is not homologous to any reported DNA sequence, and one flanking region contains the first MITE-like (miniature inverted transposable element) DNA sequence associated with gliadin genes. About a quarter of the encoded polypeptides would contain a free cysteine residue – an observation that may relate to reports that at least some gliadins can participate in wheat endosperm glutenin polymer formation. The new sequences represent both genes closely related to those previously reported and a new sub-class of γ-gliadins.     

Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin

Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations. The HMW subunit existed as a highly extended rod-like particle in solution with a length of about 69 nm and a diameter of about 6.4 nm. Specific aggregation effects were observed which may reflect mechanisms of self-assembly that contribute to the unique viscoelastic properties of wheat dough.     

Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing‐fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten‐free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α‐, γ‐, ω‐gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP‐HPLC and mass spectrometry (LC‐MS/MS), whereas gluten immunogenicity was assayed by an anti‐gliadin 33‐mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ‐ and α‐gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω‐gliadins and γ‐gliadins, and three of these also silenced α‐gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC‐MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α‐ and ω‐gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.     

Fractionation of wheat gliadin and glutenin subunits by two-dimensional electrophoresis and the role of group 6 and group 2 chromosomes in gliadin synthesis

Summary Subunits of wheat endosperm proteins have been fractionated by two-dimensional electrophoresis. To determine which subunits in the two-dimensional electrophoretic pattern belong to gliadin or glutenin the endosperm proteins have also been fractionated by a modified Osborne procedure and by gel filtration on Sephadex G-100 and Sepharose CL-4B prior to separation by two-dimensional electrophoresis.The control of production of five major grain protein subunits is shown to be determined by chromosomes 6A, 6B and 6D by comparing two-dimensional electrophoretic protein subunit patterns of aneuploid lines of the variety Chinese Spring. From these and previous studies it is concluded that some , and gliadins (molecular weights by SDS-PAGE 30,000 to 40,000) are specified by genes on the short arms of homoeologous Group 6 chromosomes, the gliadins (molecular weights by SDS-PAGE 50,000 to 70,000) are specified by genes on the short arms of homoeologous Group 1 chromosomes and the glutenin subunits (molecular weights by SDS-PAGE > 85,000) are specified by genes on the long arms of homoeologous Group 1 chromosomes.No major gliadins or glutenin subunits were absent when any of the chromosomes in homoeologous Groups 2, 3, 4, 5 or 7 were deleted. However two gliadins whose presumed structural genes are on chromosome 6D were absent in aneuploid stocks of Chinese Spring carrying two additional doses of chromosome 2A. Two out of thirty-three intervarietal or interspecific chromosome substitution lines examined, involving homoeologous Group 2 chromosomes, lacked the same two gliadins. All the subunits in the other thirty-one chromosome substitution lines were indistinguishable from those in Chinese Spring. It is therefore concluded that the major variation affecting gliadin and glutenins in wheat is concentrated on the chromosomes of homoeologous Groups 1 and 6 but Group 2 chromosomes are candidates for further study.An endosperm protein controlled by chromosome 4D in Chinese Spring is shown to be a high molecular weight globulin.     

Structural studies of wheat flour glutenin polymers by CD spectroscopy

A dissolution procedure of unreduced glutenin polymers of three wheat flour varieties (WRU 6981, Alisei 1, and Alisei 2) by sonication in the presence of SDS (sodium dodecyl sulphate), after the elimination of albumins, globulins, and gliadins, was achieved, and the molecular weight distribution of glutenin polymers obtained by this method was measured by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. A structural study by CD spectroscopy at different temperatures of WRU 6981 glutenin polymer and of 1Ax1 high-M(r) (relative molecular mass) glutenin subunit, which is the only high-M(r) subunit contained in WRU 6981 flour, was undertaken to understand if the information obtained from the single subunit were applicable to the total polymer. CD spectroscopy also has been employed to study the glutenin polymers obtained by Alisei 1 and Alisei 2 wheat flours; Alisei 1 biotype contained 1Bx7 and 1Dx2+1Dy12 high-M(r) subunits, whereas the Alisei 2 biotype contained only 1Bx7 and 1Dy12 subunits. A conformational study was undertaken by CD spectroscopy at different temperatures and in the presence of some chemical denaturant agents, such as urea and sodium dodecyl sulphate, in order to obtain information about their intrinsic stability and to verify if the 1Dx2 subunit presence determined a different structural behavior between Alisei 1 and Alisei 2 polymers. MALDI-TOF mass spectrometric experiments showed that the glutenin polymers molecular weights were in the mass range of 500000-5000000. CD spectra indicated that a single conformational state did not predominate in the temperature range studied but equilibrium between two distinct conformational states existed; moreover, all the changes induced by urea and by SDS followed a multistep transition process.     

Biochemical and genetic characterization of a monomeric storage protein (T1) with an unusually high molecular weight in Triticum tauschii

The protein named T1, present in Triticum tauschii, was previously characterized as a high-molecular-weight (HMW) glutenin subunit with a molecular size similar to that of the y-type glutenin subunit-10 of Triticum aestivum. This protein was present along with other HMW glutenin subunits named 2^t and T2, and was considered as part of the same allele at the Glu-D ^t 1 locus of T. tauschii. This paper describes a re-evaluation of this protein, involving analyses of a collection of 173 accessions of T. tauschii, by SDS-PAGE of glutenin subunits after the extraction of monomeric protein. No accessions were found containing the three HMW glutenin subunits. On the other hand, 17 lines with HMW glutenin subunits having electrophoretic mobilities similar to subunits 2^t and T2 were identified. The absence of T1 protein in these gel patterns has shown that protein T1 is not a component of the polymeric protein. Rather, the T1 protein is an ω-gliadin with an unusually high-molecular-weight. This conclusion is based on acidic polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional gel electrophoresis (A-PAGE+ SDS-PAGE), together with analysis of its N-terminal amino-acids sequence. The inheritance of ω-gliadin T1 was studied through analyses of gliadins and HMW glutenins in 106 F₂ grains of a cross between synthetic wheat, L/18913, and the wheat cv Egret. HMW glutenin subunits and gliadins derived from T. tauschii (Glu-D ^t 1 and Gli-D ^t 1) segregated as alleles of the Glu-D1 and Gli-D1 loci of bread wheat. A new locus encoding the ω-gliadin T1 was identified and named Gli-DT1. The genetic distance between this new locus and those of endosperm proteins encoded at the 1D chromosome were calculated. The Gli-DT1 locus is located on the short arm of chromosome 1D and the map distance between this locus and the Gli-D1 and Glu-D1 loci was calculated as 13.18 cM and 40.20 cM, respectively. Received: 13 October 2000 / Accepted: 18 April 2001     

Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

Background  

The gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties.     

Suppression of gliadins results in altered protein body morphology in wheat

Wheat gluten proteins, gliadins and glutenins, are of great importance in determining the unique biomechanical properties of wheat. Studies have therefore been carried out to determine their pathways and mechanisms of synthesis, folding, and deposition in protein bodies. In the present work, a set of transgenic wheat lines has been studied with strongly suppressed levels of γ-gliadins and/or all groups of gliadins, using light and fluorescence microscopy combined with immunodetection using specific antibodies for γ-gliadins and HMW glutenin subunits. These lines represent a unique material to study the formation and fusion of protein bodies in developing seeds of wheat. Higher amounts of HMW subunits were present in most of the transgenic lines but only the lines with suppression of all gliadins showed differences in the formation and fusion of the protein bodies. Large rounded protein bodies were found in the wild-type lines and the transgenic lines with reduced levels of γ-gliadins, while the lines with all gliadins down-regulated had protein bodies of irregular shape and irregular formation. The size and number of inclusions, which have been reported to contain triticins, were also higher in the protein bodies in the lines with all the gliadins down-regulated. Changes in the protein composition and PB morphology reported in the transgenic lines with all gliadins down-regulated did not result in marked changes in the total protein content or instability of the different fractions.