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.     

VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for Celiac Sprue probiotics and gluten intolerance

The native structure and distribution of gliadin epitopes responsible for Celiac Sprue (CS) may be influenced by cereal food processing. This work was aimed at showing the capacity of probiotic VSL#3 to decrease the toxicity of wheat flour during long-time fermentation. VSL#3 (10⁹ cfu/ml) hydrolyzed completely the α2-gliadin-derived epitopes 62–75 and 33-mer (750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and mass spectrometry analyses showed an almost complete degradation of gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins non-hydrolyzed during fermentation by VSL#3 were subjected to peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry). Search for several epitopes showed the only presence of α2-gliadin-fragment 62–75 at a very low concentration (sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins extracted from the chemically acidified dough (control), VSL#3 pre-digested gliadins caused a less pronounced reorganization of the intracellular F-actin which was mirrored by an attenuated effect on intestinal mucosa permeability. The release of zonulin from intestinal epithelial cells treated with gliadins was considerably lower when digested with VSL#3. Agglutination test on K 562 (S) cells showed that the PT-digest of wheat flour treated with VSL#3 increased the Minimal Agglutinating Activity of ca. 100 times. Wheat proteins were extracted from doughs and subjected to PT digestion. Compared to PT-digest from chemically acidified dough, celiac jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3⁺ intraepithelial lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance during food processing to produce pre-digested and tolerated gliadins for increasing the palatability of gluten-free products.     

VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for Celiac Sprue

The native structure and distribution of gliadin epitopes responsible for Celiac Sprue (CS) may be influenced by cereal food processing. This work was aimed at showing the capacity of probiotic VSL#3 to decrease the toxicity of wheat flour during long-time fermentation. VSL#3 (10(9) cfu/ml) hydrolyzed completely the alpha2-gliadin-derived epitopes 62-75 and 33-mer (750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and mass spectrometry analyses showed an almost complete degradation of gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins non-hydrolyzed during fermentation by VSL#3 were subjected to peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry). Search for several epitopes showed the only presence of alpha2-gliadin-fragment 62-75 at a very low concentration (sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins extracted from the chemically acidified dough (control), VSL#3 pre-digested gliadins caused a less pronounced reorganization of the intracellular F-actin which was mirrored by an attenuated effect on intestinal mucosa permeability. The release of zonulin from intestinal epithelial cells treated with gliadins was considerably lower when digested with VSL#3. Agglutination test on K 562 (S) cells showed that the PT-digest of wheat flour treated with VSL#3 increased the Minimal Agglutinating Activity of ca. 100 times. Wheat proteins were extracted from doughs and subjected to PT digestion. Compared to PT-digest from chemically acidified dough, celiac jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3(+) intraepithelial lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance during food processing to produce pre-digested and tolerated gliadins for increasing the palatability of gluten-free products.     

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.     

Functional properties of a new low-molecular-weight glutenin subunit gene from a bread wheat cultivar

Some allelic forms of low-molecular-weight glutenin subunit (LMW-GS) can greatly influence the end-use of wheat flours, understanding the function of each allele of LMW-GS is important to wheat quality breeding. A LMW-GS gene XYGluD3-LMWGS 1(AY263369) has been cloned from bread wheat cultivar Xiaoyan 6. The deduced protein contained nine cystine residues, one more than that in all other LMW-GSs reported previously, indicating that it is either a new gene or a new allele of a known LMW-GS gene. In this study, the gene was expressed in E. coil in large scale for the testing of its functional property. Reactive Red 120-Agarose resin was used efficiently to purify the expressed LMW-GS proteins from bacteria, with the lactic acid–sodium lactate buffer (pH 4.5) which contained low concentration SDS as elution solution. The purified protein (belonging to the LMW-m family, MW about 35 KDa) was supplemented into a base flour, the results of 10 g dough mixing test indicated that incorporation of the LMW-GS increased the strength of the dough, with significant increases in mixing time (MT) and peak width (PW), and decrease in breakdown in resistance (RBD) compared with the control. In addition, the dough with incorporation of the LMW-GS had more glutenin macropolyeric protein than the control, suggesting that the LMW-GS participated in forming larger glutenin polymers, and greatly contributed to dough strength. The changes in mixing parameters and the amount of glutenin macropolyeric protein were related to the quantity of incorporating subunits.     

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.     

A proteomic approach to verify in vivo expression of a novel gamma-gliadin containing an extra cysteine residue

Gliadins and glutenins are the main protein fractions present in wheat gluten. They are responsible for technological and nutritional quality of wheat based products. In particular, glutenins are mainly responsible for dough visco-elastic properties, whereas gliadins confer extensibility to dough and are the most important factor triggering celiac disease, the major human intolerance to gluten. Gliadins are monomeric proteins, whereas glutenins are polymers stabilized by disulfide bonds. Although they have distinctive structural characteristics, it is possible that some gliadins become part of the glutenin fraction because of mutations that affect cysteine number and distribution. Here, we provide evidence that a naturally mutated gamma-gliadin with an extra cysteine residue is incorporated into the polymeric fraction. This goal was achieved using an integrated approach involving heterologous expression, 2-DE, RP-HPLC and MS.     

The low-molecular-weight glutenin subunit proteins of primitive wheats. IV. Functional properties of products from individual genes

 Three genes encoding the low-molecular-weight glutenin subunits (LMW-GSs), LMWG-E2 and LMWG-E4, from A-genome diploid wheat species, and LMW-16/10 from a D-genome diploid wheat, were expressed in bacteria. The respective proteins were produced on a relatively large scale and compared with respect to their effects on flour-processing properties such as dough mixing, extensibility and maximum resistance; these are important features in the end-use of wheat for producing food products. The LMWG-E2 and LMWG-E4 proteins caused significant increases in peak resistance and mixing time, compared to the control, when incorporated into dough preparations. The LMWG-16/10 protein was qualitatively less effective in producing these changes. All three proteins also conferred varying degrees of decrease in dough breakdown. LMWG-E2 and LMWG-E4 caused significant increases in dough extensibility, and decreases in maximum resistance, relative to the control. LMW-16/10 did not show a significant effect on extensibility but showed a significant decrease in maximum resistance. The refinement of relating specific features of the structure of the LMW-GS genes to the functional properties of their respective proteins is discussed. Received: 24 November 1997 / Accepted: 18 August 1998     

小麦低分子量麦谷蛋白亚基研究进展

低分子量麦谷蛋白亚基(LMW-GS)是小麦胚乳中的一种聚合蛋白组分,LMW-GS彼此间或／和高分子量麦谷蛋白亚基(HMW-GS)间形成分子内二硫键,进而产生麦谷蛋白聚合体,决定小麦面团的加工品质。由于 LMW-GS与醇溶蛋白的提取特性和电泳迁移率相近,其研究进展缓慢。近年来随着电泳技术的提高,LMW-GS的研究也成为品质性状研究的新热点,越来越多的研究证实了LMW-GS对品质具有重要作用。然而,关于LMW-GS 的研究在我国尚处于起步阶段。本文从小麦LMW-GS的分类、染色体定位、结构及其与品质间关系等方面回顾其研究状况,并讨论研究中存在的问题。     

小麦种子贮藏蛋白质研究进展

小麦醇溶蛋白组成可以作为小麦品种鉴定的指纹图谱,其分离方法有酸性电泳、反相高压液相色谱（RP－HPLC）和毛细管电泳（CE）等手段,3种方法相互补充,而CE分辨率最高。对醇溶蛋白酸性电泳条件的改良和完善仍在进行中,利用最新的分离技术对小麦醇溶蛋白基因进行染色体定位和遗传行为分析是近年来醇溶蛋白研究的另一领域。小麦高分子量麦谷蛋白亚基（HMW－GS）与小麦面包烘烤质量密切相关,关于它的研究目前主要集中在3个方面;对各个迁3移率较近的亚基进行快速,准确分离方法的研究,HMW－GS与小麦面包烘烤质量关系的研究和通过基因工程来改良小麦的品质、提高面粉的加工特性等。低分子量麦保蛋白（LMW－Glutenin)影响小麦面粉的特性,截止目前已经获得了17个该基因的克隆,并对其基因结构进行了描述,有些低分子量麦谷蛋白亚基（LMW－GS）加入碱性面粉后改变了面筋的性质,报道了小麦醇溶蛋白,高分子量麦谷蛋白亚（HMW－GS）、低分子量麦谷蛋白亚基（LMW－GS）3个方面的最新研究进展。     

Use of selected enterococci and Rhizopus oryzae proteases to hydrolyse wheat proteins responsible for celiac disease

Aims:  This work aimed at using a pool of selected enterococci and fungal proteases to hydrolyse wheat gluten during long-time fermentation.
Methods and Results:  A liquid dough made with wheat flour (20% w/w) was fermented with three Enterococcus strains (dough A) or with the combination of enterococci and Rhizopus oryzae proteases (dough B). After 48 h of fermentation, dough A and B had a concentration of water-soluble peptides approximately threefold higher than the chemically acidified dough (CAD), used as the control. The same was found for the concentration of free amino acids, being higher in dough B with respect to dough A. SDS-PAGE analysis showed that albumin and glutenin fractions were partially hydrolysed, while gliadins almost disappeared in dough A and B, as confirmed by two-dimensional electrophoresis, RP-HPLC and R5-ELISA analyses.
Conclusions:  The combined use of enterococci and fungal proteases showed a decrease of the gluten concentration of more than 98% during long-time fermentation.
Significance and Impact of the Study:  The use of the mixture of selected enterococci and R. oryzae proteases should be considered as a potential tool to decrease gluten concentration in foods.     

Identification of several new classes of low-molecular-weight wheat gliadin-related proteins and genes

During the initial phases of a wheat endosperm Expressed-Sequence-Tag (EST) project, several clones were determined to be related to wheat gliadin sequences, but not similar enough to be classified into any of the traditional gliadin families [α-, γ-, and ω-gliadins, low-molecular-weight (LMW) glutenins]. Complete sequences of these cDNA clones revealed four new classes of gliadin-related endosperm proteins, but lacking a prominent repeat domain which until now has been characteristic of the gliadins. Two of these classes are related to different minimally described groups of Triticeae endosperm proteins. One class of proteins, which has N-terminal amino-acid sequences matching members of a reported 25-kDa globulin family from wheat, is shown by amino-acid sequencing to match to a family of 25-kDa endosperm proteins, is encoded by a multigene family, and is most similar to the LMW-glutenins. A second new class shows N-terminal homologies to LMW secalins from rye, and has an amino-acid composition similar to wheat and barley LMW proteins with extraction properties similar to prolamins. The third class is most similar to α-gliadins, and the fourth class has no close association to previously described wheat endosperm proteins. Received: 20 October 2000 / Accepted: 20 November 2000     

Overexpression of Avenin-Like b Proteins in Bread Wheat (Triticum aestivum L.) Improves Dough Mixing Properties by Their Incorporation into Glutenin Polymers

Avenin-like b proteins are a small family of wheat storage proteins, each containing 18 or 19 cysteine residues. The role of these proteins, with high numbers of cysteine residues, in determining the functional properties of wheat flour is unclear. In the present study, two transgenic lines of the bread wheat overexpressing avenin-like b gene were generated to investigate the effects of Avenin-like b proteins on dough mixing properties. Sodium dodecyl sulfate sedimentation (SDSS) test and Mixograph analysis of these lines demonstrated that overexpression of Avenin-like b proteins in both transgenic wheat lines significantly increased SDSS volume and improved dough elasticity, mixing tolerance and resistance to extension. These changes were associated with the increased proportion of polymeric proteins due to the incorporation of overexpressed Avenin-like b proteins into the glutenin polymers. The results of this study were critical to confirm the hypothesis that Avenin-like b proteins could be integrated into glutenin polymers by inter-chain disulphide bonds, which could help understand the mechanism behind strengthening wheat dough strength.     

Proteomic analysis of secretagogue-stimulated neutrophils implicates a role for actin and actin-interacting proteins in Rac2-mediated granule exocytosis

Background

Mineral nutrition during wheat grain development has large effects on wheat flour protein content and composition, which in turn affect flour quality and immunogenic potential for a commodity of great economic value. However, it has been difficult to define the precise effects of mineral nutrition on protein composition because of the complexity of the wheat flour proteome. Recent improvements in the identification of flour proteins by tandem mass spectrometry (MS/MS) and the availability of a comprehensive proteome map of flour from the US wheat Butte 86 now make it possible to document changes in the proportions of individual flour proteins that result from the application of mineral nutrition.

Results

Plants of Triticum aestivum 'Butte 86' were grown with or without post-anthesis fertilization (PAF) and quantitative 2-dimensional gel electrophoresis (2-DE) was used to analyze protein composition of the resulting flour. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, high molecular weight glutenin subunits (HMW-GS) and serpins as well as some alpha-gliadins increased in proportion with PAF. In contrast, alpha-amylase/protease inhibitors, farinins, purinins and puroindolines decreased in proportion. Decreases were also observed in several low molecular weight glutenin subunits (LMW-GS), globulins, defense proteins and enzymes. The ratio of HMW-GS to LMW-GS in the flour increased from 0.61 to 0.95 and the ratio of gliadins to glutenins increased from 1.02 to 1.30 with PAF. Because flour protein content doubled with PAF from 7 to 14%, most protein types actually increased in absolute amount (μg/mg flour protein). Data further suggest that flour proteins change with PAF according to their content of sulfur-containing amino acids Cys + Met.

Conclusions

A 2-DE approach revealed changes in the wheat flour proteome due to PAF that are important for flour quality and immunogenic potential. The work forms a baseline for further studies of the effects of environmental variables on flour protein composition and provides clues about the regulation of specific flour protein genes. The study also is important for identifying targets for breeding programs and biotechnology efforts aimed at improving flour quality.     

Genetic characterisation of dough rheological properties in a wheat doubled haploid population: additive genetic effects and epistatic interactions

Doubled haploid lines (n=160) from a cross between wheat cultivars Cranbrook (high dough extensibility) and Halberd (low dough extensibility) were grown at three Australian locations. The parents differ at all high- and low-molecular-weight glutenin loci. Dough rheological parameters were measured using small-scale testing procedures, and quantitative trait locus (QTL) mapping procedures were carried out using an existing well-saturated genetic linkage map for this cross. Genetic parameters were estimated using three software packages: QTLCartographer, Epistat and Genstat. Results indicated that environmental factors are a major determinant of dough extensibility across the three trial sites, whereas genotypic factors are the major determinants of dough strength. Composite interval mapping analysis across the 21 linkage groups revealed that as expected, the main additive QTLs for dough rheological properties are located at the high- and low-molecular-weight glutenin loci. A new QTL on chromosome 5A for M-extensibility (a mixograph-estimated measure of extensibility) was detected. Analysis of epistatic interactions revealed that there were significant conditional epistatic interactions related with the additive effects of glutenin loci on dough rheological properties. Therefore, the additive genetic effects of glutenins on dough rheological properties are conditional upon the genetic background of the wheat line. The molecular basis of the interactions with the glutenin loci may be via proteins that modify or alter the gluten protein matrix or variations in the expression level of the glutenin genes. Reverse-phase high performance liquid chromatography analysis of the molar number of individual glutenin subunits across the population showed that certain conditional epistases resulted in increased expression of the affected glutenin. The epistatic interactions detected in this study provide a possible explanation of the variable genetic effects of some glutenins on quality attributes in different genetic backgrounds and provide essential information for the accurate prediction of glutenin related variance in marker-assisted wheat breeding.     

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.     

Genetic variability and improvement of seed proteins in wheat

Summary Albumins, globulins, gliadins and glutenins presumably comprising 100 percent of the wheat seed proteins were sequentially extracted and electrophoresed on SDS-polyacrylamide gels. The SDS-electrophoretic patterns within each of the four fractions from T. boeotiaum, T. urartu, T. turgidum, T. timopheevii, T. aestivum, Ae. speltoides and Ae. squawosa were similar. They differed from one species to another only in a few minor components or density of certain components. Similarity in MW's of components, as indicated by the SDS-electrophoretic patterns, suggests that the wheats and Aegilops exhibit no variability for structural genes coding seed proteins. A minimum of 60 to 70 and a maximum of 360 to 420 structural genes with major or minor effects control the total seed protein in T. aestivum. Presumably, only one or the other homoeoallele was expressed in the polyploids. Different components of albumins and globulins presumably had distinct MW's and amino acid composition, while the components of gliadins and glutenins could be classified into a few groups each containing one or more components with the same MW and nearly identical amino acid composition. The genes for components with similar MW's and amino acid composition arose through multiplication of a single original gene and perhaps share the same regulatory mechanism. Seed protein content and quality in wheat might be improved through the incorporation of structural genes, coding for polypeptides with distinct MW's, from distantly related species, rather than by manipulation of the structural genes within the Triticum-Aegilops group. Regulatory mutants similar to opaque-2 of corn could be used to alter the proportion of gliadins in relation to albumins and globulins, to improve amino acid composition of wheat proteins.