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.     

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     

Rapid evolution and complex structural organization in genomic regions harboring multiple prolamin genes in the polyploid wheat genome

Genes encoding wheat prolamins belong to complicated multi-gene families in the wheat genome. To understand the structural complexity of storage protein loci, we sequenced and analyzed orthologous regions containing both gliadin and LMW-glutenin genes from the A and B genomes of a tetraploid wheat species, Triticum turgidum ssp. durum. Despite their physical proximity to one another, the gliadin genes and LMW-glutenin genes are organized quite differently. The gliadin genes are found to be more clustered than the LMW-glutenin genes which are separated from each other by much larger distances. The separation of the LMW-glutenin genes is the result of both the insertion of large blocks of repetitive DNA owing to the rapid amplification of retrotransposons and the presence of genetic loci interspersed between them. Sequence comparisons of the orthologous regions reveal that gene movement could be one of the major factors contributing to the violation of microcolinearity between the homoeologous A and B genomes in wheat. The rapid sequence rearrangements and differential insertion of repetitive DNA has caused the gene islands to be not conserved in compared regions. In addition, we demonstrated that the i-type LMW-glutenin originated from a deletion of 33-bps in the 5′ coding region of the m-type gene. Our results show that multiple rounds of segmental duplication of prolamin genes have driven the amplification of the ω-gliadin genes in the region; such segmental duplication could greatly increase the repetitive DNA content in the genome depending on the amount of repetitive DNA present in the original duplicate region. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Shuangcheng Gao and Yong Qiang Gu contributed equally to the work.     

Analysis of the activity of a γ-gliadin promoter in transgenic wheat and characterization of gliadin synthesis in wheat by MALDI-TOF during grain development

The wheat grain is the most important organ for human food and therefore is the target for much research focused on modifying its composition to improve nutritional and functional components. Genetic transformation provides a precise tool to alter the composition of wheat grain by expressing new genes or by down-regulating groups of proteins encoded by multigene families such as gliadins, which contain clusters of epitopes that are active groups in triggering celiac disease. For such work, specific promoters are required to express such constructs in the wheat endosperm. In the present study we report the isolation and characterization of a γ-gliadin promoter from transgenic wheat, and the analysis of gliadin synthesis during grain development in bread wheat by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI/TOF MS). The γ-gliadin promoter fragment was isolated from bread wheat by genome walking and was re-introduced, driving the expression of the gusA gene, by particle bombardment, giving fifteen independent transgenic lines. Detailed analysis of the sequence of the 885 bp promoter fragment showed that it contains three prolamin boxes but only one is conserved according to the consensus sequence reported. The AACA/TA motif is present twice in published γ-gliadin promoter sequences. The RY element i.e., CATGCAT or CATGCAC, is also present twice in the published promoter. Transgenic lines were classified as high, medium, and low expressers. The expression of the gusA gene was found only in the seeds of the transgenic lines. GUS staining was first detected in the outer endosperm of the lobes, and then it extended to the whole outer endosperm. GUS staining was not found in the aleurone layer nor in the embryo. The qRT-PCR data confirmed the data obtained by GUS staining. The expression of the gusA gene determined by qRT-PCR for the high expresser line (B281) was 4 and 8 times higher than that of medium (B282) and low (B286) expresser lines, respectively. MALDI/TOF-MS showed that gliadins exhibited different patterns of synthesis during the course of seed maturation. Thus, gliadins with masses higher than 36,000 Da were synthesised within the first 12 days post anthesis while those with masses lower than 36,000 Da were synthesised later. Results of GUS staining, qRT-PCR and MALDI/TOF-MS showed that the γ-gliadin promoter reported in this work could be a good candidate to downregulate wheat gliadins.     

Gene duplication and recombination in the evolution of mammalian Fc receptors

The immunoglobulin-related chains of cell-surface receptors for the Fc region of immunoglobulins (FCERIα, FcγRI, FcγRII, and FcγRIIIα) are encoded by members of a gene family. Phylogenetic analysis of representative members of this family from mammals revealed that FcγRIIIα genes of human, mouse, and rat are not orthologous to one another in the region of the gene encoding the Immunoglobulin C2-set domains. In phylogenetic trees of this region, FcγRIIIα and FcγRII clustered together. However, in trees based on both coding and noncoding regions 5′ and 3′ to the C2 domains, FcγRIIIα genes of human, mouse, and rat clustered together. This pattern of relationship is most easily explained as a result of two independent recombinational events occurring in the mouse and rat after these two species diverged, in each of which the exons encoding the C2 domains were donated to an FcγRIIIα gene by an FcγRII gene.     

Nucleotide sequence of a γ gliadin gene: Comparisons with other γ gliadin sequences show the structure of γ gliadin genes and the general primary structure of γ gliadins

A low stringency screening of a wheat (Triticum aestivum L.) genomic library produced three types of γ gliadin clones. The sequence of one clone, λ10–20, encoded a γ gliadin of 34.3 kDa. Comparisons of this protein with the proteins encoded by other γ gliadin DNA sequences revealed a general γ gliadin structure: a 19-residue signal peptide; 12-residue mature amino terminus; 12–16 copies of a proline- and glutamine-rich heptapeptide repeat; a 76-residue region high in glutamine containing most of the cyysteines and charged residues: a 6–16-residue polyglutamine region; and the 41-residue carboxyl terminus. Comparisons of the 5′ and 3′ flanking regions of several γ gliadins reveals the high homology and general structure of γ gliadin genes. A further comparison of the 5′ flanking regions with the 5′ flanking regions of other prolamin genes showed that γ gliadin genes contain three copies of a conserved sequence seen within approx. 600 b.p. upstream of the translation start sites of prolamin genes.     

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.     

The characterisation and mapping of a family of LMW-gliadin genes: effects on dough properties and bread volume

Analysis of a cDNA library from wheat cv Wyuna endosperm, indicated a significant size and sequence variation among seed-endosperm protein genes. In this study, a family of low-molecular-weight seed protein genes are analysed that are related to the gliadins and the low-molecular-weight glutenin subunits. Sequence analysis and comparison of these proteins showed that they are closely related to a 17-kDa protein from barley, epsilon hordein, which plays a role in beer foam stability in the brewing industry. Mapping of these genes in wheat shows that they are located on group 7 and 4 chromosomes, as opposed to a group 1 and 6 location for the glutenins and gliadins. It is possible that this family of proteins forms a new class of seed-endosperm proteins important in defining the quality characteristics of wheat flour. Therefore, a representative gene from this family was expressed in Escherichia coli and the purified protein was supplemented into a base wheat flour. Rheological analysis showed that the protein effected dough strength and resistance break down during mixing of the dough, and provided a 20% increase in loaf height after baking.     

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.     

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.     

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat

Summary Recombinant cDNA plasmids possessing the coding sequences for the -type gliadins were isolated from a cDNA library prepared from wheat seed poly (A⁺) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A⁺) RNA molecules 1 400–1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the -type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3 untranslated region as well as 245 amino acids from the C-terminus of the -type gliadin polypeptide. The 5-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the /-type and -type gliadin cDNA sequences.     

Isolation and characterization of wheat ω-gliadin genes

The DNA sequences of two full-length wheat ω-gliadin prolamin genes (ωF20b and ωG3) containing significant 5′ and 3′ flanking DNA sequences are reported. The ωF20b DNA sequence contains an open reading frame encoding a 30,460-Dalton protein, whereas the ωG3 sequence would encode a putative 39,210-Dalton protein except for a stop codon at amino-acid residue position 165. These two ω-gliadin genes are closely related and are of the ARQ-/ARE-variant type as categorized by the derived N-terminal amino-acid sequences and amino-acid compositions. The ω-gliadins were believed be related to the ω-secalins of rye and the C-hordeins of barley, and analyses of these complete ω-gliadin sequences confirm this close relationship. Although the ω-type sequences from all three species are closely related, in this analysis the rye and barley ω-type sequences are the most similar in a pairwise comparison. A comparison of ω-gliadin flanking sequences with respect to that of their orthologs and with respect to wheat gliadin genes suggests the conservation of flanking DNA necessary for gene function. Sequence data for members of all major wheat prolamin families are now available. Received: 24 August 2000 / Accepted: 15 December 2000     

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

GABA_A receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised of only α and β subunits. The pore of GABA_A receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized potentials only (E_Cl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics.     

Structure and heterogeneity of gliadin: a hydrodynamic evaluation

A study of the heterogeneity and conformation in solution [in 70% (v/v) aq. ethanol] of gliadin proteins from wheat was undertaken based upon sedimentation velocity in the analytical ultracentrifuge, analysis of the distribution coefficients and ellipsoidal axial ratios assuming quasi-rigid particles, allowing for a range of plausible time-averaged hydration values. All classical fractions (α, γ, ω_slow, ω_fast) show three clearly resolved components. Based on the weight-average sedimentation coefficient for each fraction and a weight-average molecular weight from sedimentation equilibrium and/or cDNA sequence analysis, all the proteins are extended molecules with axial ratios ranging from ~10 to 30 with α appearing the most extended and γ the least.     

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。     

Investigation of neuronal cell type-specific gene expression of Ca2+/calmodulin-dependent protein kinase II

The promoter activity of the rat Ca²⁺/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5′-flanking region of α and β isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5′-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types. Published: April 12, 2002     

Exploring the interactions of gliadins with model membranes: Effect of confined geometry and interfaces

Mechanisms leading to the assembly of wheat storage proteins into proteins bodies within the endoplasmic reticulum (ER) of endosperm cells are unresolved today. In this work, physical chemistry parameters which could be involved in these processes were explored. To model the confined environment of proteins within the ER, the dynamic behavior of γ‐gliadins inserted inside lyotropic lamellar phases was studied using FRAP experiments. The evolution of the diffusion coefficient as a function of the lamellar periodicity enabled to propose the hypothesis of an interaction between γ‐gliadins and membranes. This interaction was further studied with the help of phospholipid Langmuir monolayers. γ‐ and ω‐gliadins were injected under DMPC and DMPG monolayers and the two‐dimensional (2D) systems were studied by Brewster angle microscopy (BAM), polarization modulation infrared reflection‐absorption spectroscopy (PM‐IRRAS), and surface tension measurements. Results showed that both gliadins adsorbed under phospholipid monolayers, considered as biological membrane models, and formed micrometer‐sized domains at equilibrium. However, their thicknesses, probed by reflectance measurements, were different: ω‐gliadins aggregates displayed a constant thickness, consistent with a monolayer, while the thickness of γ‐gliadins aggregates increased with the quantity of protein injected. These different behaviors could find some explanations in the difference of aminoacid sequence distribution: an alternate repeated ‐ unrepeated domain within γ‐gliadin sequence, while one unique repeated domain was present within ω‐gliadin sequence. All these findings enabled to propose a model of gliadins self‐assembly via a membrane interface and to highlight the predominant role of wheat prolamin repeated domain in the membrane interaction. In the biological context, these results would mean that the repeated domain could be considered as an anchor for the interaction with the ER membrane and a nucleus point for the formation and growth of protein bodies within endosperm cells. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 610–622, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com