Arthur S. Tatham

Seed storage proteins: structures and biosynthesis.

in them. The presence of these groups may allow the plant to maintain high levels of storage protein synthesis despite variations in sulfur availability. The strict tissue specificity of seed storage protein synthesis contrasts with that of tuber storage proteins, which may be synthesized in vegetative tissues under unusual conditions (for example, in vitro or after removal of tubers) (Shewry, 1995). A second common property of seed storage proteins is their presence in the mature seed in discrete deposits called protein bodies, whose origin has been the subject of some dispute and may in fact vary both between and within species. Finally, all storage protein fractions are mixtures of components that exhibit polymorphism both within single genotypes and among genotypes of the same species. This polymorphism arises from the presence of multigene families and, in some cases, proteolytic processing and glycosylation.

High molecular weight subunits of wheat glutenin

The high molecular weight (HMW) subunits of wheat glutenin are of considerable interest because of their relationship to breadmaking quality. We review recent studies of their genetics, amino acid sequences and conformations, and discuss how they may be assembled to form disulphide-bonded polymers that confer elasticity on wheat dough. We also speculate on how their structure and functionality may be explored using protein engineering and expression in microorganisms or in developing seeds of transgenic plants.

Comprehensive, Quantitative Mapping of T Cell Epitopes in Gluten in Celiac Disease

Three highly immunogenic peptides from gluten are primarily responsible for celiac disease, suggesting a rational immunotherapeutic approach that could replace the need for strict, lifelong dietary gluten avoidance. Taming of the Sprue Gluten, a complex protein in wheat, barley, and rye, forms the elastic network responsible for the airy texture of bread. But gluten can also trigger a prevalent inflammatory disorder—celiac disease (sprue)—which afflicts sufferers with problems such as gastrointestinal upset, fatigue, and anemia, and confers increased risks of osteoporosis, autoimmune disease, and cancer. The current therapy consists of strict lifelong avoidance of all foods containing gluten. The development of alternatives has been hampered by the inability to fully characterize the immune response to the toxic peptides within these grains. Several immunotoxic peptides from wheat have been implicated, but it has remained unclear how they contribute to the overall immune response in celiac disease, or whether other potentially toxic peptides from barley and rye exist. Tye-Din and colleagues have now comprehensively assessed the more than 16,000 potentially toxic peptides contained within wheat, barley, and rye, and identified which ones stimulate T cells from celiac disease patients. By feeding doses of wheat, barley, or rye to more than 200 people with celiac disease, the authors were able to examine the induced T cells appearing in the bloodstream several days afterward. These T cells were then tested for recognition of peptides from large libraries encompassing every possible toxic peptide from wheat, barley, and rye. Surprisingly, they found that just three highly active peptides were responsible for most of the immune response seen in patients with celiac disease after eating any of the toxic grains. Although the range of highly stimulatory or dominant peptides was very consistent between individuals, it was dependent on which grain was consumed. A previously described peptide from wheat α-gliadin was dominant only after wheat ingestion; another distinct peptide was dominant after wheat, barley, or rye ingestion. Of most interest was the fact that a combination of these peptides, plus another from barley, could elicit 90% of the response induced by the full complement of wheat, barley, and rye proteins. Because the authors assessed every possible toxic peptide from wheat, as well as barley and rye, they can be confident that their data paint a comprehensive picture of the immune response in celiac disease. This is important because alternative therapies to the complex, costly, and inconvenient gluten-free diet are likely to require a detailed molecular understanding of the peptides driving the immune response in celiac disease. Multiple doses of peptides corresponding to immunodominant T cell epitopes are effective in treating a mouse version of celiac disease, and the discovery that a small number of peptides can elicit the disease in patients suggests that a similar approach may be successful in humans as well. Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4+ T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat α-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from ω-gliadin (wheat) and C-hordein (barley) but not α-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.

Biotechnology of breadmaking: unraveling and manipulating the multi-protein gluten complex.

Breadmaking is one of humankinds oldest technologies, being established some 4,000 years ago. The ability to make leavened bread depends largely on the visco-elastic properties conferred to wheat doughs by the gluten proteins. These allow the entrapment of carbon dioxide released by the yeast, giving rise to a light porous structure. One group of gluten proteins, the high molecular weight (HMW) subunits, are largely responsible for gluten elasticity, and variation in their amount and composition is associated with differences in elasticity (and hence quality) between various types of wheat. These proteins form elastomeric polymers stabilized by inter-chain disulphide bonds, and detailed studies of their structures have led to models for die mechanism of elasticity. This work has also provided a basis for direct improvement of wheat quality by transformation with additional HMW subunit genes.

Analysis of HMW glutenin subunits encoded by chromosome 1A of bread wheat (Triticum aestivum L.) indicates quantitative effects on grain quality.

SummaryA gene encoding the high-molecular-weight (HMW) subunit of glutenin 1Ax1 was isolated from bread wheat cv Hope. Comparison of the deduced amino acid sequence with that previously reported for an allelic subunit, 1Ax2*, showed only minor differences, which were consistent with both subunits being associated with good bread-making quality. Quantitative analyses of total protein extracts from 22 cultivars of bread wheat showed that the presence of either subunit 1Ax1 or 1Ax2*, when compared with a null allele, resulted in an increase in the proportion of HMW subunit protein from ca. 8 to 10% of the total. It is suggested that this quantitative increase in HMW subunit protein may account for the association of 1Ax subunits with good quality.

Elastomeric proteins: biological roles, structures and mechanisms

Elastomeric proteins are able to withstand significant deformations without rupture before returning to their original state when the stress is removed. Although elastomeric proteins differ considerably in their amino acid sequence, they all have a complex domain structure and share two common properties. Namely, they contain elastomeric domains, comprised of repeated sequences, and additional domains that form intermolecular crosslinks. Furthermore, several protein contain beta-turns as a structural motif within the elastomeric domains.

FTIR and NMR studies on the hydration of a high-Mr subunit of glutenin

The hydration behaviour of a purified high-M(r) subunit of glutenin has been studied using Fourier transform infra-red (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. The water-insoluble protein was examined in an unalkylated form with intermolecular disulfide bonds, and in a reduced and alkylated (unpolymerized) form. Hydration produced a marked increase in chain mobility, especially above a threshold water content of about 37% w/w. NMR experiments also showed that some parts of the chain were held in a much less mobile state, even at higher water contents. Little difference could be seen between alkylated and unalkylated subunits, implying that NMR is sensitive to localized motions, but not to any restrictions imposed by disulfide bridges close to the chain ends. FTIR spectra of the protein films have shown that increasing hydration enables changes to occur in favour of a more extended and beta-sheet-type structure. The changes in secondary structure are very noticeable at water contents corresponding to the NMR mobility threshold. The behaviour is influenced by intermolecular interactions. beta-sheet formation is enhanced by the presence of disulfide bonds in the unalkylated samples. There is little evidence of beta-structure (sheet or extended chain) either in the dry state, where protein-protein interactions are strongest, or in dilute acetic acid solution, where the interactions are weakest. The balance between protein-protein and protein-water hydrogen-bonding interactions therefore appears to influence the formation of beta-sheet and extended chain structures, and these may in turn affect the elasticity of high M(r) subunits.

Biotechnology of Wheat Quality

Wheat gluten proteins are largely responsible for the visco-elastic properties that allow doughs to be processed into bread and various other food products including cakes, biscuits (cookies), pasta and noodles. Detailed biochemical and biophysical studies are revealing details of the molecular structures and interactions of the individual gluten proteins, and their roles in determining the functional properties of gluten. In particular, one group of gluten proteins, the high molecular weight (HMW) subunits of glutenin, have been studied in detail because of their role in determining the strength (elasticity) of doughs. The development of robust transformation systems for bread wheat is now allowing the role of the HMW subunits to be explored experimentally, by manipulating their amount and composition in transgenic plants. Such studies should lead to improvement of the processing properties of wheat for traditional end uses and the development of novel end uses in food processing or as raw material for other industries.

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

Abstract.The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an Mr of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

The glass-transition behaviour of wheat gluten proteins.

The glass-transition behaviour of four hydrated wheat gluten proteins (alpha-gliadin, gamma-gliadin, omega-gliadin and high-molecular-weight (HMW) subunits of glutenin) was studied using differential scanning calorimetry (DSC). By fitting the data to the Gordon-Taylor equation, which has previously been used to describe the plasticization of polymers by diluents, the glass-transition temperatures (Tg) for the dry proteins were found by extrapolation. The values for Tg were within the range 397-418 K. Values for the heat capacity increment delta Cp at Tg for the plasticized proteins were also determined and ranged from 0.29-0.47 J g-1 K-1 with no dependence on water content. The differences in glass-transition behaviour of the proteins are discussed in relation to their secondary structure.