Genetic and environmental factors affecting pathogenicity of wheat as related to celiac disease

Gluten proteins are the basis of the rheological properties of wheat derived products, such as bread and pasta. Their particular amino acidic composition (high proline and glutamine content) is responsible for the poor gluten digestibility. Some of the high molecular weight peptides that are generated in the gastrointestinal tract are involved in an autoimmune entheropathy called celiac disease. In this work we compared the amount of peptides containing sequences involved in adaptive and immune responses, which were produced after simulated gastrointestinal digestion of prolamins extracted from different durum wheat varieties and in-bred lines. Peptides containing sequences involved in celiac disease were quantified using an isotopically labeled peptide as internal standard. The results demonstrated a very high variability in the amount of pathogenic peptides produced by different lines, showing a strong contribution of the genetic component. At the same time, the variability in total protein and gluten content was lower; the weak correlation between pathogenic peptides and the amount of gluten proteins gives rise to the possibility of a varietal selection aimed to maintain good rheological properties, but simultaneously reducing the exposure to peptides eliciting an immunological response in celiac predisposed subjects. These varieties might be useful for celiac disease prevention.     

Maize Prolamins Resistant to Peptic-tryptic Digestion Maintain Immune-recognition by IgA from Some Celiac Disease Patients

Maize is used as an alternative to wheat to elaborate foodstuffs for celiac patients in a gluten-free diet. However, some maize prolamins (zeins) contain amino acid sequences that resemble the wheat gluten immunodominant peptides and their integrity after gastrointestinal proteolysis is unknown. In this study, the celiac IgA-immunoreactivity to zeins from raw or nixtamalized grains, before and after peptic/tryptic digestion was evaluated and their possible immunogenicity was investigated by in silico methods. IgA from some celiac patients with HLA-DQ2 or DQ8 haplotypes recognized two alpha-zeins even after peptic/tryptic proteolysis. However, digestion affected zeins after denaturation, reduction, and alkylation, used for identification of prolamins as alpha-zein A20 and A30 by MS/MS sequencing. An in silico analysis indicated that other zeins contain similar sequences, or sequences that may bind even better to the HLA-DQ2/DQ8 molecules compared to the already identified ones. Results concur to indicate that relative abundance of these zeins, along with factors affecting their resistance to proteolysis, may be of paramount clinical relevance, and the use of maize in the formulation and preparation of gluten-free foods must be reevaluated in some cases of celiac disease.     

Integration of transcriptomic and proteomic data from a single wheat cultivar provides new tools for understanding the roles of individual alpha gliadin proteins in flour quality and celiac disease

The complement of alpha gliadins expressed in a single wheat cultivar was examined by assembling expressed sequence tags (ESTs) from Triticum aestivum cv. Butte 86. Twelve of 19 resulting contigs encoded complete proteins, but only two were identical to proteins reported previously. Eight contained various combinations of epitopes important in celiac disease (CD), while four lacked typical CD epitopes. One alpha gliadin contained an additional cysteine residue that could allow incorporation of the protein into glutenin polymers. In addition, two new types of alpha gliadins ending in GFFGTN and GIMSTN were identified. Based on the number of ESTs, genes encoding proteins that contained the greatest numbers of CD epitopes were expressed at the highest levels. Proteins corresponding to 12 contigs were distinguished in Butte 86 flour by tandem mass spectrometry (MS/MS). Unique peptide tags for individual alpha gliadins, including some that lack CD epitopes and the protein containing the extra cysteine, are reported. The ability to distinguish closely related alpha gliadins by MS/MS makes it possible to explore the roles of individual proteins in flour quality, better define relationships between specific proteins and celiac disease, and devise strategies to reduce the immunogenic potential of wheat flour for patients with CD.     

栽培一粒小麦α 醇溶蛋白新基因的克隆与序列分析

α-醇溶蛋白是人们生活中消费最多的蛋白,由于含有引起乳糜泻(CD)的主要毒性肽成分,也是引起CD的最活跃的蛋白。为了解一粒小麦在小麦品质育种中的潜力,利用1对α-醇溶蛋白的特异引物,采用基因组PCR法从栽培一粒小麦中克隆α-醇溶蛋白新基因,共获得片段大小为856～882bp的4个基因序列,分别命名为AA-6、AA-8、AA-9和AA-21(GenBank登录号为JN831382～JN831385)。其中,AA-8、AA-9和AA-21均在102位因C→T替换而导致TAG终止子出现成为假基因;AA-6由882个核苷酸构成,可编码293个氨基酸,与已知基因的最高同源性为99%,推断氨基酸序列具有α-醇溶蛋白的典型结构,是α-醇溶蛋白家族的新成员。AA-6的CD毒性肽分析表明,除不含A基因组所没有的glia-α和glia-α2毒性肽外,其他已知的7种毒性肽均有分布。AA-6和86个来源于小麦及其祖先供体种的α-醇溶蛋白的同源性分析表明,α-醇溶蛋白基因存在基因组来源的差异性,其中,A、D基因组来源的α-醇溶蛋白基因的相似性较高。     

Characterization of wheat gluten subunits by liquid chromatography – Mass spectrometry and their relationship to technological quality of wheat

The quality of common wheat is largely influenced by the composition of its storage proteins. The currently presented research explores factors influencing observed differences in quality and quantity between wheat cultivars, in particular in relation to gluten composition and its relationship to technological characteristics. Eight wheat cultivars (H. Wieser, Seilmeier, W., Belitz, H.D., 1994 Parsi, Sirvan, Sivand, Pishgam, Pishtaz)were selected for evaluation. Analysis results demonstrated that Morvarid and Sirvan cultivars yielded the highest quality of wheat, while the Chamran cultivar was indicated as the most favorable for baking Taftoon bread. Conversely, the Sepahan cultivar was deemed to have the worse quality in both categories. A Q Exactive LC-MS/MS system was employed to evaluate the most effective sub-fractions of gliadin and glutenin on wheat quality. Matching peptides resulting from trypsin digestion on gliadin and glutenin fractions, led to the identification of subunits α/β-gliadin, γ-gliadin, HMW-Dx5, HMW-Bx17, HMW-Dy3, HMW-Dy10, HMW-By15, LMW-m, LMW-s, and LMW-i. The obtained results indicated that the most influential subunits of glutenin on wheat quality were Dy10, Dy3 and Dx5, while the most effective gliadin subfraction was noted to be α/β-gliadin However, the most important subunit influencing the quality of flat breads in particular was identified as the x-HMW-GS, in particular the Bx17 subunit, and LMW-GS.     

Amylolysis of wheat starches. II. Degradation patterns of native starch granules with varying functional properties

Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were employed to investigate degradation patterns of native starch granules from wheat (Triticum aestivum L.) by different starch-degrading enzymes. The starches examined were from a waxy wheat and four varieties with slightly elevated amylose content, but with different functional properties. Differences in the digestion patterns after partial α-amylolysis of starch granules were noted between the starches. The waxy starch seemed to be degraded by endocorrosion, whereas the amylose-rich starches followed a slower mode of hydrolysis starting from the granular surface. X-ray diffractograms of the amylose-rich starches were not significantly altered by 2 h of α-amylolysis, whereas partial hydrolysis of the waxy starch decreased scattering intensity at higher 2θ angles, consistent with a different mode of attack by α-amylase in the initial digestion stages of granules of waxy and amylose-rich starches. We propose these differences are due to the combined effects of the change in packing density and partial preference for hydrolysis of amorphous material. The native starch granules were also attacked by beta-amylase, isoamylase and amyloglucosidase, which indicates that α-amylase is not the only starch-degrading enzyme that is able to initiate starch hydrolysis of native granules.     

Targeted proteomics-based analysis of technical enzymes from fungal origin in baked products

The application of technical enzymes is a potential tool in modulating the dough and baking quality of cereal products. No endogenous amylases (α- and β-forms) are present in mature wheat grains; they may be synthesized or activated during germination. Hence, microbial α-amylases are added to the dough, being resistant to the endogenous α-amylase/trypsin inhibitors. Here, we report on the initial identification of two technical enzymes from a commercial sample based on an in-gel tryptic digestion coupled with MALDI-MS analysis. The primary component of the protein fraction with 51.3 kDa was α-amylase from Aspergillus species. A second major protein with 24.8 kDa was identified as endo-1,4-xylanase from Thermomyces lanuginosus. In the following experimental work up, a targeted proteomics approach utilizing the combination of specific proteolytic digestion of the added amylase and xylanase in wheat flour, dough or baked products, solid phase extraction of released peptides and their detection using LC-MS/MS was optimized. The targeted (MRM) MS/MS peptide signals showed that the peptide “ALSSALHER” (MW = 983) originating from amylase and “GWNPGLNAR” (MW = 983) from xylanase can be used to identify the corresponding technical enzymes added. Consequently, locally available baked products were tested and found to contain these enzymes as supplementary ingredients.     

Tocols and carotenoids of einkorn,emmer and spring wheat varieties: Selection for breeding and production

Einkorn (Triticum monococcum L., subsp. monococcum), emmer (Triticum dicoccum Schuebl [Schrank], subsp. dicoccum) and spring bread wheat (Triticum aestivum L.) may be rich in lipophilic antioxidants (tocols and carotenoids), and therefore potential food sources with good nutritional properties. The aim of the present study was to determine the contents of major lipophilic antioxidants beneficial for human health in wheat varieties and landraces for breeding and production. In field experiments over two years, fifteen einkorn, emmer and spring wheat varieties were analysed for the contents of tocols and carotenoids in grain. A high carotenoid content (lutein, zeaxanthin, β-carotene) was typical for the selected einkorn genotypes. Among the analysed wheat species, the highest content was of β-tocotrienol, especially in the einkorn accessions. α-Tocotrienol was abundant in einkorn and emmer wheat species. Higher contents of α- and β-tocopherols were characteristic of spring and emmer wheats. δ-Tocotrienol has been detected for the first time in einkorn and some emmer accessions, although in low concentrations. Significant effects of genotype on the contents of carotenoids and tocols were found with einkorn differing from emmer and spring wheats. The year of cultivation had less effect on the contents of carotenoids and tocols. Selected accessions of einkorn with high contents of carotenoids and tocotrienols as well as spring and emmer wheats with higher contents of tocopherols are good sources of antioxidants with potential health promoting benefits for the production of nutritionally enhanced foods.     

The location of disulphide bonds in α-type gliadins

Gliadin prepared from gluten of the cultivar Rektor by extraction with 70% (v/v) aqueous ethanol adjusted to pH 5.5 was separated by RP-HPLC. Amongst 23 components obtained, two α-type gliadins (α3- and α8-gliadin) were selected for the determination of disulphide bonds. After both proteins were digested with thermolysin, differential RP-HPLC (chromatography prior to and after reduction of disulphide bonds) was used for the detection of cystine peptides. Two cystine peptides from α3-gliadin and three cystine peptides from α8-gliadin were isolated by RP-HPLC. The resulting peptides were reduced and alkylated with 4-vinylpyridine, separated by RP-HPLC and their amino acid sequences determined. The cystine peptides from both α-type gliadins had similar structures, and the corresponding fragments had homologous sequences. One cystine peptide of each gliadin was composed of three fragments linked by two disulphide bonds. The second cystine peptide consisted of two fragments linked by one disulphide bond. The third cystine peptide derived from α8-gliadin was different from the second peptide in one position of the sequences (glutamic acid instead of glutamine). Comparing complete sequences of α-type gliadins described in the literature, the cystine peptides from α3- and α8-gliadins were identical with corresponding sequences of clones A1235 and A212, respectively¹¹. The structures of the cystine peptides analysed indicate one intramolecular disulphide bond within domain III of α-type gliadins and two disulphide bonds between domains III and V. The linkages found correspond to homologous linkages determined for low M_r subunits of glutenin and glutenin-bound γ-type gliadins⁶. Obviously, these intramolecular disulphide bonds are not linked randomly, but are strongly directed.     

Benzoxazinoid and alkylresorcinol content,and their antioxidant potential,in a grain of spring and winter wheat cultivated under different production systems

The health-promoting properties of the grains of common wheat (Triticum aestivum L.) are associated with the presence of unique phytochemicals. This study determines the profile of alkylresorcinols (ARs) and benzoxazinoids (BXs) in T. aestivum spring and winter cultivars grown in Poland under the two different production systems: conventional and organic. Wheat grain extracts were subjected to qualitative and quantitative UPLC-UV-MS/MS analyses. The ARs profile consisted of five 5-n-alkylresorcinol derivatives, among which 5-n-heneicosylresorcinol (C21:0) and 5-n-nonadecanylresorcinol (C19:0) predominated; while six different BXs were determined in hydrothermally treated grains. Our research shows significant differences in the contents of ARs and BXs among wheat cultivars, as well as the two production systems used. Organically grown varieties had their total contents of ARs and BXs significantly higher than those grown conventionally. Another aim of the study was to determine the antioxidant capacity of alkylresorcinol extracts from tested wheat cultivars. The quantitative TLC-DPPH• method for determining the antiradical capacity of wheat ARs extracts was developed. We observed a positive relationship between the free radical scavenging activity of extracts and the total amount of ARs. The biological activity research is important for developing value-added wheat cultivars, having an improved profile and composition of nutritional substances.     

A ω-secalin contained decamer shows a celiac disease prevention activity

Celiac disease (CD) is an autoimmune permanent enteropathy that is triggered in susceptible individuals after the ingestion of gluten, a storage protein fraction presents in wheat, rye and barley endosperm. Specific gluten peptides can bind to HLA-DQ2/8 and induce lamina propria CD4⁺ T cell responses causing damage of the small intestine mucosa. Recent studies suggested that beside immunodominant and toxic epitopes, wheat gluten also contains epitopes capable of preventing the mucosal response in vitro. Among them, a decapeptide (QQPQDAVQPF) from wheat was reported to have an antagonist effect on the agglutination of K562(S) cells and celiac T-cell activation, although the corresponding nucleotidic sequence remained unknown. This study was therefore designed to clone the sequence encoding the protein carrying the decapetide with CD protective properties. A ω-secalin gene encoding containing the decapeptide QQPQRPQQPF was isolated. Although the decapeptide was not identical to the one previously described, QQPQRPQQPF showed the same capability to prevent K562(S) cell agglutination and celiac mucosa immune activation induced by toxic gliadins. The ω-secalin gene was found in wheat carrying the wheat–rye chromosomal translocations 1BL.1RS. The identification of this immunomodulatory gliadin sequence, naturally occurring in cultivars of wheat toxic for celiac patients, might offer new therapeutic strategies for CD.     

Characterisation of a ω-gliadin gene in Triticum tauschii

A ω-gliadin gene at the Gli-D^t1 locus of Triticum tauschii accession AUS18913 was isolated using PCR primers, designed from published sequences of ω-gliadin genes of bread wheat cv Cheyenne, and deduced sequences of the N-terminal amino acids of ω-gliadin proteins. Further, the derived protein was isolated from A-PAGE and was sequenced. The protein sequence contained a signal peptide of 19 amino acids followed by a short N-terminal sequence of 11 amino acids, a central repetitive domain that covers approximately 90% of the sequence and a short C-terminal domain of 12 amino acids. The sequence comparison with other ω-gliadins showed a high level of similarities between them. Further analysis of the ω-gliadins using A-PAGE revealed that there are three ω-gliadin proteins in AUS18913 accession. Comparison of N-terminal sequences of these proteins revealed that two of these proteins have very high homologies with ω-gliadins of Cheyenne while the third one was significantly different.     

Proteomic analysis of the mature kernel aleurone layer in common and durum wheat

The aleurone layer (AL) is one of inner tissues removed from the grain with the wheat bran. It is the main source of vitamins, minerals and antioxidants of potential nutritional value in the wheat kernel. The AL of three varieties of each of the two main species of wheat, Triticum aestivum (ABD) and Triticum durum (AB), were manually dissected and analysed using two-dimensional gel-based proteomics. A total of 1258 and 1109 Coomassie-stained spots were detected in the AL of representatives of the ABD and AB genomes. In two varieties (T. aestivum Chinese Spring and T. durum Bidi17), grown in two different years with full fungicide protection, no quantitative or qualitative (presence/absence) differences in spots were detected, suggesting that AL proteome is strongly genetically controlled. Comparison within and between species revealed a total of 339 AL significant protein spots. Among these spots, 30.8% differed within T. aestivum and 56.5% within T. durum varieties, whereas only 12.7% differed between the two species. Among the 142 AL proteins identified using MALDI-TOF and LC-MS/MS, 57% were globulin type storage proteins (Glo-3, Glo-3B, Glo-3C, Glo-2), 16.2% were involved in carbohydrate metabolism and 17.6% in defence/stress pathways. These variations in AL proteome are discussed.     

不同粒色小麦籽粒维生素E含量及其与籽粒色素的相关性

为了提高小麦中维生素E的含量,应用超高效液相色谱串联质谱法(大气压电喷雾离子源,负离子模式,多反应检测)测定24个不同有色小麦品种(系)籽粒中α-VE、β-VE、γ-VE、δ-VE及总VE含量,用分光光度法测定小麦籽粒色素含量,并分析小麦籽粒中维生素E含量与籽粒色素含量的相关性.结果表明,不同籽粒颜色品种(系)的VE含量和色素含量均存在显著差异,籽粒色素含量与总维生素E含量呈极显著正相关,在彩色小麦育种中可以用籽粒色素含量作为高维生素E品种(系)的一个选择指标.     

Content and relative composition of some phytochemicals in diploid,tetraploid and hexaploid Triticum species with potential nutraceutical properties

Seven cultivated Triticum species or subspecies (Triticum monococcum, Triticum turgidum ssp dicoccum, T. turgidum ssp. durum, T. turgidum ssp turanicum, Triticum timopheevii, Triticum aestivum and Triticum zhukovskyi) were compared for their contents of bioactive compounds, mainly 5-n-alkylresorcinols (ARs). Multivariate analysis of variance and principal component analysis were used to evaluate the differences in the phytochemical profiles and to establish the relationships among variables. Significant differences were observed for both total phenol (TP) and AR content. The highest AR level (377 μg/g) was observed in Triticum dicoccum, which also exhibited the highest variability for these compounds (298–436 μg/g). By contrast, the lowest AR content (286 μg/g) was found in Triticum durum. C21:0 was the main homologue chain in all the samples, its value ranging between 54.5% in T. durum and 41.2% in T. aestivum. The T. dicoccum and T. monococcum wheats had relatively low amounts of TP, whereas Triticum turanicum (215 mg/kg), T. timopheevii (250 mg/kg) and T. zhukovskyi (286 mg/kg) had approximately 3-fold higher TP levels. These results suggest that there are opportunities for breeding wheat varieties with superior health benefits and for promoting the use of ancient Triticum crops as novel sources of healthy food.     

Gluten and non-gluten proteins of wheat as target antigens in autism,Crohn’s and celiac disease

Studies show that patients with celiac disease react not only with gluten wheat proteins but also with non-gluten wheat components. Our goal was to measure IgG or IgA antibodies against wheat proteins or peptides that would provide the most sensitive method for the detection of wheat immune reaction in children with autism spectrum disorder, and patients with Crohn’s and celiac disease (CD). Using ELISA, we measured these antibodies against various gluten and non-gluten wheat proteins. Compared to controls in all three conditions, the strongest reaction was against CXCR3-binding gliadin peptide, followed by a wheat protein mixture, and α-gliadin 33-mer peptide. We determined that a sample that strongly reacted against non-gluten proteins also reacted strongly against gluten proteins. We also found that IgA antibodies against CXCR3-binding gliadin peptide were strongly reactive in a subgroup of 33% in the autism group, 42% in the Crohn’s group, and all tested samples with CD. The results indicate that measuring IgG and IgA antibodies against non-gluten proteins adds nothing to the pathologic relevance of these antibodies. Further research is needed on CXCR3-binding gliadin peptide antibodies as a possible biomarker and as a guide for dietary elimination in CD, Crohn’s disease and a subgroup of children with ASD.     

Effects of species and breeding on wheat protein composition

Wheat proteins are characterized by their excellent contribution to technological and baking properties. However, wheat proteins, especially gluten and amylase-trypsin inhibitors (ATIs), are also known to be responsible for a broad range of intolerances and allergies. In order to evaluate the impact of genetic variability on the composition of these functional but immunogenic protein types, a set of different Triticum species, including common wheat, durum, spelt, emmer and einkorn, was examined regarding ATI and gluten concentrations by RP-HPLC. Additionally, inhibition towards trypsin was determined by an enzymatic assay. Based on the results, none of the investigated wheat species can be considered to be less ‘immunogenic’. Nevertheless, due to the large variability of ATI and gluten amounts among different genotypes, the selection of less-immunoreactive wheat varieties for individuals that suffer from wheat related diseases (WRDs) might be possible. The impact of breeding was assessed for different varieties of common wheat from different breeding periods. Results revealed significant improvement of technologically valuable parameters such as the amount of high molecular weight (HMW) glutenins and the gliadin to glutenin ratio, but no increase in immunogenic proteins.     

Amylolysis of wheat starches. I. Digestion kinetics of starches with varying functional properties

The susceptibility of wheat (Triticum aestivum L.) starches to hydrolysis by pancreatic α-amylase in vitro was investigated using a series of 35 starches with slightly enriched amylose content within a narrow range (36–43%), but widely differing functional properties. After 2 h of incubation with α-amylase, native starch granules were digested to different extents, but there were no differences between any of the starches once they were gelatinized. Cooling the starch for 72 h at 4 °C after cooking reduced the susceptibility of all of the starches to enzymic digestion by a similar extent, whereas addition of monopalmitin decreased the digestibility of the starches that contained amylose, but did not affect the digestibility of waxy starches that were also included in the study. Amylopectin chain length distribution of partly digested starch granules displayed increased proportion of short and medium chains and decreased proportion of long chains in comparison to native granules. Separated large (A) and small (B) starch granules from three of the starches differed significantly in their susceptibility to in-vitro digestion. A predictive model of the susceptibility of starch in the different forms was developed from the physico-chemical and functional properties of the starches.     

Evaluation of wheat and emmer varieties for artisanal baking,pasta making,and sensory quality

Identifying varieties best suited to local food systems requires a comprehensive understanding of varietal performance from field to fork. After conducting four years of field trials to test which varieties of ancient, heritage, and modern wheat grow best on organically managed land, we screened a subset of varieties for bread, pastry, pasta, and cooked grain quality. The varieties evaluated were three lines of emmer (T. turgidum L. ssp. dicoccum Schrank ex Schübl) and eleven lines of common wheat (Triticum aestivum L.), including two modern soft wheat varieties, four soft heritage wheat varieties, four hard modern wheat varieties, and one hard heritage wheat variety. A diverse group of bakers, chefs, researchers, and consumers compared varieties for qualities of interest to regional markets. Participants assessed differences in sensory profiles, pasta making ability, and baking quality for sourdough, matzah crackers, yeast bread, and shortbread cookies. In addition to detecting significant differences among varieties for pasta, sourdough, and pastry quality, participants documented variation in texture and flavor for the evaluated products. By demonstrating which varieties perform best in the field, in the bakery, and on our taste buds, these results can support recommendations that strengthen the revival of local grain economies.     

Relationships between dough strength,polymeric protein quantity and composition for diverse durum wheat genotypes

Five different Glu-B1 HMW-GS patterns were identified among a collection of diverse durum wheat genotypes grown in 2001 in two locations in western Canada. The durum wheat lines exhibited a wide range of dough and gluten strength characteristics as measured by alveograph and 2 g mixograph parameters, gluten index (GI), and protein composition as measured by unextractable polymeric protein (UPP) content and the ratio of high-molecular weight (HMW) glutenin subunits (GS) to low-molecular weight (LMW) GS. HMW-GS subunits patterns represented within the genotypes were 6+8, 7+8, 7+16, 14+15 and 20. Two of the genotypes expressed Glu-A1 HMW-GS 2* in combination with other HMW-GS. Approximately 95% of the durum genotypes were γ-gliadin 45 types. Analysis of variance indicated that genotype was a greater source of variation in all measurements than was growing location, with the exception of protein content which showed less variation contributed by genotype and more contributed by location than for other quality parameters. UPP was strongly associated with all strength measurements. All of the γ-gliadin 42 types were low in UPP and weak. Among the γ-gliadin 45 types, those possessing HMW-GS 20 were typically in the lower half of the UPP and strength range. There was no clear evidence of an association between any of the other HMW-GS patterns and gluten strength. The majority exhibited HMW to LMW-GS ratios that were within the relatively narrow range of 0.15–0.25, yet there were wide variations in dough strength among genotypes within that range. Increasing proportions of HMW-GS resulting in ratios of greater than 0.30 were generally associated with weak dough and gluten and low UPP content.