Influence of pH and lipid membrane on the liquid-liquid phase separation of wheat γ-gliadin in aqueous conditions.

Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid-liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.

Comparative evaluation of gliadins from four extraction protocols using advanced analytical techniques.

Gliadin, a major component of gluten, is known to trigger celiac disease; therefore, its extraction is important to study its properties as well as its presence in gluten-free products. Four gliadin extraction procedures Osborne (1924), Weiss (1993), Wallace (1989) and DuPont (2005), were investigated on six wheat cultivars using advanced analytical techniques such as dynamic light scattering (DLS), X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Higher zeta potential of extracted gliadin was observed in DuPont (23.53-27), followed by Weiss (16.17-20.80), Osborne (16.17-20.13), and Wallace (14.60-19.47). Particle Z-average size (15.74-184.83 nm) was found to have an inverse relationship with the Polydispersity index (0.17-184.83). The surface morphological structure of TEM studies revealed the compact globular particle arrangement of gliadin, besides rod-shaped arrangement, was also found in DuPont and Wallace extracted gliadin. XRD pattern of gliadin exposed the crystalline domain at 44.1°, 37.8°, and 10.4° diffraction peaks. The d-spacing obtained from XRD and TEM-SAED analysis supports the presence of crystalline domains in gliadin apart from the amorphous domain. The insight obtained from this work will provide a better understanding of morphology and other properties of the same protein extracted with different extraction procedures.

Variable Immunogenic Potential of Wheat: Prospective for Selection of Innocuous Varieties for Celiac Disease Patients via in vitro Approach.

Celiac Disease (CD) is a multifactorial, autoimmune enteropathy activated by cereal proteins in genetically predisposed individuals carrying HLA DQ2/8 genes. A heterogenous gene combination of the cereal prolamins is documented in different wheat genotypes, which is suggestive of their variable immunogenic potential. In the current study, four wheat varieties (C591, C273, 9D, and K78) identified via in silico analysis were analyzed for immunogenicity by measuring T-cell proliferation rate and levels of inflammatory cytokines (Interferon-γ and Tumor Necrosis Factor-α). Peripheral Blood Mononuclear Cells and biopsy derived T-cell lines isolated from four CD patients in complete remission and two controls were stimulated and cultured in the presence of tissue transglutaminase activated pepsin-trypsin (PT) digest of total gliadin extract from test varieties. The immunogenicity was compared with PBW 621, one of the widely cultivated wheat varieties. Phytohaemagglutinin-p was taken as positive control, along with unstimulated cells as negative control. Rate of cell proliferation (0.318, 0.482; 0.369, 0.337), concentration of IFN- γ (107.4, 99.2; 117.9, 99.7 pg/ml), and TNF- α (453.8, 514.2; 463.8, 514.2 pg/ml) was minimum in cultures supplemented with wheat antigen from C273, when compared with other test varieties and unstimulated cells. Significant difference in toxicity levels among different wheat genotypes to stimulate celiac mucosal T-cells and PBMC's was observed; where C273 manifested least immunogenic response amongst the test varieties analyzed.

Efecto de la alimentación del lactante en la excreción de inmunopéptidos del gluten en heces / Influence of infant feeding on the excretion of gluten immunopeptides in feces

Introducción: la secreción de antígenos de la dieta en la leche materna ha sido ampliamente documentada. La transferencia de gliadina podría ser crítica para el desarrollo de una respuesta inmune. Objetivos: investigar la presencia de péptidos inmunogénicos de gluten en heces de lactantes alimentados con diversos regímenes. Material y métodos: estudio ciego, prospectivo, controlado, colaborativo entre tres centros hospitalarios y desarrollado entre septiembre de 2016 y enero de 2017. El protocolo del estudio fue aprobado previamente por el Comité de Ética de los hospitales de Sevilla. Resultados: se reclutaron 90 niños divididos en tres grupos de 30 niños. Un grupo experimental (edad media de 9,2 ± 2,8 semanas) con lactancia materna exclusiva; un grupo control1 (edad media 10,3 ± 3,3 semanas), alimentados con fórmula de inicio de forma exclusiva y en el que en ninguno de sus integrantes se detectó un test positivo del péptido 33-mer de gliadina en heces; y un grupo control2 con lactantes que consumían gluten en forma habitual (edades promedio 56 semanas ± 3,7 semanas) y que en un 23% (siete casos) tampoco excretaron en sus heces el PIG 33-mer. No se apreció diferencia en la cantidad de gluten ingerida por estos niños y por los que sí excretaron el péptido 33-mer en sus heces. Conclusiones: el hecho de no detectar gluten en heces de lactantes alimentados con leche materna exclusiva indica que probablemente este se encuentre por debajo de los límites para su detección. Hay niños sanos de un año de edad que, aunque consumen gluten, no lo excretan en heces

Introduction: the secretion of antigens from the diet into breast milk has been extensively documented. The transfer of gliadin could be critical for the development of an immune response. Objectives: to investigate the presence of immunogenic gluten peptides in the feces of infants fed with different diets. Material and methods: a blind, prospective, controlled, collaborative study was performed in three hospitals, between September 2016 and January 2017. The study protocol was approved by the Ethics Committee of the hospitals in Seville prior to starting the study. Results: the cohort was divided into three groups of 30 infants: an experimental group (average age 9.2 ± 2.8 weeks) with exclusive breastfeeding, a control group 1 (average age 10.3 ± 3.3 weeks) exclusively fed with onset formula and a control group 2 (average age 56 ± 3.7 weeks) with infants that consumed gluten on a regular basis. The peptide 33-mer of gliadin was negative in all feces samples from both the experimental and control group 1. With regard to control group 2, the peptide 33-mer of gliadin was negative in 23% of cases (seven children). There was no difference in the amount of gluten ingested by these children compared to those who excreted the 33-mer peptide. Conclusions: the failure to detect gluten in the feces of infants that were exclusively breastfed indicates that it is probably below the limits of detection. Healthy children who consume gluten may not excrete it in feces

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

Dynamics of liquid-liquid phase separation of wheat gliadins.

During wheat seeds development, storage proteins are synthetized and subsequently form dense protein phases, also called Protein Bodies (PBs). The mechanisms of PBs formation and the supramolecular assembly of storage proteins in PBs remain unclear. In particular, there is an apparent contradiction between the low solubility in water of storage proteins and their high local dynamics in dense PBs. Here, we probe the interplay between short-range attraction and long-range repulsion of a wheat gliadin isolate by investigating the dynamics of liquid-liquid phase separation after temperature quench. We do so using time-resolved small angle light scattering, phase contrast microscopy and rheology. We show that gliadins undergo liquid-liquid phase separation through Nucleation and Growth or Spinodal Decomposition depending on the quench depth. They assemble into dense phases but remain in a liquid-like state over an extended range of temperatures and concentrations. The analysis of phase separation kinetics reveals that the attraction strength of gliadins is in the same order of magnitude as other proteins. We discuss the respective role of competing interactions, protein intrinsic disorder, hydration and polydispersity in promoting local dynamics and providing this liquid-like behavior despite attractive forces.

Report- Immunological study of different fraction of wheat proteins.

Wheat allergy specifically refers to the adverse reaction involving IgE antibody to one or more protein fraction of wheat such as albumin, globulin, gliadin and glutenin (gluten). The majority of IgE-mediated reactions to wheat involve albumin and globulin fraction while gluten (gliadin & glutenin) also cause allergy (Celiac disease). Allergic reactions to wheat may be caused by ingestion of wheat containing foods or inhalation of flour (Bakers asthma). The present study was an effort to explore the antibody response of different proteins present in wheat. ELISA results revealed that the antibody response for albumin varied from 0.92-1.78, whereas, for globulin ranged from 1.39-1.60. Antibody response against glutenin and gliadin ranged from 0.57-1.05 and 0.98-1.95 respectively, among the different varieties of wheat. All the tested wheat varieties showed the significant difference antibody response against the different fractions of protein.

Insights into gliadin supramolecular organization at digestive pH 3.0.

Alpha-gliadin is a highly immunogenic protein from wheat, which is associated with many human diseases, like celiac disease and non-celiac gluten sensitivity. Because of that, gliadin solution is subject to intense biomedical research. However, the physicochemical nature of the employed gliadin solution at physiological pH is not understood. Herein, we present a supramolecular evaluation of the alpha-gliadin protein in water at pH 3.0 by dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM) and small-angle-.X-ray scattering (SAXS). We report that at 0.5 wt% concentration (0.1 mg/ml), gliadin is already a colloidal polydisperse system with an average hydrodynamic radius of 30 ±â€¯10 nm. By cryo-TEM, we detected mainly large clusters. However, it was possible to visualise for the first time prolate oligomers of around 68 nm and 103 nm, minor and major axis, respectively. SAXS experiments support the existence of prolate/rod-like structures. At 1.5 wt% concentration gliadin dimers, small oligomers and large clusters coexist. The radius of gyration (Rg1) of gliadin dimer is 5.72 ±â€¯0.23 nm with a dimer cross-section (Rc) of 1.63 nm, and an average length of around 19 nm, this suggests that gliadin dimers are formed longitudinally. Finally, our alpha-gliadin 3D model, obtained by ab initio prediction and analysed by molecular dynamics (MD), predicts that two surfaces prone to aggregation are exposed to the solvent, at the C-terminus. We hypothesise that this region may be involved in the dimerisation process of alpha-gliadin.

Aromatic and proteomic analyses corroborate the distinction between Mediterranean landraces and modern varieties of durum wheat.

In this paper volatile organic compounds (VOCs) from durum wheat cultivars and landraces were analyzed using PTR-TOF-MS. The aim was to characterize the VOC's profile of the wholemeal flour and of the kernel to find out if any VOCs were specific to varieties and sample matrices. The VOC data is accompanied by SDS-PAGE analyses of the storage proteins (gliadins and glutenins). Statistical analyses was carried out both on the signals obtained by MS and on the protein profiles. The difference between the VOC profile of two cultivars or two preparations of the same sample - matrices, in this case kernel vs wholemeal flour - can be very subtle; the high resolution of PTR-TOF-MS - down to levels as low as pptv - made it possible to recognize these differences. The effects of grinding on the VOC profiles were analyzed using SIMPER and Tanglegram statistical methods. Our results show that it is possible describe samples using VOC profiles and protein data.

Protective effects of ID331 Triticum monococcum gliadin on in vitro models of the intestinal epithelium.

A growing interest in developing new strategies for preventing coeliac disease has motivated efforts to identify cereals with null or reduced toxicity. In the current study, we investigate the biological effects of ID331 Triticum monococcum gliadin-derived peptides in human Caco-2 intestinal epithelial cells. Triticum aestivum gliadin derived peptides were employed as a positive control. The effects on epithelial permeability, zonulin release, viability, and cytoskeleton reorganization were investigated. Our findings confirmed that ID331 gliadin did not enhance permeability and did not induce zonulin release, cytotoxicity or cytoskeleton reorganization of Caco-2 cell monolayers. We also demonstrated that ID331 ω-gliadin and its derived peptide ω(105-123) exerted a protective action, mitigating the injury of Triticum aestivum gliadin on cell viability and cytoskeleton reorganization. These results may represent a new opportunity for the future development of innovative strategies to reduce gluten toxicity in the diet of patients with gluten intolerance.

Macromolecular changes and nano-structural arrangements in gliadin and glutenin films upon chemical modification: Relation to functionality.

Protein macromolecules adopted for biological and bio-based material functions are known to develop a structured protein network upon chemical modification. In this study, we aimed to evaluate the impact of chemical additives such as, NaOH, NH4OH and salicylic acid (SA), on the secondary and nano-structural transitions of wheat proteins. Further, the effect of chemically induced modifications in protein macromolecular structure was anticipated in relation to functional properties. The gliadin-NH4OH-SA film showed a supramolecular protein organization into hexagonal structures with 65 Å lattice parameter, and other not previously observed structural entities having a characteristic distance of 50 Å. Proteins in gliadin-NH4OH-SA films were highly polymerized, with increased amount of disulfide crosslinks and ß-sheets, causing improved strength and stiffness. Glutenin and WG proteins with NH4OH-SA showed extensive aggregation and an increase in ß-sheet content together with irreversible crosslinks. Irreversible crosslinks hindered a high order structure formation in glutenins, and this resulted in films with only moderately improved stiffness. Thus, formation of nano-hierarchical structures based on ß-sheets and disulfide crosslinks are the major reasons of high strength and stiffness in wheat protein based films.

Rapid separation of seed gliadins by reversed-phase ultra performance liquid chromatography (RP-UPLC) and its application in wheat cultivar and germplasm identification.

To separate gliadin from wheat flour, a novel and stability-indicating reversed-phase ultra performance liquid chromatography (RP-UPLC) method is established and optimized. A comparative analysis of routine capillary electrophoresis (CE), reversed-phase high-performance liquid chromatography (RP-HPLC), and RP-UPLC was performed and the results showed that the resolution and efficiency of RP-UPLC were significantly higher than those of CE and RP-HPLC. Characteristic RP-UPLC patterns of different bread wheat variety and related species were readily identified. These results demonstrated that our RP-UPLC procedure resulted in significant improvements in sensitivity, speed, and resolution, and thus is highly useful in wheat cultivar and germplasm identification.

Effects of protein in wheat flour on retrogradation of wheat starch.

Albumins, globulins, gliadins, and glutenins were isolated from wheat flour and the effects of those proteins on retrogradation of wheat starch were investigated. The results showed that only glutenins retarded retrogradation of wheat starch and other 3 proteins promoted it. The results of IR spectra proved that no S-S linkage formed during retrogradation of wheat starch blended with wheat proteins. Combination of wheat starch and globulins or gliadins through glucosidic bonds hindered the hydrolysis of wheat starch by α-amylase. The melting peak temperatures of retrograded wheat starch attached to different proteins were 128.46, 126.14, 132.03, 121.65, and 134.84 °C for the control with no protein, albumins, glutenins, globulins, gliadins groups, respectively, and there was no second melting temperature for albumins group. Interaction of wheat proteins and starch in retrograded wheat starch greatly decreased the endothermic enthalpy (△H) of retrograded wheat starch. Retrograded wheat starch bound to gliadins might be a new kind of resistant starch based on glycosidic bond between starch and protein.

The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly.

Brachypodium distachyon, is a new model plant for most cereal crops while gliadin is a class of wheat storage proteins related with wheat quality attributes. In the published B. distachyon genome sequence databases, no gliadin gene is found. In the current study, a number of gliadin genes in B. distachyon were isolated, which is contradictory to the results of genome sequencing projects. In our study, the B. distachyon seeds were found to have no gliadin protein expression by gel electrophoresis, reversed-phase high-performance liquid chromatography and Western blotting analysis. However, Southern blotting revealed a presence of more than ten copies of α-gliadin coding genes in B. distachyon. By means of AS-PCR amplification, four novel full-ORF α-gliadin genes, and 26 pseudogenes with at least one stop codon as well as their promoter regions were cloned and sequenced from different Brachypodium accessions. Sequence analysis revealed a few of single-nucleotide polymorphisms among these genes. Most pseudogenes were resulted from a C to T change, leading to the generation of TAG or TAA in-frame stop codon. To compare both the full-ORFs and the pseudogenes among Triticum and Triticum-related species, their structural characteristics were analyzed. Based on the four T cell stimulatory toxic epitopes and two ployglutamine domains, Aegilops, Triticum, and Brachypodium species were found to be more closely related. The phylogenetic analysis further revealed that B. distachyon was more closely related to Aegilops tauschii, Aegilops umbellulata, and the A or D genome of Triticum aestivum. The α-gliadin genes were able to express successfully in E. coli using the functional T7 promoter. The relative and absolute quantification of the transcripts of α-gliadin genes in wheat was much higher than that in B. distachyon. The abundant pseudogenes may affect the transcriptional and/or posttranscriptional level of the α-gliadin in B. distachyon.

A combined biochemical, biophysical and immunological approach towards the identification of celiac disease-specific wheat antigens.

Celiac disease (CD) is an inflammatory affliction of the small bowel caused by an immunological hypersensitivity to ingested wheat antigens affecting almost 1 % of the population. The gliadin fraction of wheat has been shown to contain the pathogenic antigens which react with antibodies and T cells. However, there is only limited knowledge regarding the precise nature of the wheat antigens recognized by IgA antibodies from CD patients and diagnostic tests based on the gliadin fraction have been demonstrated to give frequently false positive results. The aim of this study was the characterization of wheat antigens specifically recognized by IgA antibodies of CD patients. We developed a combined biochemical, biophysical, and immunological approach for the identification of celiac disease-specific wheat antigens. It is based on sub-fractionation of the wheat gliadin fraction using two ion exchange chromatography steps, the localization of CD-specific antigens by immunoblotting with IgA antibodies from CD patients, subsequent digestion followed by electro spray ionization-liquid chromatography/mass spectrometry (LC-ESI-MS/MS) and N-terminal sequencing by Edman degradation. Through the sub-fractionation procedure it was possible to separate CD-specific IgA-reactive wheat antigens from other wheat antigens which were also recognized by IgA antibodies of individuals without CD or by CD patients on gluten-free diet. Analysis by LC-ESI-MS/MS and N-terminal sequencing of the sub-fractions and the proteins specifically recognized by CD patients identified certain γ-gliadins with molecular mass of 37,000 and 45,000 as CD-specific wheat antigens. The CD-specific γ-gliadins with the molecular mass of 37,000 and 45,000 should be useful to study pathomechanisms of the disease and to improve the specificity of diagnostic tests for CD.

Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.

Anticancer drug-loaded gliadin nanoparticles induce apoptosis in breast cancer cells.

Nanoscale drug carriers play an important role in regulating the delivery, permeability, and retention of the drugs. Although various carriers have been used to encapsulate anticancer drugs, natural biomaterials are of great benefit for delivery and controlled release of drugs. We used the electrospray deposition system to synthesize gliadin and gliadin-gelatin composite nanoparticles for delivery and controlled release of an anticancer drug (e.g., cyclophosphamide). The size profile and synthesis of nanoparticles was characterized by dynamic light scattering and X-ray diffractometry. Cyclophosphamide was gradually released from the gliadin nanoparticles for 48 h. In contrast, the gliadin-gelatin composite nanoparticles released cyclophosphamide in a rapid manner. Furthermore, we demonstrated that breast cancer cells cultured with cyclophosphamide-loaded 7% gliadin nanoparticles for 24 h became apoptotic, confirmed by Western blotting analysis. Therefore, the gliadin-based nanoparticle could be a powerful tool for delivery and controlled release of anticancer drugs.

Validation of a new enzyme-linked immunosorbent assay to detect the triggering proteins and peptides for celiac disease: interlaboratory study.

The performance of Gluten-Tec (EuroProxima, Arnhem, The Netherlands) was tested through an interlaboratory study in accordance with AOAC guidelines. Gluten-Tec is a competitive ELISA that detects an immunostimulatory epitope of a-gliadin in dietary food for celiacs. Fifteen laboratories, representing 14 different countries, announced their interest in taking part in this study. Of the 12 laboratories that sent the results within the established timeframe, two submitted inappropriate standard curves and were excluded from the statistical analysis. Four different food matrixes (rice-based baby food, maize bread, chocolate cake mix, and beer) were selected for preparing the test samples. Two gliadin extraction procedures were used: the conventional 60% ethanol, and a new method based on the reducing reagent dithiothreitol. The 38 samples (19 blind duplicates) tested in this study were prepared by diluting the different extracts in order to cover a wide range of gliadin levels. Both sample extraction and dilution were performed by EuroProxima; the present interlaboratory study was focused only on testing the ELISA part of the Gluten-Tec kit protocol. Repeatability values (within-laboratory variance), expressed as RSD(r) ranged from 6.2 to 25.7%, while reproducibility values (interlaboratory variance), expressed as RSD(R), ranged from 10.6 to 45.9%. Both statistical parameters were in the acceptable range of ELISAs under these conditions, and the method will be presented to the Codex Alimentarius as a preferred method for gluten analysis.

Molecular and immunological characterization of gluten proteins isolated from oat cultivars that differ in toxicity for celiac disease.

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.

Impact of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis in mice.

The effects of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis (WDEIA) were investigated by using a mouse model. The gliadin fraction was prepared as a 70% ethanol-soluble solution, and ω-5 gliadin was purified by chromatography. Purified ω-5 gliadin was run on SDS-PAGE gel to reveal three bands with a molecular mass of 53-60 kDa and had the characteristic N-terminal sequence of ω-5 gliadin. The mice were sensitized to the gliadin fraction, and the anaphylactic response was assessed by measuring the body temperature and voluntary physical activity. An oral administration of ω-5 gliadin evoked a significant drop in both the body temperature and voluntary physical activity, similar to the effects of the whole gliadin fraction. ELISA and immunoblotting analyses revealed that the IgE expression from sensitized mice reacted most strongly to ω-5 gliadin. Taken together, these results indicate ω-5 gliadin to be a major allergen responsible for stimulating WDEIA in mice, with the characteristic potential for stimulating IgE production.