Antibody reactivity in patients with IgE-mediated wheat allergy to various subunits and fractions of gluten and non-gluten proteins from ω-gliadin-free wheat genotypes

Introduction and objective Gluten proteins (gliadins and glutenins) are polymorphic wheat storage proteins of allergenic properties. Significant differences in chemical composition between both protein groups allow to expect highly specific immunological response of individual subunits and fractions in reactions with IgE sera of people allergic to wheat. The aim of these studies was to identify and characterize the most allergenic gluten proteins (GP) and nongluten proteins (NGP) occurred in two closely related wheat hybrid genotypes. Material and Methods 3xC and 3xN wheat hybrids, which differ strongly in regard of gliadin composition, were analyzed. Seven people manifesting different symptoms of wheat allergy donated sera for the experiment. The technique of immunoblotting after SDS-PAGE was used for identification of allergenic subunits and fractions among GP and NGP. Immunologically active protein bands were visualized by chemiluminescence. Results Great variation of immunodetection spectra was observed. Results of immunoblotting showed LMW glutenins to be of highest, gliadins of medium, while NGP of lowest allergenicity for selected patients. The 43-kDa and 47-kDa LMW glutenin subunits, 40-kDa and 43-kDa γ-gliadin fractions and 49-kDa NGP can be considered as the most immunoreactive among all protein bands [b]separated by SDS-PAGE. Conclusions The observed differentiation of immunodetection spectra allows to model highly specific IgE-binding profiles of allergenic wheat proteins attributed to individual patients with symptoms of gluten intolerance. Highly immunoreactive subunits and fractions among GP and NGP were identified. The observed immunoreactivity of 49 kDa NGP is worth to emphasize, as it has never been reported as wheat allergenic protein before

Does nitrogen fertilization affect the secondary structures of gliadin proteins in hypoallergenic wheat?

One of the macronutrients indispensable for plant growth and development is nitrogen (N). It is responsible for starch and storage protein (gliadins and glutenins) biosynthesis and, in consequence, influences kernels’ quality and yields. However, applying N-fertilizers increases gluten content in wheat, and it may intensify the risk of developing allergy symptoms in gluten-sensitive individuals. The purpose of our research was to analyse whether and how the elimination of N-fertilizers during the cultivation of wasko.gl− wheat (modified genotype lacking ω-gliadins) changes the secondary structures of gliadin proteins. To this aim, using the FT-Raman technique, we examined flour and gliadin protein extracts obtained from kernels of two winter wheat lines: wasko.gl+ (with a full set of gliadin proteins) and wasko.gl− (without ω-gliadin fraction) cultivated on two different N-fertilization levels—0 and 120 kg N·ha(−1). On the basis of the obtained results, we proved that nitrogen fertilization does not have a major impact on the stability of the secondary structures of gliadin proteins for wasko.gl− wheat line with reduced allergenic properties. Furthermore, the results presented herein suggest the possibility of increasing the stability of glutenin structures as a result of the N-fertilization of wasko.gl− wheat line, which gives hope for its use in the production of wheat articles devoted to people suffering from diseases related to gluten sensitivity

Antibody reactivity in patients with IgE-mediated wheat allergy to various subunits and fractions of gluten and non-gluten proteins from ω-gliadin-free wheat genotypes

Introduction and objective Gluten proteins (gliadins and glutenins) are polymorphic wheat storage proteins of allergenic properties. Significant differences in chemical composition between both protein groups allow to expect highly specific immunological response of individual subunits and fractions in reactions with IgE sera of people allergic to wheat. The aim of these studies was to identify and characterize the most allergenic gluten proteins (GP) and nongluten proteins (NGP) occurred in two closely related wheat hybrid genotypes. Material and Methods 3xC and 3xN wheat hybrids, which differ strongly in regard of gliadin composition, were analyzed. Seven people manifesting different symptoms of wheat allergy donated sera for the experiment. The technique of immunoblotting after SDS-PAGE was used for identification of allergenic subunits and fractions among GP and NGP. Immunologically active protein bands were visualized by chemiluminescence. Results Great variation of immunodetection spectra was observed. Results of immunoblotting showed LMW glutenins to be of highest, gliadins of medium, while NGP of lowest allergenicity for selected patients. The 43-kDa and 47-kDa LMW glutenin subunits, 40-kDa and 43-kDa γ-gliadin fractions and 49-kDa NGP can be considered as the most immunoreactive among all protein bands [b]separated by SDS-PAGE. Conclusions The observed differentiation of immunodetection spectra allows to model highly specific IgE-binding profiles of allergenic wheat proteins attributed to individual patients with symptoms of gluten intolerance. Highly immunoreactive subunits and fractions among GP and NGP were identified. The observed immunoreactivity of 49 kDa NGP is worth to emphasize, as it has never been reported as wheat allergenic protein before

Genetic diversity of winter wheat cultivars and strains determined by electrophoregrams of gliadin and glutenin proteins

Based on the polymorphism of gliadin and glutenin proteins relationships of 45 cultivars and strains of winter wheat were evaluated. The cluster analysis showed a considerable variation of the investigated genotypes. The similarity indices were calculated using the Nei and Li formula. The genetic distances between the cultivars ranged from 1.00 to 0.12. The highest similarity index - SI=1.00- being proof of the identical physicochemical composition of storage proteins, was found for the pair Farmer and Elena. The groups of similar and genetically distant cultivars have been presented in the form of a dendrogram. The possibility of using the results obtained from the cluster analysis in breeding programmes has been discussed

Relationship between wheat storage proteins and winter hardiness in chosen hybrids of winter wheat

Celem badań było poszukiwanie związków pomiędzy markerami biochemicznymi a stopniem przezimowania u 2 grup pszenic ozimych: 19 wieloliniowych genotypów (90 linii F5) pochodzących z 6 kombinacji mieszańcowych pszenicy zwyczajnej (T. aestivum ssp. vulgare) oraz trzech par genotypów wytworzonych z mieszańca orkiszu (odmiany Oberkummer Rotkorn (Triticum aestivum spelta L.) i rodu LAD 480 (T. aestivum ssp. vulgare). Materiał poddano analizom elektro¬foretycznym. W pierwszej grupie badanego materiału stwierdzono silne zróżnicowanie pod względem zimotrwałości wahające się od 4 do 9 stopni (w skali 9°). Uzyskane wyniki sugerują, że kombinacja podjednostek HMW glutenin [Glu A1(Null), Glu B1(7+9), Glu D1 (5+10)] jest powiązana z podwyższoną zimotrwałością pszenicy ozimej. Zimotrwałość drugiej grupy mieszańców oddalonych była niższa i wahała się od 1 do 5 stopnia. Markerami wysokiej zimotrwałości u tej grupy mieszańców okazał się blok białek gluteninowych z podjednostką HMW warunkowanych przez locus Glu A1 z podjednostką „2”i locus Glu D1 z podjednostką „5+10/2+12” oraz blok białek gliadynowych z podjednostkami zawierającymi markery białkowe linii z null allelem w locus Gli B2.The aim of these studies was to find out whether polymorphic wheat storage proteins (gliadins and HMW glutenins) and winter hardiness are interrelated. Two sets of winter wheats were analyzed. In the first set 19 groups of hybrid genotypes (90 lines in total) of F5 generation from cross combinations between common wheat (Triticum aestivum ssp. vulgare) cultivars and strains were analyzed. The second set comprised four pairs of hybrid genotypes originating from crossing between spelt (Triticum aestivum ssp. spelta, cv. Obercummer Rotkorn) and common wheat breeding strain LAD 480. Gliadins and HMW glutenins extracted from individual kernels of analyzed hybrid genotypes were separated using electrophoretic techniques: A-PAGE and SDS-PAGE. The obtained results suggested close relationships between HMW glutenin subunits: Glu A1-1, Glu B1-7+9/6+8, Glu D1-5+10 and gliadin null alleles localized on the chromosome 6B (locus Gli B2) and winter hardiness of winter wheat

Changes in qualitative and quantitative traits of birch (Betula pendula) pollen allergenic proteins in relation to the pollution contamination

Birch (Betula pendula) pollen causes inhalant allergy in about 20% of human population in Europe, most of which is sensitive to the main birch allergen, Bet v1. The aim of the study was to find out (i) whether and how the analysed birch individuals differ in regard to composition of individual subunits of pollen proteins and to protein content in these subunits; (ii) whether the level of particulate matter relates to concentration of Bet v1 allergen. Study was performed in Southern Poland, in 2017–2019. Pollen material was collected at 20 sites, of highly or less polluted areas. Protein composition was analysed by SDS-PAGE, while the concentration of Bet v1 was evaluated by ELISA. The obtained results were estimated at the background of the particulate matter (PM10) level and the birch pollen seasons in Kraków. The electrophoregrams of pollen samples collected at different sites showed huge differences in staining intensities of individual protein subunits, also among important birch allergens: Bet v1, Bet v2, Bet v6 and Bet v7. The level of Bet v1 was significantly higher in the pollen samples collected at the more polluted sites. While the birch pollen allergenic potential is determined, the both pollen exposure and the content of the main allergenic components should be considered, as factors causing immunological response and clinical symptoms manifestation in sensitive individuals

Efficiency of Poroshell type chromatogrphic columns for wheat gliadin separation using RP-HPLC

Wysokosprawna chromatografia cieczowa w odwróconej fazie (RP-HPLC) jest efektywną metodą analizy polimorficznych białek roślin uprawnych, w tym również białek zapasowych zbóż. Możliwość jej powszechnego wykorzystania w hodowli roślin ogranicza jednak znaczny koszt aparatury, wyposażenia i odczynników. W ostatnich latach opracowano nowy rodzaj kolumn chromatograficznych o złożach C18 osadzonych na powierzchniowo porowatej krzemionce (tzw. Fused-Core technology). Główną zaletą tych kolumn jest wysoki wzrost zdolności rozdzielczej (zwiększenie ilości tzw. półek teoretycznych), co daje możliwość znacznego skrócenia czasu, a tym samym obniżenia kosztów analizy. Celem badań było oszacowanie dokładności i efektywności rozdziału polimorficznych białek gliadynowych metodą RP-HPLC przy użyciu kolumn typu Fused-Core w porównaniu do kolumn C18 o klasycznych parametrach ziaren krzemionki. Materiał roślinny stanowiły linie mieszańcowe pszenicy ozimej różniące się pod względem alleli kontrolujących syntezę gliadyn. Analizowano pary blisko spokrewnionych linii mieszańcowych, z których jedna zawierała null allel natomiast druga stanowiła genotyp kontrolny. Porównywano także rozdziały mieszańców z ich komponentami rodzicielskimi. Punktem odniesienia dla oceny obrazów chromatograficznych kolumny typu Fused-Core — 300SB-C18 Poroshell były rozdziały uzyskane na tradycyjnej kolumnie SB300 Zorbax C18. Wyniki badań potwierdziły, iż analiza chromatograficzna gliadyn na kolumnie Poroshell jest metodą szybką i ekonomiczną. Wysokiej jakości obrazy rozdzielonych białek uzyskiwano już w czasie 8 minut. Jednakże krótki czas rozdziału obniżył ich rozdzielczość, jedną z najważniejszych zalet chromatografii RP-HPLC. Wyniki badań sugerują, iż kolumny typu Fused-Core mogą być przede wszystkim efektywnym narzędziem selekcji masowej natomiast tradycyjne kolumny, pozwalające identyfikować subtelne szczegóły obrazu chromato¬graficznego, mogą okazać się bardziej przydatne w badaniach genetycznych np. w analizie rozszczepienia genów kontrolujących syntezę białek prolaminowych.RP-HPLC is an efficient tool for wheat storage proteins separation. Its extensive utilization in plant breeding is limited, however, by heavy expenses of apparatus, equipment and reagents. Recently a new type of chromatographic columns was introduced where stationary phase is based on the superficially porous silca (so called Fused-Core Particles). The main advantage of the Fused-Core stationary phase is a high increase of separation power (theoretical plates) which enables shortening of the separation time (up to few minutes) and consequently the reduction of the analysis costs. The aim of this study was a comparative evaluation of the accuracy and efficacy of the Fused-Core C18 column with a classic C18 column in the separation of wheat gliadins — one of the most complex and polymorphic plant proteins. Closely related winter wheat hybrid lines, differentiated in respect of gliadin controlling alleles, were analysed. Gentypes containing gliadin null alleles and corresponding control lines as well as parental forms and their progenies were compared using Fused-Core Poroshell 300SB-C18 and traditional Zorbax 300SB-C18 columns. The obtained results proved that RP-HPLC separation on the Poroshell column are indeed very fast and economical as the high quality chromatograms were obtained after 8 minutes of separation. However, their resolution was considerably lower as compared to the traditional column. In conclusion, the rapid PR-HPLC appears to be very well suited for mass selection of breeding materials, where large number of genotypes must be evaluated in a very short time period. However in a genetic research, where identification of discrete details of the chromatograms could be important for genotype classification, traditional columns seem to be more useful

FT-Raman spectroscopy as a tool to study the secondary structures of wheat gliadin proteins

Raman spectroscopy is a useful method in biological, biomedical, food, and agricultural studies, allowing the simultaneous examination of various chemical compounds and evaluation of molecular changes occurring in tested objects. The purpose of our research was to explain how the elimination of ω-fractions from the wheat gliadin complex influences the secondary structures of the remaining αβγ-gliadins. To this aim, we analyzed the endosperm of wheat kernels as well as gliadin proteins extracted from two winter wheat genotypes: wasko.gl+ (control genotype containing the full set of gliadins) and wasko.gl− (modified genotype lacking all ω-gliadins). Based on the decomposition of the amide I band, we observed a moderate increase in β-forms (sheets and turns) at the expense of α-helical and random coil structures for gliadins isolated from the flour of the wasko.gl− line. Since ω-gliadins contain no cysteine residues, they do not participate in the formation of the disulfide bridges that stabilize the protein structure. However, they can interact with other proteins via weak, low-energetic hydrogen bonds. We conclude that the elimination of ω-fractions from the gliadin complex causes minor modifications in secondary structures of the remaining gliadin proteins. In our opinion, these small, structural changes of proteins may lead to alterations in gliadin allergenicity