Barley γ3-hordein: glycosylation at an atypical site, disulfide bridge analysis, and reactivity with IgE from patients allergic to wheat.

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.

A recombinant ω-gliadin-like D-type glutenin and an α-gliadin from wheat (Triticum aestivum): two immunoglobulin E binding proteins, useful for the diagnosis of wheat-dependent allergies.

Among the wheat prolamins, D-type glutenins display a highly repetitive sequence similar to ω-gliadins, but they contain a cysteine, that allows them to be included in the gluten macropolymers. An ω-gliadin-like D-type glutenin, an α-gliadin, and an ω5-gliadin-like D-type glutenin were obtained as recombinant proteins and compared using synchrotron radiation circular dichroism. This technique evidenced the strong thermostability of the ω5-gliadin-like protein. The IgE reactivity of recombinant proteins was evaluated using 45 sera from wheat-allergic patients. The sera from patients diagnosed with cutaneous hypersensitivity to hydrolyzed wheat proteins often reacted with the ω-gliadin-like D-type glutenin and α-gliadin, whereas the IgE reaction was less frequent after dietary sensitization. So, these two proteins could be useful to diagnose these diseases. The sera from patients with exercise-induced anaphylaxis recognized the ω5-gliadin-like protein as a positive control and, less frequently, the other proteins tested. Only some sera from patients with baker's asthma reacted with the proteins tested.

Low molecular weight glutenins in wheat-dependant, exercise-induced anaphylaxis: allergenicity and antigenic relationships with omega 5-gliadins.

BACKGROUND: Adults suffering from wheat-dependant, exercise-induced anaphylaxis (WDEIA) develop IgE directed against wheat omega5-gliadins (major allergens for this allergy) and against wheat low-molecular weight glutenin subunits (LMW-GS). However, the ability of LMW-GS to trigger an inflammatory response is still unknown. It also remains to be determined if IgE from these patients bind the same epitopes on LMW-GS and omega5-gliadins or if the epitopes are independent. METHODS: WDEIA patients were selected and skin prick tests (SPTs) were performed on them using commercial gluten, wheat flour extracts, prolamin fractions and a purified natural LMW-GS P42. The IgE-binding ability of natural and recombinant wheat prolamins was verified by immunoblot experiments. Cross-reactivity between LMW-GS and omega5-gliadins was studied by immunoblot inhibition experiments, using purified natural omega5-gliadin as an inhibitor. RESULTS: Patients developed positive SPTs with natural LMW-GS fractions and/or with the purified LMW-GS P42. Natural and recombinant LMW-GS were highly reactive with patient IgE in immunoblot experiments, as was omega5-gliadin. However, differences in reactivity were evident within the LMW-GS group. Except for one recombinant LMW-GS (P73), IgE cross-reactivity between LMW-GS and natural omega5-gliadin was only partial. CONCLUSION: LMW-GS are able to promote local inflammation and they share common epitopes with omega5-gliadins. The nature of these epitopes is discussed. LMW-GS also carried specific epitopes, completely independent from the omega5-gliadin epitopes. Thus, LMW-GS behaved partly as independent allergens.

High molecular weight entities in industrial wheat protein hydrolysates are immunoreactive with IgE from allergic patients.

Hydrolyzed wheat proteins (HWP) can induce immediate hypersensitivity through skin contact and/or food ingestion. Such patients develop IgE against unmodified wheat proteins without allergy to wheat. Our objective was to study the IgE-reacting content of HWP. We compared the reactivity of HWP and unmodified wheat proteins with IgE from patients suffering from immediate hypersensitivity to HWP. We studied the cross-reactivity between one HWP preparation and wheat proteins using immunoblot inhibition experiments. This showed that the tested HWP carried mainly unmodified epitopes originating from wheat proteins. The size distribution of polypeptides from two HWP preparations was analyzed by size-exclusion-high performance liquid chromatography (SE-HPLC), and their reactivity with IgE was studied. This showed that they contained highly IgE-reacting high molecular weight entities, likely resulting in a rearrangement of peptides issued from gluten processes. These multiepitopic entities could explain the high immunogenicity of HWP for sensitized people.

Physical and biochemical properties of airborne flour particles involved in occupational asthma.

Aerosol particles which deeply penetrate the human airways and which trigger baker's asthma manifestations are known to represent only a part of flour and of airborne particles found in bakeries. They were a major focus of this study. To this end, aerosols were produced from different wheat and rye flours, using an automatic generator designed for bronchial challenge. Particles were characterized for their size distribution, their ability to be deposited in the airways, their protein content, their histological composition and their reactivity with immunoglobulin E (IgE) present in sera from asthmatic bakers. Like dust particles collected in the bakery, the aerosols produced showed increased protein content but decreased IgE reactive protein content when compared to the corresponding bulk flours. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of these particles showed a predominance of endosperm gluten proteins. Under scanning electron microscopy, flour particles displayed various tissue fragments with entrapped large A-starch and small B- or C-starch granules, whereas aerosol particles appeared primarily as a mixture of the endosperm intracellular interstitial protein matrix and small B- or C-starch granules free or still associated. These observations showed that aerosols supposed to penetrate deeply the airways, mainly correspond to intracellular fragments of endosperm cells enriched in gluten proteins but with lower amount of allergens belonging to albumins or globulins.

Recombinant proteins and peptides as tools for studying IgE reactivity with low-molecular-weight glutenin subunits in some wheat allergies.

Two genes of wheat low-molecular-weight glutenin subunits (LMW-GS), B16 and P73, were cloned and expressed in E. coli. They were homologous to proteins encoded respectively at Glu-B3 and Glu-D3 loci. The N-terminal and C-terminal halves of B16 (NB16 and B16C) and the two chimeras combining the halves of the two genes (B16-P73 and P73- B16) were also expressed. All these constructs were compared for their reactivity with IgE from 24 patients suffering from different forms of wheat allergies. The results confirmed that LMW-GSs bound IgE in all adult allergies tested. Strong differences in reactivity between all the constructs were observed. They were disease-dependent. In wheat-dependent exercise-induced anaphylaxis (WDEIA), the reactivity of the constructs depended partly on common epitopes with omega-5 gliadins but also on differences in molecule conformation. The presence of NB16 in the constructs greatly influenced their IgE reactivity.

Influence of the allelic variants encoded at the Gli-B1 locus, responsible for a major allergen of wheat, on IgE reactivity for patients suffering from food allergy to wheat.

Wheat presents an important genetic diversity that could be useful to look for cultivars with reduced allergencity. omega5-Gliadins have been described as major allergens for wheat allergic patients suffering from wheat-dependent exercise-induced anaphylaxis (WDEIA) and some cases of chronic urticaria (U). Our objective was to study the influence of genetic variability at the Gli-B1 locus encoding for omega5-gliadins on the reactivity of IgE antibodies from these patients. We selected cultivars expressing 13 alleles at Gli-B1 including a wheat/rye translocation and studied the reactivity to gliadins of a rabbit antiserum specific for omega5-gliadins and of IgE from 10 patients. The antiserum and IgE from nine patients with WDEIA and U strongly detected omega5-gliadins expressed by most of the Gli-B1 alleles but showed no or faint responses to the gliadins and secalins extracted from the translocated wheat. The selection of genotypes lacking the Gli-B1 locus may reduce wheat allergenicity.

Hydrolysed wheat proteins present in cosmetics can induce immediate hypersensitivities.

Cosmetics containing hydrolysed wheat proteins (HWP) can induce rare but severe allergic reactions. 9 patients, all females without common wheat allergy, but with contact urticaria to such cosmetics, were studied. 6 of them also experienced generalized urticaria or anaphylaxis to foods containing HWP. All patients had low to moderate levels of immunoglobulin (Ig)E specific of wheat flour (f4) or gluten (f79). Their sensitivity to HWP and their tolerance to unmodified wheat proteins extracted from grains were confirmed using skin tests. Immunoblotting analyses showed that IgE from all patients reacted with almost all HWP tested. Reactions generally occurred with large random peptide aggregates. IgE reacted also with unmodified grain proteins, which contrasted with skin tests results. They reacted always with salt soluble proteins but variably with gluten proteins. No reaction occurred with gliadins in patients without associated immediate hypersensitivity to food containing HWP. These results show the role of hydrolysis on the allergenicity of wheat proteins, both through skin or digestive routes. At least part of the epitopes involved is pre-existing in unmodified wheat proteins. The aggregation of peptide bearing these epitopes and others created by hydrolysis, along with the increased solubility and the route of exposure, are possible factors of the allergenicity of HWP.

Study of IgE antigenic relationships in hypersensitivity to hydrolyzed wheat proteins and wheat-dependent exercise-induced anaphylaxis.

BACKGROUND: Wheat is involved in different forms of respiratory, food and contact allergy. The IgE of patients generally reacts with various flour proteins. It is not known if antigenic relationships could explain some of these reactions and if proteins could be involved in different pathologies. METHODS: Two sera were selected as representative of patients with either wheat-dependent exercise-induced anaphylaxis (WDEIA) or hypersensitivity to hydrolyzed wheat proteins (HHWP). Their IgE specificity was studied with wheat, barley and rye proteins, using immunoblot, and immunoblot inhibition with recombinant gamma-3 hordein. This protein was chosen for its cross-reactivity with omega-5 gliadin, a major allergen in WDEIA. RESULTS: The IgE from both sera strongly reacted with natural and recombinant gamma-3 hordein but displayed different patterns of reactivity with wheat, barley and rye proteins. Those from the WDEIA patient showed expected reactions with omega-5 gliadin, gamma-35 and gamma-75 secalins, but also with wheat low-molecular-weight glutenin subunits (LMW-GS), and not with C hordeins. On the contrary, IgE from a HHWP patient reacted with C hordeins, various omega gliadins, and gamma-75 secalin, but very weakly with gamma-35 secalin and LMW-GS. Recombinant gamma-3 hordein inhibited strongly but not totally the WDEIA patient's IgE binding to prolamins. No such inhibition could be observed for the HHWP patient's IgE. CONCLUSIONS: At least part of the reactions of prolamins with the IgE from the WDEIA patient was due to antigenic homologies. The occurrence of cross-reacting carbohydrates was unlikely. These common IgE epitopes were not involved in the pathology of the HHWP patient.

Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot.

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.