Study of IgE antigenic relationships in hyper sensitivity 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 -3 hordein. This protein was chosen for its cross-reactivity with -5 gliadin, a major allergen in WDEIA. Results: The IgE from both sera strongly reacted with natural and recombinant -3 hordein but displayed different patterns of reactivity with wheat, barley and rye proteins. Those from the WDEIA patient showed expected reactions with -5 gliadin, -35 and -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 gliadins, and -75 secalin, but very weakly with -35 secalin and LMW-GS. Recombinant -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

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 ω5-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 ω5-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 ω5-gliadins was studied by immunoblot inhibition experiments, using purified natural ω5-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 ω5-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 ω5-gliadin was only partial. Conclusion: LMW-GS are able to promote local inflammation and they share common epitopes with ω5-gliadins. The nature of these epitopes is discussed. LMW-GS also carried specific epitopes, completely independent from the ω5-gliadin epitopes. Thus, LMW-GS behaved partly as independent allergens

Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase.

It has now been clearly shown that lipid droplets (LDs) play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Delta mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+) ATPase (V-ATPase). Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS) complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective. 2015. Published by The Company of Biologists Ltd

Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis

In oleaginous seeds, lipids-stored in organelles called oil bodies (OBs)-are degraded post-germinatively to provide carbon and energy for seedling growth. To date, little is known about how OB coat proteins, known as oleosins, control OB dynamics during seed germination. Here, we demonstrated that the sequential proteolysis of the five Arabidopsis thaliana oleosins OLE1-OLE5 begins just prior to lipid degradation. Several post-translational modifications (e.g. phosphorylation and ubiquination) of oleosins were concomitant with oleosin degradation. Phosphorylation occurred only on the minor OLE5 and on an 8 kDa proteolytic fragment of OLE2. A combination of immunochemical and proteomic approaches revealed ubiquitination of the four oleosins OLE1-OLE4 at the onset of OB mobilization. Ubiquitination topology was surprisingly complex. OLE1 and OLE2 were modified by three distinct and predominantly exclusive motifs: monoubiquitin, K48-linked diubiquitin (K48Ub2) and K63-linked diubiquitin. Ubiquitinated oleosins may be channeled towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin-proteasome pathway. A proteasome inhibitor (MG132) reduced oleosin degradation and induced cytosolic accumulation of K48Ub2-oleosin aggregates. These results indicate that K48Ub2-modified oleosins are selectively extracted from OB coat and degraded by the proteasome. Proteasome inhibition also reduced lipid hydrolysis, providing in vivo evidence that oleosin degradation is required for lipid mobilization

Probing cytokinin homeostasis in Arabidopsis thaliana by constitutively overexpressing two forms of the maize cytokinin oxidase/dehydrogenase 1 gene

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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

Immunoblotting analysis of wheat allergens: control of side reactions through wheat polypeptides naturally present in dried cow milk

Immunoblotting analysis of wheat allergens can be a two-step procedure: an incubation with a human serum and one with an anti-IgE-horseradish peroxidase (HRP) conjugate, visualised by chemiluminescence. Different procedures produced side reactions that appeared as non-specific additional bands which hampered the identification of specific IgE-reacting proteins. We then showed that the rabbit anti-human IgE-HRP conjugate most likely contains anti-wheat antibodies that bind with the proteins. We investigated another procedure to accurately identify wheat specific IgE-reacting proteins. The use of polyvinylpyrrolidone-40 throughout the procedure and 3% cow milk for the conjugate incubation provided signals clear-cut enough to detect proteins up to the allelic level. Tested against patients, the procedure identified IgE-reacting proteins. It also provided new data in terms of gliadins for patients suffering from hypersensitivity to hydrolysed wheat proteins. Lastly, we investigated why dried cow milk blocks. Using rabbit serum containing anti-wheat antibodies, we detected wheat cross-reacting polypeptides in it

A Recombinant omega-Gliadin-like D-Type Glutenin and an alpha-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 omega-gliadins, but they contain a cysteine, that allows them to be included in the gluten macropolymers. An omega-gliadin-like D-type glutenin, an alpha-gliadin, and an omega 5-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 omega 5-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 omega-gliadin-like D-type glutenin and alpha-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 omega 5-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