Michel Laurière

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.

Is the application of cosmetics containingprotein-derived products safe?

Plant, mainly wheat, proteins hydrolyzed or not, are common substitutes for animal proteins in cosmetics for safety reasons. Contact urticaria induced by cosmetics is not common. The conjunction of contact urticaria and oral allergy to the same plant proteins is even less frequent. Gluten is the major group of proteins in wheat and other cereal grains. We describe here a case of urticaria to hydrolyzed wheat proteins in cosmetics associated with generalized urticaria induced by preserved foods containing wheat proteins.

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

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.

Antigenic homologies between oat and wheat globulins

Total globulins of oat and wheat were compared by SDS‐electophoresis immunoblotting and in vitro protein synthesis experiments. Numerous homologies were found. The same 4 protein subgroups, with respect to molecular mass, were found in the two cereals. Immunoblotting with antibodies for total oat globulins allowed the detection of homologous proteins in each wheat protein group, especially in the 60‐kDa group, which corresponds to the major 12 S oat globulin. The same antibodies also reacted with in vitro synthesized proteins of the two cereals. However, compared to mature proteins, only a limited number of polypeptides react, suggesting that wheat globulins, like oat globulins, undergo post‐translational processes.

Polyacrylamide gel-urea electrophoresis of cereal prolamins at acidic pH

Abstract An electrophoretic procedure is described for vertical polyacrylamide gel-urea electrophoresis of cereal prolamins. The polyacrylamide gel is directly polymerized in potassium lactate buffer, pH 3.6, with an ammonium persulfate-silver nitrate system of catalysts, which enables one to control the polymerization of acrylamide. Electrophoresis is performed in aluminum lactate buffer, pH 3.6. Thus proteins are separated in a discontinuous system of buffers, which allows sample concentration at the beginning of the electrophoresis. Because the system acts in a very dissociating medium (6 m urea), any type of prolamins (e.g., gliadin, secalin, hordein, avenin, or zein) can be simultaneously analyzed and compared on the same gel slab. On the other hand, as is shown with barley prolamins, improved resolution is obtained, without any sample-reducing requirement during the same run, for both typical hordeins (B- and C-hordeins) and fast-moving low-molecular-weight proteins (A-polypeptides).

ACCURATE DETECTION OF BOTH GLYCOPROTEINS AND TOTAL PROTEINS ON BLOTS : CONTROL OF SIDE REACTIONS OCCURRING AFTER PERIODATE OXIDATION OF PROTEINS

The detection of glycoproteins based on the periodate oxidation of their carbohydrate moiety, and their conjugation to digoxigenin hydrazide directly on blots, leads to high background staining, especially with polyvinylidene difluoride (PVDF) membranes. The additon of Tween‐20 to all incubation solutions, except at the oxidation step, strongly reduced the background staining and allowed us to detect lower amounts of glycans fixed on the blots through the protein moiety. The presence of polysaccharides during the oxidation step was shown to produce side reactions that led to the staining of nonglycosylated proteins; it demonstrated risks that may occur with crude extracts when periodate oxidation is performed in solution. The phenomenon was used to design a total protein‐staining assay that can be included along with positive and negative controls in a general strategy based on blotting, which was delineated for the identification of glycoproteins in complex tissue extracts.

Nature et fractionnement des protéines de l'Orge extraites par l'éthanol, l'isopropanol et le n-propanol à des titres différents

Summary Barley alcoholsoluble protein extractibilities by aqueous ethanol, isopropanol and n-propanol were measured at room temperature. The quantities extracted by each alcohol strongly depend on the concentration of the alcohol. The most efficient concentration for the three alcohols were by increasing order: 45 per cent ethanol, 40 per cent isopropanol, 35 per cent (w/w) n-propanol. Hordein preparations extracted by these three alcoholic solutions and by 75 per cent (w/w) ethanol were compared by means of the flour nitrogen percentage they contain and by electrophoresis, amino-acid analysis and Sephadex G 100 gel filtration. The preparations studied do not differ markedly in their amino-acid composition or electrophoretic pattern which shows at least 17 different bands. On the other hand, Sephadex G 100 gel filtration separates two main groups of proteins. The first one is present at the same level in all preparations studied and consists of electrophoretically typical hordeins (already described hordeins). The other group represents a fraction of the preparation, the more abundant as the solvent is more effective. This second group is excluded on Sephadex G 100 chromatography and does not give well defined bands by starch gel electrophoresis. Consequently it is related to some glutelins. Nevertheless its amino-acid composition is very close to the mean hordein composition. Electrophoretic comparison with glutelins extracted by acetic acid and with hordeins, all reduced and alkylated, discloses a great similitude between this fraction, the glutelins and some hordein fast components α, β and γ.

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 bakers asthma reacted with the proteins tested.

PRESENCE OF GLYCOSYLATED POLYPEPTIDES IN GLIADIN AND GLUTENIN FRACTIONS

1 SUMMARY Glycans covalently bound to proteins, were investigated on gliadin or glutenin fractions, using specific chemical derivatization of glycans, anti-carbohydrate antibodies and lectins. Glycosylated proteins were evidenced in both gliadin and glutenin fractions. All high molecular weight glutenin subunits produced hydrazide conjugates after periodate oxidation; a reaction specific of bound carbohydrates. On the contrary, only some gliadins or low molecular weight glutenin subunits developed the same response or reacted with anti-carbohydrate antibodies. None of them specifically reacted with the tested lectins. Glycosylated polypeptides differed from one variety to an other and seemed to be present in low amount among wheat storage proteins.