Structural Basis of IgE Binding to α- and γ-Gliadins: Contribution of Disulfide Bonds and Repetitive and Nonrepetitive Domains.

Wheat products cause IgE-mediated allergies. The present study aimed to decipher the molecular basis of α- and γ-gliadin allergenicity. Gliadins and their domains, the repetitive N-terminal and the nonrepetitive C-terminal domains, were cloned and expressed in Escherichia coli. Their secondary structures and their IgE binding capacity were compared with those of natural proteins before and after reduction/alkylation. Allergenicity was evaluated with sera from patients who had a wheat food allergy or baker's asthma. The secondary structures of natural and recombinant proteins were slightly different. Compared with natural gliadins, recombinant proteins retained IgE binding but with reduced reactivity. Reduction/alkylation decreased IgE binding for both natural and recombinant gliadins. Although more continuous epitopes were identified in the N-terminal domains of α- and γ-gliadins, both the N-terminal and C-terminal domains contributed to IgE binding. As for other members of the prolamin superfamily, disulfide bonds appear to be of high importance for IgE binding.

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.

Immunoglobulin-E reactivity and structural analysis of wheat low-molecular-weight glutenin subunits and their repetitive and nonrepetitive halves.

The IgE reactivity of the recombinant glutenin subunits P73 and B16, and of their repetitive N-terminal and nonrepetitive C-terminal halves, was analyzed using dot-blot with sera from patients diagnosed with baker's asthma, wheat-dependent exercise-induced anaphylaxis, or allergy to hydrolyzed wheat proteins. The linear epitopes of B16 were identified using the Pepscan method. Except for one common epitope, the IgE binding domains of glutenins differ from those of ω5-gliadins. Secondary structure content of the proteins was determined using synchrotron radiation circular dichroism (SRCD): while α structures were predominant in all glutenin subunits, fragments, or chimeras, a high IgE reactivity was associated with proteins rich in ß structures. Mixing B16 halves induced conformational interaction, as evidenced by dynamic light scattering and SRCD. IgE reactivity was correlatively increased, as when the halves were associated in the B16-P73 chimera. These results suggest that structural interaction between N- and C-terminal halves may promote epitope presentation.

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.

Carboxylate and aromatic active-site residues are determinants of high-affinity binding of ω-aminoaldehydes to plant aminoaldehyde dehydrogenases.

The crystal structures of both isoforms of the aminoaldehyde dehydrogenase from pea (PsAMADH) have been solved recently [Tylichováet al. (2010) J Mol Biol396, 870-882]. The characterization of the PsAMADH2 proteins, altered here by site-directed mutagenesis, suggests that the D110 and D113 residues at the entrance to the substrate channel are required for high-affinity binding of ω-aminoaldehydes to PsAMADH2 and for enzyme activity, whereas N162, near catalytic C294, contributes mainly to the enzyme's catalytic rate. Inside the substrate cavity, W170 and Y163, and, to a certain extent, L166 and M167 probably preserve the optimal overall geometry of the substrate channel that allows for the appropriate orientation of the substrate. Unconserved W288 appears to affect the affinity of the enzyme for the substrate amino group through control of the substrate channel diameter without affecting the reaction rate. Therefore, W288 may be a key determinant of the differences in substrate specificity found among plant AMADH isoforms when they interact with naturally occurring substrates such as 3-aminopropionaldehyde and 4-aminobutyraldehyde.

Biochemical characterization of pea ornithine-delta-aminotransferase: substrate specificity and inhibition by di- and polyamines.

Ornithine-delta-aminotransferase (OAT, EC 2.6.1.13) catalyzes the transamination of L-ornithine to L-glutamate-gamma-semialdehyde. The physiological role of OAT in plants is not yet well understood. It is probably related to arginine catabolism resulting in glutamate but the enzyme has also been associated with stress-induced proline biosynthesis. We investigated the enzyme from pea (PsOAT) to assess whether diamines and polyamines may serve as substrates or they show inhibitory properties. First, a cDNA coding for PsOAT was cloned and expressed in Escherichia coli to obtain a recombinant protein with a C-terminal 6xHis tag. Recombinant PsOAT was purified under native conditions by immobilized metal affinity chromatography and its molecular and kinetic properties were characterized. Protein identity was confirmed by peptide mass fingerprinting after proteolytic digestion. The purified PsOAT existed as a monomer of 50 kDa and showed typical spectral properties of enzymes containing pyridoxal-5'-phosphate as a prosthetic group. The cofactor content of PsOAT was estimated to be 0.9 mol per mol of the monomer by a spectrophotometric analysis with phenylhydrazine. L-Ornithine was the best substrate (K(m)=15 mM) but PsOAT also slowly converted N(alpha)-acetyl-L-ornithine. In these reactions, 2-oxoglutarate was the exclusive amino group acceptor (K(m)=2mM). The enzyme had a basic optimal pH of 8.8 and displayed relatively high temperature optimum. Diamines and polyamines were not accepted as substrates. On the other hand, putrescine, spermidine and others represented weak non-competitive inhibitors. A model of the molecular structure of PsOAT was obtained using the crystal structure of human OAT as a template.

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.

Structural and functional characterization of plant aminoaldehyde dehydrogenase from Pisum sativum with a broad specificity for natural and synthetic aminoaldehydes.

Aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the large aldehyde dehydrogenase (ALDH) superfamily, namely, the ALDH9 family. They oxidize polyamine-derived omega-aminoaldehydes to the corresponding omega-amino acids. Here, we report the first X-ray structures of plant AMADHs: two isoenzymes, PsAMADH1 and PsAMADH2, from Pisum sativum in complex with beta-nicotinamide adenine dinucleotide (NAD(+)) at 2.4 and 2.15 A resolution, respectively. Both recombinant proteins are dimeric and, similarly to other ALDHs, each monomer is composed of an oligomerization domain, a coenzyme binding domain and a catalytic domain. Each subunit binds NAD(+) as a coenzyme, contains a solvent-accessible C-terminal peroxisomal targeting signal (type 1) and a cation bound in the cavity close to the NAD(+) binding site. While the NAD(+) binding mode is classical for PsAMADH2, that for PsAMADH1 is unusual among ALDHs. A glycerol molecule occupies the substrate binding site and mimics a bound substrate. Structural analysis and substrate specificity study of both isoenzymes in combination with data published previously on other ALDH9 family members show that the established categorization of such enzymes into distinct groups based on substrate specificity is no more appropriate, because many of them seem capable of oxidizing a large spectrum of aminoaldehyde substrates. PsAMADH1 and PsAMADH2 can oxidize N,N,N-trimethyl-4-aminobutyraldehyde into gamma-butyrobetaine, which is the carnitine precursor in animal cells. This activity highly suggests that in addition to their contribution to the formation of compatible osmolytes such as glycine betaine, beta-alanine betaine and gamma-aminobutyric acid, AMADHs might participate in carnitine biosynthesis in plants.

Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum.

Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Aminoaldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 A resolution at 100 K. Crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 A, beta = 98.1 degrees. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit.

Ornithine delta-aminotransferase: An enzyme implicated in salt tolerance in higher plants.

This review deals with biochemical and physiological aspects of plant ornithine d-aminotransferase (OAT, EC 2.6.1.13). OAT is a mitochondrial enzyme containing pyridoxal-5'-phosphate as a cofactor, which catalyzes the conversion of L-ornithine to L-glutamate gamma-semialdehyde using 2-oxoglutarate as a terminal amino group acceptor. It has been described in humans, animals, insects, plants and microorganisms. Based on the crystal structure of human OAT, both substrate binding and reaction mechanism of the enzyme are well understood. OAT shows a large structural and mechanistic similarity to other enzymes from the subgroup III of aminotransferases, which transfer an amino group from a carbon atom that does not carry a carboxyl function. In plants, the enzyme has been implicated in proline biosynthesis and accumulation (via pyrroline-5-carboxylate), which represents a way to regulate cellular osmolarity in response to osmotic stress. However, the exact metabolic pathway involving OAT remains a subject of controversy.

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.

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.