Wheat DOF transcription factors TaSAD and WPBF regulate glutenin gene expression in cooperation with SPA.

Grain storage proteins (GSPs) quantity and composition determine the end-use value of wheat flour. GSPs consists of low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins. GSP gene expression is controlled by a complex network of DNA-protein and protein-protein interactions, which coordinate the tissue-specific protein expression during grain development. The regulatory network has been most extensively studied in barley, particularly the two transcription factors (TFs) of the DNA binding with One Finger (DOF) family, barley Prolamin-box Binding Factor (BPBF) and Scutellum and Aleurone-expressed DOF (SAD). They activate hordein synthesis by binding to the Prolamin box, a motif in the hordein promoter. The BPBF ortholog previously identified in wheat, WPBF, has a transcriptional activity in expression of some GSP genes. Here, the wheat ortholog of SAD, named TaSAD, was identified. The binding of TaSAD to GSP gene promoter sequences in vitro and its transcriptional activity in vivo were investigated. In electrophoretic mobility shift assays, recombinant TaSAD and WPBF proteins bound to cis-motifs like those located on HMW-GS and LMW-GS gene promoters known to bind DOF TFs. We showed by transient expression assays in wheat endosperms that TaSAD and WPBF activate GSP gene expression. Moreover, co-bombardment of Storage Protein Activator (SPA) with WPBF or TaSAD had an additive effect on the expression of GSP genes, possibly through conserved cooperative protein-protein interactions.

Critical structural elements for the antigenicity of wheat allergen LTP1 (Tri a 14) revealed by site-directed mutagenesis.

Lipid transfer proteins (LTPs) were identified as allergens in a large variety of pollens and foods, including cereals. LTPs belong to the prolamin superfamily and display an α-helical fold, with a bundle of four α-helices held together by four disulfide bonds. Wheat LTP1 is involved in allergic reactions to food. To identify critical structural elements of antibody binding to wheat LTP1, we used site-directed mutagenesis on wheat recombinant LTP1 to target: (i) sequence conservation and/or structure flexibility or (ii) each disulfide bond. We evaluated the modifications induced by these mutations on LTP1 secondary structure by synchrotron radiation circular dichroism and on its antigenicity with patient's sera and with mouse monoclonal antibodies. Disruption of the C28-C73 disulfide bond significantly affected IgE-binding and caused protein denaturation, while removing C13-C27 bond decreased LTP1 antigenicity and slightly modified LTP1 overall folding. In addition, we showed Lys72 to be a key residue; the K72A mutation did not affect global folding but modified the local 3D structure of LTP1 and strongly reduced IgE-binding. This work revealed a cluster of residues (C13, C27, C28, C73 and K72), four of which embedded in disulfide bonds, which play a critical role in LTP1 antigenicity.

The bZIP transcription factor SPA Heterodimerizing Protein represses glutenin synthesis in Triticum aestivum.

The quality of wheat grain is mainly determined by the quantity and composition of its grain storage proteins (GSPs). Grain storage proteins consist of low- and high-molecular-weight glutenins (LMW-GS and HMW-GS, respectively) and gliadins. The synthesis of these proteins is essentially regulated at the transcriptional level and by the availability of nitrogen and sulfur. The regulation network has been extensively studied in barley where BLZ1 and BLZ2, members of the basic leucine zipper (bZIP) family, activate the synthesis of hordeins. To date, in wheat, only the ortholog of BLZ2, Storage Protein Activator (SPA), has been identified as playing a major role in the regulation of GSP synthesis. Here, the ortholog of BLZ1, named SPA Heterodimerizing Protein (SHP), was identified and its involvement in the transcriptional regulation of the genes coding for GSPs was analyzed. In gel mobility shift assays, SHP binds cis-motifs known to bind to bZIP family transcription factors in HMW-GS and LMW-GS promoters. Moreover, we showed by transient expression assays in wheat endosperm that SHP acts as a repressor of the activity of these gene promoters. This result was confirmed in transgenic lines overexpressing SHP, which were grown with low and high nitrogen supply. The phenotype of SHP-overexpressing lines showed a lower quantity of both LMW-GS and HMW-GS, while the quantity of gliadin was unchanged, whatever the nitrogen availability. Thus, the gliadin/glutenin ratio was increased, which suggests that gliadin and glutenin genes may be differently regulated.

A chimeric IgE that mimics IgE from patients allergic to acid-hydrolyzed wheat proteins is a novel tool for in vitro allergenicity assessment of functionalized glutens.

BACKGROUND: Acid-hydrolyzed wheat proteins (acid-HWPs) have been shown to provoke severe allergic reactions in Europe and Japan that are distinct from classical wheat allergies. Acid-HWPs were shown to contain neo-epitopes induced by the deamidation of gluten proteins. However, products with variable rates of deamidation can be found. OBJECTIVES: In this work, we studied the effect of the extent of wheat proteins deamidation on its allergenicity. A recombinant chimeric IgE was produced and compared to patients' IgE for its capacity to assess the IgE-mediated triggering potential of acid-HWPs. METHODS: Sera from acid-HWP allergic patients were analyzed via ELISA and a functional basophil assay for their IgE reactivity to wheat proteins with different deamidation levels. A chimeric mouse/human IgE (chIgE-DG1) specific for the main neo-epitope, QPEEPFPE, involved in allergy to acid-HWPs was characterized with respect to its functionality and its reactivity compared to that of patients' IgE. RESULTS: Acid-HWPs with medium (30%) and high (50-60%) deamidation levels displayed a markedly stronger IgE binding and capacity to activate basophils than those of samples with weak (15%) deamidation levels. The monoclonal chIgE-DG1 allowed basophil degranulation in the presence of deamidated wheat proteins. ChIgE-DG1 was found to mimic patients' IgE reactivity and displayed the same ability to rank acid-HWP products in a degranulation assay. CONCLUSION: Increasing the deamidation level of products from 15% to 60% resulted in an approximately 2-fold increase in their antigenicity and a 100-fold increase in their eliciting potential. The chimeric ChIgE-DG1 may be a useful tool to evaluate functionalized glutens for their allergenic potential. By mimicking patient sera reactivity, chIgE-DG1 also provided data on the patients' IgE repertoire and on the functionality of certain repeated epitopes in gluten proteins.

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.

MS analysis and molecular characterization of Botrytis cinerea protease Prot-2. Use in bioactive peptides production.

Prot-2 protease previously purified to homogeneity from Botrytis cinerea showed potentiality to be used in detergency and for production of bioactive peptides. To extend the characterization of Prot-2 protease, antifungal and antibacterial assays were performed in vitro using protein hydrolysates prepared from muscle of mackerel (Scomber scomborus) treated with this enzyme. The most active hydrolysate (degree of hydrolysis of 8 %) exhibited inhibition effect towards bacteria and phytopathogenic fungi, demonstrating that Prot-2 proteolysis generated bioactive peptides. Biochemical and molecular characterization of the purified Prot-2, by SDS-PAGE/Tryptic in gel-digestion and LC-MS/MS analysis, was investigated. The peptide amino acid sequence alignment search in database revealed a moderate homology between the determined amino acid sequence of Prot-2 protease and the known fungal trypsin/chymotrypsin in particular from Glomerella, Metarhizium and Streptomyces. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 786 bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 262 amino acid residues. The deduced amino acid sequence of Prot-2 showed moderate identity with trypsin of Glomerella graminicola (74 %) and with chymotrypsin from Metarhizium anisopliae (71 %). Prot-2 exhibited a Ser protease homology and showed in addition the specific His motif of trypsin/chymotrypsin family.

Molecular and immunological characterization of wheat Serpin (Tri a 33).

SCOPE: Several wheat proteins are responsible for food and respiratory allergies. Due to their large polymorphism, the allergenic potential of a number of them has not yet been precisely established. The aim of this work was to perform a thorough assessment of serpin (Tri a 33) allergenicity. METHODS AND RESULTS: Recombinant wheat Serpin-Z2B isoform (rSerpin-Z2B) was expressed in Escherichia coli. Synchrotron radiation circular dichroism data indicated that the recombinant serpin contains slightly more ß-strands than α-helix structures. IgE reactivity of sera from 103 patients with food allergy and 29 patients with Baker's asthma was evaluated using ELISA, a model of basophil activation and linear epitope mapping (Pepscan). Twenty percent of patients with food allergy to wheat and 31% of those with Baker's asthma displayed rSerpin-Z2B-specific IgE in ELISA. The protein was able to induce IgE-dependent basophil degranulation. The Pepscan experiment identified four regions involved in IgE binding to serpin. Heating the protein induced its irreversible denaturation and impaired IgE binding, revealing the predominance of conformational epitopes. CONCLUSION: This study confirms wheat serpin allergenicity and shows that recombinant serpin may be a marker of a broad spectrum of sensitization to wheat proteins.

Wheat gliadins modified by deamidation are more efficient than native gliadins in inducing a Th2 response in Balb/c mice experimentally sensitized to wheat allergens.

SCOPE: Wheat gluten proteins such as gliadins constitute major food allergens. Gluten can be modified industrially by deamidation which increases its solubility and enhances its use as a food ingredient. Sensitization to deamidated gluten has been reported to cause severe allergic reactions with anaphylaxis. The aim of this study was therefore to compare the sensitization and elicitation potentials of native (NG) and deamidated (DG) gliadins. The reactivity pattern of mice IgE was also compared with that of DG-allergic patients. METHODS AND RESULTS: The ability of DG to sensitize Balb/c mice using intra-peritoneal administration with aluminium hydroxide as an adjuvant, and to elicit an allergic response after a challenge, was tested in comparison with NG. Mice sensitized with DG secreted higher levels of total IgE, IL-4, gliadin-specific IgE and IgG1 than mice sensitized with NG. By contrast, mice sensitized with NG produced higher levels of gliadin-specific IgG2a and INFγ. After a challenge, histamine levels were higher in mice sensitised with DG. CONCLUSIONS: DG can sensitize mice much more efficiently than NG. Moreover, this mouse model of allergy to DG revealed an IgE reactivity pattern against purified gliadins which was very similar to that of DG-allergic patients.

Micellisation and immunoreactivities of dimeric beta-caseins.

Bovine beta-casein (beta-CN) is a highly amphiphilic micellising phospho-protein showing chaperone-like activity in vitro. Recently, existence of multiple sequential epitopes on beta-CN polypeptide chain in both hydrophilic-polar (psi) and hydrophobic-apolar domains (phi) has been evidenced. In order to clarify specific contribution of polar and apolar domains in micellisation process and in shaping immunoreactivity of beta-CN, its dimeric/bi-amphiphilic "quasi palindromic" forms covalently connected by a disulfide bond linking either N-terminal (C4 beta-CND) or C-terminal domain (C208 beta-CND) were produced and studied. Depending on the C- or N-terminal position of inserted cysteine, each dimeric beta-CN contains one polar/apolar region at the centre and two external hydrophobic/hydrophilic ends. Consequently, such casein dimers have radically different polarities/hydrophobicities on their outside surfaces. Dynamic light scattering (DLS) measurements indicate that these dimeric casein molecules form micelles of different sizes depending on arrangement of polar fragments of the beta-CN mutants in their constrained dimers. Non-aggregated dimers have different hydrodynamic diameters that could be explained by their different geometries. Measurements of fluorescence showed more hydrophobic environment of Trp residues of C208 beta-CND, while in similar experimental conditions Trp residues of C4 beta-CND and native beta-CN were more exposed to the polar medium. Both fluorescence and DLS studies showed greater propensity for micellisation of the dimeric beta-CNs, suggesting that the factors inducing the formation of micelles are stronger in the bi-amphiphilic dimers. 1-anilino-naphthalene-8-sulfonate (ANS) binding studies showed different binding of ANS by these dimers as well as different exposition of ANS binding (hydrophobic) regions in the micellar states. The differences in fluorescence resonance energy transfer (FRET) profiles of C4 beta-CND and C208 beta-CND can be explained by differences of distances and/or by differences of relative orientations of the donor (Trp) and acceptor (ANS), as well as by differences in quenching properties of the disulfide bridges and intra-molecular hydrophobic interactions. The immunoreactivity assays showed somewhat lower IgE response to C208 beta-CND than to C4 beta-CND. Thus, dimerization of C208 beta-CN, connecting two C-terminal hydrophobic domains of two monomers doubling long-range hydrophobic interactions, possibly may hide a part of epitopes in the hydrophobic interface/core of C208 beta-CND that is consistent with the results of DLS and fluorescence studies. The obtained results indicate structural differences of dimers - possibly the formation of Y- and U-shaped structures for C208 beta-CND and C4 beta-CND, respectively. This study not only demonstrated the importance of the organization of polar and hydrophobic regions during micellisation of the constrained and oriented beta-CN dimers but also confirmed a possible role of C-terminal hydrophobic domain in the immunoreactivity profile of native beta-CN.

Effects of heating and glycation of beta-lactoglobulin on its recognition by IgE of sera from cow milk allergy patients.

beta-Lactoglobulin (beta-LG) is one of the cow's major milk proteins and the most abundant whey protein. This globular protein of about 18 kDa is folded, forming a beta-barrel (or calyx) structure. This structure is stabilized by two disulfide bonds and can be altered by heating above 65 degrees C. beta-LG is also one of the major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. During heating in the presence of reducing sugars, beta-LG is also submitted to the Maillard reaction, which at the first stage consists of the covalent fixation of sugars on the epsilon-amino groups of lysyl residues. The following steps are condensation and polymerization reactions leading to the formation of melanoidins (brown pigments). Despite the frequency of use of heating during milk transformation, the effects of heat-induced denaturation and of glycation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) patients are not fully understood. The objectives of our work were to evaluate the effect of heat-induced denaturation of bovine beta-LG on binding of IgE from CMA patients and to determine the effect of moderate glycation on the degree of recognition by IgE. We showed that heat-induced denaturation (loss of tertiary and secondary structures) of beta-LG is associated with weaker binding of IgE from CMA patients. It was also shown that moderate glycation of beta-LG in early stages of Maillard reaction has only a small effect on its recognition by IgE, whereas a high degree of glycation has a clear "masking" effect on the recognition of epitopes. This demonstrates the importance of epsilon-amino groups of lysines in the definition of epitopes recognized by IgE.

Engineering of caseins and modulation of their structures and interactions.

Beta-casein (beta-CN) is a milk protein widely used in food industries because of its mild emulsifying properties due to its amphiphilicity. However, the elements determining its micellization behavior in solution and interfacial behavior at the air-water interface are not well known. In order to study how the forced dimerisation influences functional properties of beta-CN, recombinant wild-type beta-CN was produced and distal cysteinylated forms of recombinant beta-CN were engineered. We show that 1) cysteinylated beta-CN formed mainly dimers bridged by disulfide bonds; 2) the process of dimerization adds to the micellization process with temperature and is poorly reversible; 3) covalent disulfide linkage forms at the air-water interface at a lower temperature than in bulk. In conclusion, the location of the cysteinylation in the C-terminus or N-terminus or both is of importance for the properties of beta-CN.

Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

Do G protein-coupled receptors expressed in human lingual epithelium interact with HPV11?

Human papillomaviruses infect epithelia but little is known about the nature of cell surface receptors interacting with the viral particles. It has been proposed that glycosaminoglycans and integrins may be involved in the attachment process. In the present study, the putative interactions of virus-like particles of human papillomavirus type 11 (HPV11), which present a tropism for nasopharyngeal epithelia, with olfactory and taste receptors expressed in the human lingual epithelium were studied. The L1 protein of HPV11 was produced in insect cells. The presence of L1 virus-like particles was analyzed by ELISA using monoclonal antibodies specific for full-size particles and by electron microscopy. Using immunofluorescence, it was observed that virus-like particles interacted with taste buds from murine tongue, with the tagged human olfactory receptor hJCG5 expressed in HEK-293 but not with the tagged taste receptor hT2R4. This therefore suggests that hJCG5 may be involved in the adsorption process of HPV11 to lingual epithelium serving as a so-called "adsorption-adhesive molecule."

Impact of Maillard type glycation on properties of beta-lactoglobulin.

The Maillard reaction occurs during many thermal treatments of foods. It is used because of its role in creating colors, flavors, textures and other functional properties in foods. Glycated beta-lactoglobulin (BLG) can improve techno-functional properties as heat stability, emulsifying and foaming properties. Among the six common sugars used, arabinose and ribose induce the highest degree of modification of proteins. Glycation induced also the oligomerization of BLG monomers. Depending on the reactivity of the sugar, the population of oligomers produced showed smaller or larger heterogeneity in molecular masses. Antiradical properties of glycated BLG were estimated using a radical scavenging activity test. Glycation induced a radical scavenging activity; the intensity depended on the sugar used for modification.

Mouse orthologs of human olfactory-like receptors expressed in the tongue.

Olfactory-like receptors (OLRs) have been shown previously to be expressed in adult and foetal human tongue. This prompted us to verify the hypothesis if OLRs were involved in taste perception. In the present work, the mouse orthologs of OLR genes expressed in adult and/or foetal human tongue were identified. Analysis of their genomic localization and of their primary sequence features using bioinformatics did not reveal any shared remarkable characteristic. The expression of eight of these orthologs (S25/mJCG1, K42, mJCG2, mJCG3, P2/mJCG5, P3/mJCG6, mT09m/mJCG9, K21/mTPCR85 and mTPCR06) was studied in three types of mouse papillae as well as in the olfactory epithelium. It was found that all of them are expressed in olfactory epithelium and that only three of them (S25/mJCG1, K42/mJCG2 and mTPCR06) are expressed in papillae. However, despite many efforts it was impossible to detect without ambiguity the presence of OLR mRNAs in taste receptor cells nor in surrounding tissues by in-situ hybridization. Hence, the studied OLRs very likely play in taste papillae other roles than taste perception.

Olfactory-like receptor cDNAs are present in human lingual cDNA libraries.

Olfactory and pheromone receptors (ORs) constitute a large family of G-protein-coupled receptors involved in the detection and transduction of odorant signals. Using degenerated primers complementary to the highly conserved transmembrane domains II, III, VI, and VII within this protein family, Gaudin et al. have recently described the expression of several OR genes in foetal human tongue. Among the nine genes identified in human foetal tongue (HTPCR06, HGMP07I, JCG6, TPCR85, JCGI1, JCG2, JCG3, JCG5, and JCG9), only four (HTPCR06, HGMP07I, JCG3, and JCG5) were found to be expressed in adult tongue, suggesting that ORs might perform developmental functions in this organ. The objective of our work was to obtain additional information about the expression of olfactory-like genes in human tongue. In the present study, the synthesis and the screening of a cDNA library from epithelial cells of human adult tongue is reported. Two kinds of PCR analysis were performed. First, partial olfactory-like receptor cDNAs amplified with the degenerated primers used by Gaudin et al. were cloned and described. Second, a comparison of the expression profiles of the olfactory-like receptor genes previously identified before was carried out using specific primers. Among the genes studied we found that four genes (HTPCR06, JCG3, JCG5, and JCG6) are expressed in epithelial cells of the surface of the adult tongue. Additionally, we show that three olfactory-like receptor genes OR7A5/HTPCR2, OR6Q1, and OR7C1/TPCR86 are also expressed in these cells.

A recombinant C121S mutant of bovine beta-lactoglobulin is more susceptible to peptic digestion and to denaturation by reducing agents and heating.

The lipocalin beta-lactoglobulin (BLG) is the major whey protein of bovine milk and is homodimeric at physiological conditions. Each monomer contains two disulfide bonds and one cysteine at position 121 (C121). This free thiol plays an important role in the heat-induced aggregation of BLG and, possibly, in its conformational stability. We describe here the expression in the yeast Pichia pastoris of a mutant bovine BLG, in which C121 was changed into Ser (C121S). Circular dichroism and high-performance liquid chromatography experiments, together with the X-ray crystal structure, show that the C121S mutant retains a nativelike fold at both neutral and acid pH. The mutation completely blocks the irreversible aggregation induced by heat treatment at 90 degrees C. Compared to the recombinant wild-type protein, the mutant is less stable to temperature and disulfide reducing agents and is much more sensitive to peptic digestion. Moreover, its affinity for 1-anilino-8-naphthalenesulfonate is increased at neutral and acid pH. We suggest that the stability of the protein arising from the hydrophobic effect is reduced by the C121S mutation so that unfolded or partially unfolded states are more favored.