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Abstract: Plants may trigger hypersensitivity reactions when individuals with allergies consume foods derived from plant materials or inhale plant pollen. As each plant food or pollen contains multiple allergens, proteomics is a powerful tool to detect the allergens present. Allergen-targeted proteomics, termed allergenomics, has been used for comprehensive identification and/or quantification of plant allergens, because it is a simple and inexpensive tool for rapid detection of proteins that bind to IgE. There are increasing numbers of reports on the applications of allergenomics. In this review, we outline some of the applications of proteomics, including: (i) identification of novel allergens, (ii) allergic diagnoses, (iii) quantification of allergens, and (iv) natural diversity of allergens, and finally discuss (v) the use of allergenomics for safety assessment of genetically modified (GM) plants. Biological significance: Recently, the number of allergic patients is increasing. Therefore, a comprehensive analysis of allergens (allergenomics) in plants is highly important for not only risk assessment of food plants but also diagnosis of allergic symptoms. In this manuscript, we reviewed the recent progress of allergenomics for identification, quantification and profiling of allergens. This article is part of a Special Issue entitled: Translational Plant Proteomics. [Copyright &y& Elsevier]

Abstract: Salt-soluble proteins extracted from non-transgenic and transgenic rice were evaluated for the presence of known and potential allergens by proteomic techniques. The salt-soluble proteins were extracted, separated by 1D and 2D electrophoresis, and analyzed by Western blotting. 1D immunoblot analysis with patients’ sera revealed few qualitative differences between the IgE-binding proteins of the non-transgenic and transgenic rice. 1D immunoblot with antigen-specific-animal sera revealed no qualitative or quantitative differences in two known allergens, RAG2 and glyoxalase I, between non-transgenic and transgenic rice. Multiple spots containing known and novel IgE-binding proteins were detected among the salt-soluble proteins of non-transgenic rice by 2D immunoblotting. Two globulin-like proteins, a 52kDa protein and a 63kDa protein, were identified as novel IgE-binding proteins that are candidates for rice allergens. These globulin-like proteins were homologous to Cupin superfamily allergens. Quantitative analysis of 19, 52, and 63kDa globulins with protein-specific-animal sera showed no significant differences in the expression of these proteins between the transgenic rice and non-transgenic rice. These results indicate that none of the known or novel endogeneous IgE-binding proteins detected in this study appear to be altered by genetic modification. [Copyright &y& Elsevier]

Abstract: Genetically modified (GM) foods are beneficial from the standpoint of ensuring a constant supply of foodstuffs, but they must be tested for safety before being released on the market, including by allergenicity tests to ensure that they do not contain new allergens or higher concentrations of known allergens than the same non-GM foods. In this study we used GM-amago salmon into which a growth hormone gene had been introduced and compared the allergens contained in the GM and the non-GM-amago salmons. We used a combination of Western blotting with allergen-specific antibodies and a proteomic analysis of their allergens with patients’ sera, a so-called allergenome analysis, to analyze allergens. Western blotting with specific antibodies showed no increase in the content of the known allergens fish parvalbumin and fish type-I collagen in GM-amago salmon, in comparison with their content in non-GM-amago salmon. The allergenome analysis of two fish-allergic patients allowed us to identify several IgE-binding proteins in amago salmon, including parvalbumin, triose-phosphate isomerase, fructose-bisphosphate aldolase A, and serum albumin, and there were no qualitative differences in these proteins between GM and non-GM-amago salmons. These results indicate that amago salmon endogenous allergen expression does not seem to be altered by genetic modification. [Copyright &y& Elsevier]

Abstract: The 2D-DIGE (2-Dimensional Fluorescence Difference Gel Electrophoresis) method was applied to proteomic phenotyping of natural variants in 10 varieties of rice (Nipponbare, Koshihikari, Sasanishiki, Akitakomachi, Hitomebore, Hinohikari, Kasalath, Rexark, Bleiyo, Cho-ko) from the world rice collection (WRC) in the GenBank of the National Institute of Agrobiological Sciences (NIAS), Japan. Salt-soluble protein extracts of Nipponbare brown rice were labeled with Cy2 fluorochrome and used as an internal standard. Protein extracts from nine other rice varieties were labeled with Cy3 or Cy5 fluorochrome and applied to 2D-PAGE (13cm gel length) analysis. Approximately 700 spots of rice proteins were observed. Fluorescence intensities of each of these spots for the nine rice varieties were expressed as relative ratios to that of Nipponbare. Statistical analysis revealed the spot numbers of five Japanese rice varieties above threshold five (relative ratio of each variety to Nipponbare exceeded 5-fold or was less than 1/5) to be less than three, while those of four varieties from other countries were more than five (especially, Kasalath which was 29 and Bleiyo which was 23). The 2D-DIGE method seems to be useful for analyzing natural varieties of different cultivars and also for comparing the expression of allergen proteins. [Copyright &y& Elsevier]

Key message: RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. Abstract: A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2. [ABSTRACT FROM AUTHOR]

Background: An in vitro elicitation test employing human high-affinity IgE receptor-expressing rat mast cell lines appears to be a useful method for measuring mast cell activation using a patient's IgE and an allergen; however, such cell lines are sensitive to human complements in the serum. We have recently developed a new luciferase-reporting mast cell line (RS-ATL8) to detect IgE crosslinking-induced luciferase expression (EXILE) with relatively low quantities of serum IgE. Methods: A total of 30 patients suspected of having egg white (EW) allergy were subjected to an oral food challenge (OFC) test; then, the performances of EW-specific serum IgE (CAP-FEIA), EW-induced degranulation, and EXILE responses in RS-ATL8 cells were compared using receiver-operating characteristic (ROC) curve analysis. The patients' sera were diluted to 1 : 100, which causes no cytotoxicity when sensitizing the RS-ATL8 cells for the degranulation and EXILE tests. Results: The area under the ROC curves was highest in the EXILE test (0.977), followed by CAP-FEIA (0.926) and degranulation (0.810). At an optimal cutoff range (1.648-1.876) calculated from the ROC curve of the EXiLE test, sensitivity and specificity were 0.944 and 0.917, respectively. A 95% positive predictive value was given at a cutoff level of 2.054 (fold increase in luciferase expression) by logistic regression analysis. Conclusions: In contrast to in vivo tests, the EXILE test appears to be a useful tool in diagnosing patients suspected of having IgE-dependent EW allergy without the risk of severe systemic reactions. [ABSTRACT FROM AUTHOR]

Background: The buckwheat 16-kDa protein (BWp16), as reported in our previous study, is a major allergen in buckwheat; however, the IgE-binding epitopes of BWp16 have not as yet been identified. Methods: We screened candidates for IgE-binding epitopes on BWp16 by using arrays of overlapping peptides synthesized on activated cellulose membranes (SPOTs membrane). The mimotope method was also used to analyze IgE-binding epitopes of BWp16. Nine single alanine (Ala) mutants of BWp16 expressed in Escherichia coli were used to confirm the epitopes of BWp16. The IgE-binding activity of single Ala mutants of BWp16 was determined by ELISA with mouse anti-BWp16 polyclonal antiserum or ELISA inhibition with sera from buckwheat allergic patients. Results: The SPOTs assay identified amino acid residues 99–110, i.e. EGVRDLKELPSK, as a candidate for the linear IgE-binding epitope of BWp16. The mimotope method indicated that peptides similar to EGVRDLKE were candidate sequences for epitopes of BWp16. Ala scanning of rBWp16 revealed that all EGVRDLKE peptides containing a single amino acid mutation had weaker IgE-binding activity than rBWp16 WT. An ELISA inhibition assay for rBWp16 WT revealed the inhibitory effect of rBWp16 D103A to be less than that of rBWp16 WT. Conclusions: We identified the peptide EGVRDLKE as a very likely candidate for the IgE-binding epitope of BWp16, and Asp103 as the critical amino acid in BWp16. This is the first report on the identification of IgE-binding epitopes of BWp16. Our findings will contribute to the production of BWp16 hypoallergens, and to allergen-specific immunotherapy for buckwheat allergy. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]