Rie Satoh

2D-DIGE analysis of rice proteins from different cultivars.

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.

Identification of an IgE-binding epitope of a major buckwheat allergen, BWp16, by SPOTs assay and mimotope screening.

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.

Purification, crystallization and preliminary X‐ray analysis of a deletion mutant of a major buckwheat allergen

A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. An immunologically active mutant of BWp16 was prepared and a three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled mutant protein.

Proteomic Approaches for Allergen Analysis in Crop Plants

Crop plants are important foods that provide necessary energy in human diets but can cause allergic reactions resulting in serious health problems. Therefore, it is necessary to identify allergens in crop plants to determine causative allergenic foods in patients and facilitate allergen diagnosis. Recent advances in proteomic approaches have significantly contributed to the detection and identification of food allergens. Proteomic techniques can also reveal the qualitative and quantitative variability of allergens among crops and changes in the proteome constitution of crops following genetic modification. Moreover, allergen identification and characterization are important for the diagnosis of food allergies and risk assessment of genetically modified crops as food sources. In the present review, we discuss how proteomics can be applied to detect and identify allergens in plant-derived foods.

Allergen Analysis in Plants and Use in the Assessment of Genetically Modified Plants

Recent technological advances in molecular biology have facilitated the generation of genetically modified (GM) plants to meet the increasing needs of the ever-increasing global population. On the other hand, food allergy continues to be a serious health concern, with an increasing frequency worldwide. Allergenicity is a major concern of food safety regarding newly expressed proteins in foods derived from GM crops. Identification and characterization of allergenic molecules are important for not only diagnosis of food allergies but also risk assessment of GM crops as food. In this chapter, we describe methods for identifying and characterizing plant food allergens and the application of proteomics for the evaluation of the allergenicity of proteins in GM crops.

Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52–63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14–16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens. Graphical abstract Representative multiplexed detection and 2D-DIGE profile of rice endogenous allergens.