title: Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.
abstract: To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application.
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan; International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan. Electronic address: yukiy@ims.u-tokyo.ac.jp.
authors: Yoshikazu
authors: Y
authors: Yuki
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan.
authors: Shiho
authors: S
authors: Kurokawa
authors: Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan.
authors: Hiroko
authors: H
authors: Kozuka-Hata
authors: Department of Pediatrics, Osaka City University Graduate School of Medicine, 545-8585, Osaka, Japan.
authors: Daisuke
authors: D
authors: Tokuhara
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan.
authors: Mio
authors: M
authors: Mejima
authors: Crop Development Division, NARO Agriculture Research Center, 943-0193, Niigata, Japan.
authors: Masaharu
authors: M
authors: Kuroda
authors: Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan.
authors: Masaaki
authors: M
authors: Oyama
authors: Division of Biochemistry, National Institute of Health Sciences, 158-8501, Tokyo, Japan.
authors: Tomoko
authors: T
authors: Nishimaki-Mogami
authors: Division of Foods, National Institute of Health Sciences, 158-8501, Tokyo, Japan.
authors: Reiko
authors: R
authors: Teshima
authors: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan; International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, 108-8639, Tokyo, Japan.
authors: Hiroshi
authors: H
authors: Kiyono
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D052179:Antigens, Plant; D018506:Gene Expression Regulation, Plant; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D013058:Mass Spectrometry; D016253:Microscopy, Immunoelectron; D012275:Oryza / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D018570:Risk Assessment; D012401:Rotavirus / Q000235:genetics / Q000276:immunology*; D022243:Rotavirus Vaccines / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D057825:Two-Dimensional Difference Gel Electrophoresis
chemical_list: D000485:Allergens; D000914:Antibodies, Viral; D052179:Antigens, Plant; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D010940:Plant Proteins; C079480:RAG2 protein, Oryza sativa; D022243:Rotavirus Vaccines
keywords: 2D-DIGE; Immunoelectron microscopy; Rice allergens; Rice-based antibody; Shotgun MS