Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.)

Abstract

Glutelin, the major storage protein of rice seed, consists of microheterogenous subunits and partially exists in a macromolecular form that is polymerized by intersubunit disulfide bonds. In order to analyze the glutelin subunits using high-throughput CE, we first identified a sample preparation procedure suitable for CE. The polymerized glutelin treated with a reductant could not dissociate into its constituent monomer subunits when it was dissolved in an acidic solution. However, the glutelin dissociated into its subunits and component α and β polypeptides when it was denatured and reduced by an appropriate amount of urea and 2-mercaptoethanol at a specific incubation time and temperature. The molecular species of the completely dissociated α and β polypeptides were identified and quantitatively analyzed by CE using glutelin mutants. The CE analysis also demonstrated that the actual subunit variation in terms of the charge and/or size of the β polypeptides is much smaller than predicted when compared with that of α polypeptides, even under denaturing and reducing condition. Thus, the combined analytical system described here will be useful for basic and applied research, such as the kinetic characterization of higher-order structure and the quantitative evaluation of glutelin in a large number of diverse rice varieties.

Glutelin is partially degraded in globulin-less mutants of rice (Oryza sativaL.)

“…For example, our previous analysis on some glutelin subunit mutants using capillary electrophoresis, a technique suitable for accurate quantification, has demonstrated that mutants Type1, Type2, Type3, and a-123less, in which GluB4, GluA2, GluA1, and their three subunits are deleted, respectively, had apparently no other changes in protein composition and total protein content, suggesting the deletions were compensated evenly by other storage proteins (Katsube-Tanaka et al, 2010).…”

Glutelin subtype-dependent protein localization in rice grain evidenced by immunodetection analyses

“…Glutelin polypeptide is cleaved into an N-terminal half (acidic subunit) and a C-terminal half (basic subunit) in the vacuole by an aspartic protease (Wang et al 2009 ; Kumamaru et al 2010 ). They are conjugated intra-molecularly and inter-molecularly by disulfide bonds to form a higher structural conformation (Katsube-Tanaka et al 2004 , 2010 ; Kawagoe et al 2005 ; Motoyama et al 2009 ), and they accumulate with α-globulin in PSVs. Glutelin is encoded by a multigene family, and the molecular type has been studied by many researchers before the whole rice genome was revealed (Takaiwa et al 1987 , 1991 ; Masumura et al 1989 ; Takaiwa and Oono 1991 ; Mitsukawa et al 1998 ; Qu et al 2002 ; Kusaba et al 2003 ).…”

Identification of Rice Proteins Recognized by the IgE Antibodies of Patients with Food Allergies

“…Herein, we could only speculate about the IgEbinding capacity of the protein encoded by locus Os02gO453600, because both spots 111 and 112 were mixed with the already known IgE-binding proteins, glutelin type-A 1 or glutelin type-B 1, respectively. 40 Glutelin type-B 1 was also identified in the separated spot 113. Spots 114, 115, and 116 corresponded to a 19 kDa globulin, the seed allergenic protein RAG2, and a protein encoded by the gene locus Os07g0216700, respectively.…”