title: O-glycosylation of protein subpopulations in alcohol-extracted rice proteins.
abstract: Mucin-type O-glycosylation has been well characterized in mammalian systems but not in plants. In this study, the purified alcohol-soluble, non-reduced protein (prolamin) fraction from rice seed was investigated for the occurrence of O-linked oligosaccharides. As storage prolamins are unlikely to be O-glycosylated, any O-glycosylation found was likely to belong to co-extracted proteins, whether because of association with the protein body or solubility. SDS-PAGE and MS analyses revealed 14 and 16kDa protein families in fractions that bound to the lectins peanut agglutinin (PNA), Vicia villosa lectin (VVL) and Jacalin, indicative of the presence of O-linked saccharides. Enzymatic cleavage, fluorescent labeling and high-performance liquid chromatography (HPLC) analysis demonstrated a peak consistent with Gal-beta-(1-->3)-GalNAc, with similar MS/MS fragmentation. Additionally, upon chemical analysis, a GlcNAc-containing O-linked carbohydrate moiety was discovered. Protein blotting with anti-O-GlcNAc antibody (clone CTD110.6) was positive in a subpopulation of the 14kDa alcohol-soluble protein fraction, but a hot capping experiment was negative. Therefore, the GlcNAc residue in this case is unlikely to be terminal. Additionally, a positive reaction with CTD110.6mAb cannot be taken as absolute proof of O-GlcNAc modification and further confirmatory experiments should be employed. We hypothesize that O-glycosylation may contribute to protein functionality or regulation. Further investigation is required to identify the specific proteins with these modifications. This 'reverse' approach could lead to the identification of proteins involved in mRNA targeting, signaling, translation, anchoring or maintenance of translational quiescence and may be applied to germinating rice seed extracts for further elucidation of protein function and regulation.
authors.affiliation: Center for Glycosciences and Technology, Biodesign Institute at Arizona State University, 1001 S McAllister Avenue, Tempe, AZ 85287, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Kilcoyne
authors.forename: Miti
authors.initials: M
authors.lastname: Shah
authors.forename: Jared Q
authors.initials: JQ
authors.lastname: Gerlach
authors.forename: Veer
authors.initials: V
authors.lastname: Bhavanandan
authors.forename: Vinay
authors.initials: V
authors.lastname: Nagaraj
authors.forename: Amy D
authors.initials: AD
authors.lastname: Smith
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
authors.forename: Ulf
authors.initials: U
authors.lastname: Sommer
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
authors.forename: Neil
authors.initials: N
authors.lastname: Olszewski
authors.forename: Lokesh
authors.initials: L
authors.lastname: Joshi
mesh_terms: D000438:Alcohols / Q000737:chemistry*; D000595:Amino Acid Sequence; D000941:Antigens / Q000378:metabolism; D001681:Biological Assay; D019921:Biotinylation; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017124:Conserved Sequence; D005944:Glucosamine / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000032:analysis; D012275:Oryza / Q000737:chemistry* / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D055315:Prolamins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000438:Alcohols; D000941:Antigens; D009005:Monosaccharides; D037121:Plant Lectins; D010940:Plant Proteins; D055315:Prolamins; C043696:jacalin; D005944:Glucosamine