TY - JOUR
T1 - O-glycosylation of protein subpopulations in alcohol-extracted rice proteins
AU - Kilcoyne, Michelle
AU - Shah, Miti
AU - Gerlach, Jared Q.
AU - Bhavanandan, Veer
AU - Nagaraj, Vinay
AU - Smith, Amy D.
AU - Fujiyama, Kazuhito
AU - Sommer, Ulf
AU - Costello, Catherine E.
AU - Olszewski, Neil
AU - Joshi, Lokesh
PY - 2009/2/15
Y1 - 2009/2/15
N2 - 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 16 kDa 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-β-(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 14 kDa 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 CTD110.6 mAb 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.
AB - 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 16 kDa 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-β-(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 14 kDa 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 CTD110.6 mAb 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.
KW - Mucin type
KW - O-GlcNAc
KW - O-glycosylation
KW - Prolamin
KW - Rice
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UR - http://www.scopus.com/inward/citedby.url?scp=58349094932&partnerID=8YFLogxK
U2 - 10.1016/j.jplph.2008.05.007
DO - 10.1016/j.jplph.2008.05.007
M3 - Article
C2 - 18639953
AN - SCOPUS:58349094932
VL - 166
SP - 219
EP - 232
JO - Journal of Plant Physiology
JF - Journal of Plant Physiology
SN - 0176-1617
IS - 3
ER -