O-glycosylation of protein subpopulations in alcohol-extracted rice proteins

Michelle Kilcoyne; Miti Shah; Jared Q. Gerlach; Veer Bhavanandan; Vinay Nagaraj; Amy D. Smith; Kazuhito Fujiyama; Ulf Sommer; Catherine E. Costello; Neil Olszewski; Lokesh Joshi

doi:10.1016/j.jplph.2008.05.007

O-glycosylation of protein subpopulations in alcohol-extracted rice proteins

Michelle Kilcoyne, Miti Shah, Jared Q. Gerlach, Veer Bhavanandan, Vinay Nagaraj, Amy D. Smith, Kazuhito Fujiyama, Ulf Sommer, Catherine E. Costello, Neil Olszewski, Lokesh Joshi

Plant and Microbial Biology

Research output: Contribution to journal › Article › peer-review

22 Scopus citations

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 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.

Original language	English (US)
Pages (from-to)	219-232
Number of pages	14
Journal	Journal of Plant Physiology
Volume	166
Issue number	3
DOIs	https://doi.org/10.1016/j.jplph.2008.05.007
State	Published - Feb 15 2009

Bibliographical note

Funding Information:
We thank the Wallace Research Foundation and the Biodesign Institute at Arizona State University for financial support. The BUSM MSR is supported by NIH P41 RR10888 and S10 RR15942. Undergraduate student workers Ms. Hoai-Huang Tran, Ms. Meghan Soulvie and Mr. Bobby Keyvan-Jafari are acknowledged for help with protein extractions. We thank Prof. Paul Haynes for initial MudPIT analysis of crude prolamin extract.

Keywords

Mucin type
O-GlcNAc
O-glycosylation
Prolamin
Rice

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10.1016/j.jplph.2008.05.007

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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 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.",

keywords = "Mucin type, O-GlcNAc, O-glycosylation, Prolamin, Rice",

author = "Michelle Kilcoyne and Miti Shah and Gerlach, {Jared Q.} and Veer Bhavanandan and Vinay Nagaraj and Smith, {Amy D.} and Kazuhito Fujiyama and Ulf Sommer and Costello, {Catherine E.} and Neil Olszewski and Lokesh Joshi",

note = "Funding Information: We thank the Wallace Research Foundation and the Biodesign Institute at Arizona State University for financial support. The BUSM MSR is supported by NIH P41 RR10888 and S10 RR15942. Undergraduate student workers Ms. Hoai-Huang Tran, Ms. Meghan Soulvie and Mr. Bobby Keyvan-Jafari are acknowledged for help with protein extractions. We thank Prof. Paul Haynes for initial MudPIT analysis of crude prolamin extract. ",

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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

N1 - Funding Information: We thank the Wallace Research Foundation and the Biodesign Institute at Arizona State University for financial support. The BUSM MSR is supported by NIH P41 RR10888 and S10 RR15942. Undergraduate student workers Ms. Hoai-Huang Tran, Ms. Meghan Soulvie and Mr. Bobby Keyvan-Jafari are acknowledged for help with protein extractions. We thank Prof. Paul Haynes for initial MudPIT analysis of crude prolamin extract.

PY - 2009/2/15

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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.

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O-glycosylation of protein subpopulations in alcohol-extracted rice proteins

Abstract

Bibliographical note

Keywords

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