title: Detecting O-GlcNAc using in vitro sulfation.
abstract: O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation, the covalent attachment of N-acetylglucosamine to serine and threonine residues of proteins, is a post-translational modification that shares many features with protein phosphorylation. O-GlcNAc is essential for cell survival and plays important role in many biological processes (e.g. transcription, translation, cell division) and human diseases (e.g. diabetes, Alzheimer's disease, cancer). However, detection of O-GlcNAc is challenging. Here, a method for O-GlcNAc detection using in vitro sulfation with two N-acetylglucosamine (GlcNAc)-specific sulfotransferases, carbohydrate sulfotransferase 2 and carbohydrate sulfotransferase 4, and the radioisotope (35)S is described. Sulfation on free GlcNAc is first demonstrated, and then on O-GlcNAc residues of peptides as well as nuclear and cytoplasmic proteins. It is also demonstrated that the sulfation on O-GlcNAc is sensitive to OGT and O-β-N-acetylglucosaminidase treatment. The labeled samples are separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. Overall, the method is sensitive, specific and convenient.
authors.affiliation: R&D Systems, Inc., 614 McKinley Place N.E., Minneapolis, MN 55413, USA leon.wu@rndsystems.com jzaia@bu.edu.
authors.forename: Zhengliang L
authors.initials: ZL
authors.lastname: Wu
authors.affiliation: R&D Systems, Inc., 614 McKinley Place N.E., Minneapolis, MN 55413, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Robey
authors.affiliation: R&D Systems, Inc., 614 McKinley Place N.E., Minneapolis, MN 55413, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Tatge
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry, Boston University, Boston, MA 02118, USA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Lin
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry, Boston University, Boston, MA 02118, USA.
authors.forename: Nancy
authors.initials: N
authors.lastname: Leymarie
authors.affiliation: R&D Systems, Inc., 614 McKinley Place N.E., Minneapolis, MN 55413, USA.
authors.forename: Yonglong
authors.initials: Y
authors.lastname: Zou
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry, Boston University, Boston, MA 02118, USA leon.wu@rndsystems.com jzaia@bu.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Zaia
mesh_terms: D000117:Acetylglucosamine / Q000032:analysis* / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
chemical_list: D000117:Acetylglucosamine; D000118:Acetylglucosaminidase; D013431:Sulfates; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases
keywords: CHST; O-GlcNAc; OGA; OGT; glycosylation