title: O-GlcNAc modification of the extracellular domain of Notch receptors.
abstract: Epidermal growth factor (EGF) domains are posttranslationally modified with unique O-linked glycans. The classical types of O-glycans on EGF domains are O-fucose and O-glucose glycans, found on many plasma glycoproteins and signaling molecules, whose biological functions have been demonstrated especially in the context of the Notch signaling pathway. We recently discovered O-GlcNAc modification as a new modification of the EGF domain that occurs on the conserved Ser/Thr residue located between the fifth and sixth cysteine residues within the EGF domain of Notch receptors in Drosophila. Here, we describe the methods employed to detect the O-GlcNAc modification of EGF repeats of Notch receptors. These methods include mass spectrometric analysis, galactosyltransferase labeling, immunoblotting with a specific antibody, and beta-N-acetyl-hexosaminidase digestion experiments. We also describe a method to detect O-GlcNAc transferase activity from crude membrane fraction proteins prepared from cultured S2 cells.
authors.affiliation: Department of Biochemistry II, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Sakaidani
authors.forename: Koichi
authors.initials: K
authors.lastname: Furukawa
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Okajima
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002623:Chemistry Techniques, Analytical / Q000379:methods; D004330:Drosophila / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism* / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology
chemical_list: D051880:Receptors, Notch; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D000117:Acetylglucosamine