title: A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation.
abstract: Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Westmead, Australia. mgraham@cmri.org.au
authors.forename: Mark E
authors.initials: ME
authors.lastname: Graham
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bache
authors.forename: George E
authors.initials: GE
authors.lastname: Craft
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D034041:Monomeric Clathrin Assembly Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D013403:Sugar Phosphates / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D034041:Monomeric Clathrin Assembly Proteins; D010446:Peptide Fragments; D013403:Sugar Phosphates; C439023:clathrin assembly protein AP180; D000117:Acetylglucosamine