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.