O-GlcNAc glycosylation sites bracket phosphorylation sites in synapsin I

Numerous cytoplasmic and nuclear proteins are modified by the attachment of N-acetylglucosamine to serine or threonine residues (O-GlcNAc). The abun(lance and dynamic nature of O-GlcNAc glycosylation (O-GlcNAcylation) is similar to that of phosphorylation. A reciprocal relationship between phosphorylation and O-GlcNAcylation is indicated by (1) the identical glycosylation and phosphorylation site in c-Myc and (2) the mutually exclusive O-GlcNAc and phospho-forms of RNA polymerase II. Synapsin I, a major phosphoprotein in the nerve terminal, anchors synaptic vesicles to the cytoskeleton (Greengard etal, 1993 Science 259:780). Phosphorylation of synapsin I lowers its affinity for synaptic vesicles and actin. Synapsin I also bears O-GlcNAc. Elucidating synapsin I function requires understanding both its phospho- and glyco- modifications. We mapped 50-GIcNAc sites in synapsin I by reverse phase HPLC analysis coupled with gas phase sequencing, mass spectrometry (LC-EMS/MS. MALDI-TOF) and manual adman degradation of [3H]galactose-labeled OGIcNAc on synapsin I tryptic peptides. We found that the O-GIcNAc sites in synapsin I are (1) as abundant as its phosphorylation sites, (2) clustered in its B and D domains and (:3) bracket its sites of phosphorylation. We are currently using synthetic unmodified and gtyco-peptides to address the effect of O-GIcNAcylation on the phosphorytation of synapsin I by purified kinases.