Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions

Synapsin I is concentrated in nerve terminals, where it appears to anchor synaptic vesicles to the cytoskeleton and thereby ensures a steady supply of fusion-competent synaptic vesicles. Although phosphorylation- dependent binding of synapsin I to cytoskeletal elements and synaptic vesicles is well characterized, little is known about synapsin I's O-linked N-acetylglucosamine (O-GlcNAc) modifications. Here, we identified seven in vivo O-GlcNAcylation sites on synapsin I by analysis of HPLC-purified digests of rat brain synapsin I. The seven O-GlcNAcylation sites (Ser⁵⁵, Thr⁵⁶, Thr⁸⁷, Ser⁵¹⁶, Thr⁵²⁴, Thr⁵⁶², and Ser⁵⁷⁶) in synapsin I are clustered around its five phosphorylation sites in domains B and D. The proximity of phosphorylation sites to O-GlcNAcylation sites in the regulatory domains of synapsin I suggests that O-GlcNAcylation may modulate phosphorylation and indirectly affect synapsin I interactions. With use of synthetic peptides, however, the presence of an O-GlcNAc at sites Thr⁵⁶² and Ser⁵⁷⁶ resulted in only a 66% increase in the K(m) of calcium/calmodulin-dependent protein kinase II phosphorylation of site Ser⁵⁶⁶ with no effect on its V(max). We conclude that O-GlcNAcylation likely plays a more direct role in synapsin I interactions than simply modulating the protein's phosphorylation.