Abstract
Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn(60). Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn(70)/Asn(104) flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn(60) reduces adhesion, N-glycans at Asn(70)/Asn(104) of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.
Highlights
Receptor tyrosine kinases, including EphB receptors, instruct synaptogenesis through trans-synaptic signaling [15, 16]. These synapse-inducing proteins act in conjunction with N-cadherins that set the pace of synaptic maturation [17, 18]. Among these synapse-organizing proteins, SynCAMs4 form a family of four Ig superfamily members that are single-spanning membrane proteins with three extracellular Ig-like domains [19]
Surface Expression Control—COS7 cells expressing synaptic cell adhesion molecules (SynCAMs) constructs tagged with an extracellular FLAG epitope were fixed, labeled with anti-FLAG antibodies to detect surface-expressed epitopes, washed, and permeabilized using 0.1% Triton X-100 to perform immunostaining for total SynCAM protein
Surface-expressed SynCAM proteins were detected in these live cells by simultaneously adding anti-FLAG and secondary antimouse antibodies conjugated to Alexa 488 (Invitrogen) (1:1000)
Summary
We address the molecular properties that underlie and regulate SynCAM adhesion and function. Mass spectrometric, and biochemical analyses of SynCAM 1 and 2 demonstrate that they carry N-glycans adjacent to and within the first Ig domain that provides their extracellular binding interface. The glycosylation of these two SynCAM family members serves different roles. N-glycans within the Ig1 binding interface of SynCAM 2 reduce its binding, glycosylation at the SynCAM 1 Ig1 domain promotes its adhesion. The ability to N-glycosylate SynCAM 1 increases its trans-synaptic interactions and synapse inducing activity. Together, these results identify glycosylation as a novel mechanism for positively and negatively regulating the trans-synaptic SynCAM adhesion complex in the brain
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