Synaptic Membrane Glycoproteins gp65 and gp55 Are New Members of the Immunoglobulin Superfamily

Abstract

Glycoproteins gp65 and gp55 are major components of synaptic membranes prepared from rat forebrain. Both are recognized by the monoclonal antibody SMgp65. We have used SMgp65 to screen a rat brain cDNA expression library. Two sets of overlapping cDNAs that contain open reading frames of 397 and 281 amino acids were isolated. The deduced proteins are members of the immunoglobulin (Ig) superfamily containing three and two Ig domains, respectively. The common part has ϳ40% sequence identity with neurothelin/basigin. The identity of the proteins as gp65 and gp55 was confirmed by production of new antisera against a common recombinant protein fragment. These antisera immunoprecipitate gp65 and gp55. Furthermore, expression of gp65 and gp55 cDNAs in human 293 cells treated with tunicamycin results in the production of unglycosylated core proteins of identical size to deglycosylated gp65 and gp55. Northern analysis revealed that gp65 transcripts are brain-specific, whereas gp55 is expressed in most tissues and cell lines examined. The tissue distribution was confirmed at the protein level though the pattern of glycosylation of gp55 varies between tissues. In situ hybridization experiments with a common and a gp65-specific probe suggest differential expression of gp65 and gp55 transcripts in the rat brain.Synaptic junctions are highly specialized areas of contact and communication between neurons. They comprise the membranes and the underlying cytoskeleton of the pre-and postsynaptic neurons. In particular, the postsynaptic nerve ending is characterized by the presence of an electron-dense cytoskeletal structure, the postsynaptic density (PSD) 1 , which underlies the postsynaptic membrane. It is well established that synaptic structures and biochemically isolated synaptic subfractions, particularly synaptic membrane (SM) and PSD fractions, are enriched in distinct sets of glycoproteins that bind the lectin concanavalin A (1, 2). Some of these glycoproteins have been identified and their functions established. These include the major 180-kDa PSD glycoprotein that corresponds to the NR2B subunit of the N-methyl-D-aspartate receptor (3) and the SMenriched glycoprotein, gp50, that has been identified as the ␤2 subunit of the Na ϩ /K ϩ -ATPase (4, 5). SM and PSD glycoproteins, which are oriented with their oligosaccharide-containing domains facing into the synaptic cleft, are ideally placed to mediate adhesive interactions between the pre-and postsynaptic nerve endings. Indeed, the PSD-enriched PAC 1 glycoproteins (6, 7) have been identified as members of the cadherin family (8), providing evidence for such a role. Furthermore, integrin-type adhesion molecules (9) and NCAM (10), a member of the immunoglobulin (Ig) superfamily, have been recognized as SM components. Considering the complex interplay between stabilization and plasticity at synaptic connections, it is likely that more synaptic glycoproteins with adhesive functions are to be identified.Here we describe the molecular cloning of cDNAs encoding two synaptic glycopr...

The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses

“…To express the F c region of human IgG alone, the BM40 signal sequence was fused directly to the F c sequence of pIgPlus. Cell lines stably expressing and secreting the fusion proteins were generated as described (23). Fusion proteins were purified from culture supernatants by affinity chromatography using GammaBind Plus Sepharose.…”

“…Transcripts of both isoforms are widely distributed in rat brain. However, whereas np55 is present in all brain regions, np65 is concentrated in subpopulations of predominantly forebrain neurons and is enriched in neuropil regions (23,24).…”

“…The 65-kDa, but not the 55-kDa, isoform is enriched in rat forebrain postsynaptic density (PSD) preparations. Whereas np55 is expressed in most rat tissues, np65 is brain-specific (23,26). Transcripts of both isoforms are widely distributed in rat brain.…”

“…Both proteins are highly glycosylated and occur as multiple glycoforms (23)(24)(25)(26). Interestingly, a fraction of np65 carries fucose ␣ (1-2)-linked to galactose in its carbohydrate moiety (27).…”

“…The neuroplastins are major glycoprotein components of synaptic membrane (SM) preparations belonging to the Ig superfamily (23). They originally were identified by using the mAb SMgp65, which was generated against SM glycoprotein preparations (24,25).…”