Molecular characterisation and structural relationship of the synapse-enriched glycoproteins gp65 and gp55.
gp65 and gp55 are glycoprotein components of CNS synapses that are recognised by a single monoclonal antibody, SMgp65. This antibody has now been used to investigate the molecular properties of these two glycoproteins and the structural relationship between them. Both gp65 and gp55 occur in most brain regions as doublets of apparent molecular masses of 63 and 67 kDa, and 52 and 57 kDa, respectively. Striatal samples, however, are enriched in a novel gp65 isoform of 69 kDa. Removal of oligosaccharide residues from gp65 and gp55 with trifluoromethanesulphonic acid shows that gp65 and gp55 are composed of single polypeptide chains of 40 and 28 kDa, respectively. Removal of sialic acid residues with neuraminidase lowers the apparent molecular mass of both glycoproteins by 5-6 kDa. Triton X-114 phase partitioning and alkaline extraction of synaptic membranes indicate that both gp65 and gp55 are integral membrane glycoproteins. Treatment of synaptic membranes with phosphatidylinositol-specific phospholipase C does not solubilise either glycoprotein. One-dimensional peptide and epitope maps obtained by digestion of gp65 and gp55 with endoproteinase lys C or subtilisin are consistent with a close structural relationship between the two molecules. Tryptic digestion of samples enriched in gp65 and/or gp55 results in the formation of a novel immunoreactive 53-kDa species that is resistant to further trypsin degradation except in the presence of 0.1% (wt/vol) sodium dodecyl sulphate. Trypsin treatment of cultures of forebrain neurones in situ lowers the apparent molecular mass of gp65 to 53 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Willmott
T
T
Department of Biochemistry, Royal Holloway and Bedford New College, Egham, Surrey, England.
Skitsa
I
I
Hill
I
I
Mummery
R
R
Beesley
P W
PW
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D002478:Cells, Cultured; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D008970:Molecular Weight; D016501:Neurites / Q000737:chemistry / Q000648:ultrastructure; D009474:Neurons / Q000737:chemistry / Q000648:ultrastructure; D010449:Peptide Mapping; D016548:Prosencephalon / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D013569:Synapses / Q000737:chemistry* / Q000648:ultrastructure; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D008562:Membrane Glycoproteins; D014357:Trypsin