N-cadherin is a major glycoprotein component of isolated rat forebrain postsynaptic densities.
We have previously described a monoclonal antibody, PAC 1, that recognises two postsynaptic density (PSD)-enriched glycoproteins (pgps) of apparent M(r) 130,000 (pgp130) and 117,000 (pgp117). Immunodevelopment of western blots of rat forebrain homogenate, synaptic membrane (SM), and PSD samples with PAC 1 and an N-cadherin antiserum shows that pgp130 and N-cadherin are of identical apparent M(r) and show identical patterns of enrichment in these fractions. The apparent molecular masses of pgp130 and N-cadherin are both lowered by 11 kDa following removal of N-linked carbohydrate with endoglycosidase-F containing N-glycopeptidase. The two molecules show an identical pattern of migration when separated by two-dimensional electrophoresis. A single 130-kDa band immunoprecipitated from solubilised PSD preparations by the N-cadherin antiserum is recognised by PAC 1 on western blots. We conclude that pgp130 is N-cadherin. Development of western blots of two-dimensional gel separations of SM and PSD glycoproteins shows that N-cadherin is a major glycoprotein component of PSDs. The immunoprecipitation experiments show that the M(r) of N-cadherin is greater than that of the major pgp, PSD gp116. The PAC 1 antibody recognises two concanavalin A-binding glycoproteins with apparent molecular masses of 136 and 127 kDa in liver samples. The 136-kDa band is also recognised by the N-cadherin antiserum. These observations, together with data showing that the PAC 1 epitope is intracellular, suggest that PAC 1 is a pan-cadherin antibody and recognises an epitope on the conserved cadherin intracellular carboxyl-terminal domain.
Beesley
P W
PW
Department of Biochemistry, Royal Holloway University of London, Egham, Surrey, England.
Mummery
R
R
Tibaldi
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D015820:Cadherins / Q000032:analysis* / Q000276:immunology; D003208:Concanavalin A / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D007163:Immunosorbent Techniques; D007526:Isoelectric Point; D008099:Liver / Q000737:chemistry; D008970:Molecular Weight; D016548:Prosencephalon / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D013569:Synapses / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D015820:Cadherins; D006023:Glycoproteins; D003208:Concanavalin A