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The monoclonal antibody PAC 1 (postsynaptic density and cytoskeleton enriched) recognizes an epitope present on two postsynaptic density-enriched glycoproteins of 130,000 (postsynaptic density-enriched glycoprotein 130) and 117,000 mol. wt (postsynaptic density-enriched glycoprotein 117), and a cytoskeleton-enriched polypeptide of 155,000 mol. wt (cp155). The PAC 1 antibody has been used to study the development of the PAC 1 antigens in the developing rat forebrain in vivo and in tissue culture. cp155 is detected by embryonic day 14 and its level continues to rise until the sixth postnatal week. Postsynaptic density-enriched glycoproteins 130 and 117 are also expressed in embryonic brain although the level of postsynaptic density-enriched glycoprotein 130 initially increases more rapidly than that of postsynaptic density-enriched glycoprotein 117. Peak values are observed at postnatal days 4 (postsynaptic density-enriched glycoprotein 117) and 9 (postsynaptic density-enriched glycoprotein 130). The level of post synaptic density-enriched glycoprotein 117 subsequently decreases to some 50% of the peak value by postnatal day 42. Immunocytochemical studies show that PAC 1 immunoreactivity in developing cerebral cortex, detectable by postnatal day 0, is primarily associated with the perikarya and dendrites of pyramidal cells. The immunoreactivity develops as patches of PAC 1-positive neurons, uniform staining of the cortex only being fully established after postnatal day 9. Double-immunofluorescence labelling studies of forebrain cultures prepared from embryonic day 18 animals shows that many, but not all, growth-associated protein 43-positive neurons exhibit PAC 1 immunoreactivity. Some non-neuronal cells also stain with the PAC 1 monoclonal antibody. The growth cones of cultured neurons exhibit PAC 1 immunoreactivity and the PAC 1 antigens are detected on immunodeveloped western blots of isolated growth cones. The PAC 1 epitope is intracellular, but immunoreactivity does not co-localize with F-actin as detected by rhod-amine-phalloidin or with tubulin immunoreactivity. Postsynaptic density-enriched glycoprotein 130 is readily detected on PAC 1 immunodeveloped western blots of forebrain cultures maintained for up to 14 days in vitro. Postsynaptic density-enriched glycoprotein 117 is only poorly expressed by these cultures. The PAC 1 glycoproteins are present in forebrain synaptic membranes and postsynaptic densities at an early stage of development. The synaptic membrane level of postsynaptic density-enriched glycoprotein 130 and postsynaptic density-enriched glycoprotein 117 increases markedly between postnatal days 3 and 8. The level of both glycoproteins detected in postsynaptic densities remain virtually constant from postnatal days 9-90. These results are consistent with functional roles for these molecules in neuronal and synapse development.

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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)

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A monoclonal antibody has been raised which recognizes an epitope, PAC 1 (postsynaptic density and cytoskeleton enriched), which is specifically associated with two novel glycoprotein components of forebrain postsynaptic density preparations and a novel neuronal cytoskeletal-associated polypeptide. The monoclonal antibody has been used to study the cellular and subcellular localization of these molecules and for the partial characterization of all three PAC 1 antigens in the rat. The PAC 1 epitope is present on two concanavalin A binding glycoproteins of apparent molecular weights 130,000 (pgp130) and 117,000 (pgp117). Both species are enriched in preparations of rat forebrain postsynaptic densities and to a lesser extent in synaptic membranes. The epitope is also expressed by a polypeptide of 155,000 mol. wt, cp155. This molecule is highly enriched in cytoskeleton rather than membrane preparations. Enzymic removal of N-linked carbohydrate lowers the molecular weights of the PAC 1 glycoproteins pgp130 and pgp117 by 11,000 and 14,000 respectively, and suggests that cp155 is not glycosylated. Detergent, alkaline and salt extractions of postsynaptic densities and synaptic membranes indicate that pgp130 and pgp117 are integral membrane glycoproteins and are tightly bound components of postsynaptic density preparations. Immunocytochemical studies of adult rat forebrain show prominent staining of pyramidal cell dendrites and perikarya. There is no evidence of glial staining. Electron microscope studies show staining of microtubules together with punctate deposits of plasma membrane-associated reaction product. Several criteria have been used to show that pgp130 and pgp117 do not correspond to other known neuronal glycoproteins of similar molecular weight. We conclude that the PAC 1 epitope is expressed by two novel synaptic glycoproteins which are very probably integral components of the postsynaptic density and by a novel neuronal cytoskeleton-associated protein.

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