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Neuron-glia cell adhesion molecule (Ng-CAM) has previously been shown to be present exclusively on neurons and to mediate adhesion between neuronal membranes and glial cells. In the present study, its chain structure, binding functions, and relation to N-CAM (the other known CAM on neurons) were investigated further. Three polypeptide components of chicken Ng-CAM (Mr 200,000, 135,000, and 80,000) have been isolated. By using specific antisera against each component, the Mr 135,000 and Mr 80,000 components were found to cross-react antigenically with the Mr 200,000 component but not with each other. The conclusion that the Mr 135,000 and 80,000 components are structurally related to different regions of the Mr 200,000 component was further supported by the finding that 32P could be incorporated in vitro into the Mr 200,000 and 80,000 components but not into the Mr 135,000 component. Ng-CAM appears to be involved in both neuron-glia adhesion and neuron-neuron adhesion by distinguishable mechanisms that appear to involve different sites or conformations of the molecule. Polyclonal antibodies and a monoclonal antibody against Ng-CAM both inhibited adhesion between glia and neurons derived from brain, cerebellum, and retina. In contrast, antibodies against N-CAM (which inhibit neuron-neuron adhesion) did not inhibit neuron-glia adhesion. These findings confirm the proposed function of Ng-CAM in neuron-glia adhesion. In addition, however, Ng-CAM was found to be involved directly or indirectly in neuron-neuron adhesion. Non-cross-reactive polyclonal anti-Ng-CAM and anti-N-CAM antibodies each inhibited the aggregation of neurons from whole brain and cerebellum and the inhibition was greater when both antibodies were present together. In contrast, monoclonal anti-Ng-CAM antibodies were found that inhibited neuron-glia adhesion but did not inhibit neuronal cell aggregation. The amount of Ng-CAM expressed on neurons was not directly predictive of the effect of anti-Ng-CAM antibodies on their homotypic aggregation. Although Ng-CAM and N-CAM can be expressed simultaneously on individual neurons, the ratio of N-CAM to Ng-CAM ranged from 1.5 for cerebellar cells to 10.0 for retinal cells. While, as expected, retinal cell aggregation was inhibitable only by anti-N-CAM, cerebellar cells, which expressed at least as much Ng-CAM as brain cells, showed significantly less inhibition by anti-Ng-CAM antibodies. These findings raise the possibility that Ng-CAM may actually interact with N-CAM to yield non-linear effects. That Ng-CAM and N-CAM may function differently in vivo was suggested by their distribution in sections of brain regions. Within the cerebellum, for example, immunofluorescent anti-N-CAM staining was relatively uniform in all layers; in contrast anti-Ng-CAM staining was absent on dividing external granule cells and was present in greatest abundance on processes of post-mitotic migratory cells in the molecular layer. These observations are consistent with the hypothesis that Ng-CAM mediates neuron-glia adhesion and is thereby also involved in neuronal migration along radial glial cells.

Previous studies in this laboratory have led to the identification of the neural cell adhesion molecule, N-CAM, a homophilic ligand that mediates adhesion between neurons as well as between neurons and striated muscle precursors. By means of a similar immunological approach but with different assays, we have now identified a cell adhesion molecule on neurons (Ng-CAM) that mediates the heterotypic adhesion between neuronal membranes and glial cells. In this paper, we compare certain aspects of the structure and function of Ng-CAM and embryonic N-CAM from the chicken. Ng-CAM was localized by specific antibodies on neurons but not on glia, and double-staining methods showed that individual neurons contained both Ng-CAM and N-CAM. Embryonic Ng-CAM migrates primarily as a single component of Mr 135,000; its apparent Mr shifted to 127,000 after neuraminidase treatment. In contrast, the embryonic form of N-CAM migrates on NaDodSO4/polyacrylamide gels in the apparent Mr range of 200,000-250,000; after neuraminidase treatment, N-CAM migrates as two components of Mr 170,000 and Mr 140,000. Although both Ng-CAM and N-CAM have calcium-independent binding mechanisms, immunologically based cell adhesion assays suggested that they have different specificities in mediating cell adhesion. Whereas 0.25 micrograms of Ng-CAM partially neutralized the ability of 0.5 mg of polyspecific antineural Fab' fragments to inhibit the heterotypic binding of neuronal membrane vesicles to glial cells and larger amounts of Ng-CAM completely neutralized this inhibition, 20 micrograms of N-CAM had no neutralization activity in this assay. Reciprocally, 0.25 micrograms of N-CAM partially neutralized the ability of 0.5 mg of the same Fab' fragments to inhibit the direct homotypic aggregation of neuronal cells, but 20 micrograms of Ng-CAM had no detectable activity. Although peptide maps of the two cell adhesion molecules differed considerably and despite the differences in binding specificity of these molecules, two independently derived monoclonal antibodies were found to crossreact with both Ng-CAM and N-CAM. Therefore, these different neuronal cell adhesion molecules with distinct binding specificities share at least one antigenic determinant, raising the possibility that they arose from a common evolutionary precursor.

An extracellular matrix protein, cytotactin, with widespread tissue distribution has been identified, isolated, and partially characterized. Cytotactin mediates glia-neuron adhesion in vitro, but unlike Ng-CAM, the neuron-glia cell-adhesion molecule, it is absent from neurons. Cytotactin was isolated from 14-day embryonic chicken brains as structurally related polypeptides of Mr 220,000, 200,000, and 190,000. These polypeptides were efficiently extracted in the absence of detergent and appeared to be disulfide-linked into higher polymers. Immunofluorescence staining with specific antibodies indicated that cytotactin is found in extracellular spaces and in basement membranes of a variety of non-neural tissues including smooth muscle, lung, and kidney. In the cerebellum, it appears on glial end-feet, on Bergmann glial fibers, and in extracellular spaces. The molecule is synthesized by glia and cells from smooth muscle, lung, and kidney. It is found at the surface of glia in culture in a cell-associated fibrillar pattern. A survey of the times and sites of its appearance during embryogenesis is consistent with the hypothesis that cytotactin is a cell-substrate adhesion molecule that may mediate cell migration in a site-restricted fashion.