Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule.

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Abstract
By means of a multistage quantitative assay, a new kind of cell adhesion molecule (CAM) was identified on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which is named neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAM separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously inhibited by Fab'' fragments prepared from antisera against neuronal membranes but not by Fab'' fragments against N-CAM, the neural cell adhesion molecule. Neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAM. To verify this, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and .gamma. spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab'' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells inhibited neuronal vesicle binding to glial cells. The inhibitory activity of the Fab'' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab'' fragment inhibition; after chromatography, neutralizing activity was enriched 50-fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an MW = 135,000 band in immunoblots of embryonic chick brain membranes. This molecule is present on the surfaces of neurons and it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody and the original polyspecific antibodies that were active in the assay did not bind to glial cells, neuron-glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.