Monoclonal antibody to neural cell surface protein: Identification of a glycoprotein family of restricted cellular localization

Abstract

A monoclonal antibody, designated anti-NSP-4 (anti-Neural cell Surface Protein-4), was obtained from a hybridoma generated by fusing rat myeloma cells with splenocytes of a rat immunized with membranes from the cerebella of weaver mutant mice. This antibody reacted with several high-molecular weight polypeptides in extracts prepared from the newborn and adult CNS of wild-type mice. The main NSP-4-reactive bands from neonatal cerebellum and spinal cord migrated with apparent molecular weights of 220,000 and 140,000. Major bands of 160,000 and of 175,000, 160,000 and 140,000 molecular weight were revealed in the adult cerebellum and spinal cord, respectively. Reaction of the antibodies with concanavalin A-binding proteins demonstrated the glycoprotein nature of the antigen. Cell types expressing NSP-4 antigen were determined using indirect immunofluorescence on monolayer cultures of early postnatal mouse cerebellar and dorsal root ganglion cells and on sections of developing and adult mouse cerebellum. In cerebellar cultures, the antibody reacted with the surface membrane of a subpopulation of astrocytes and of a small subset of neurones. In dorsal root ganglion cultures, anti-NSP-4 antibodies were highly specific for a subclass of small neurones. Staining for NSP-4 in sections of adult cerebellum was confined to the granular layer where the antibody seemed to label astroglia. In the developing cerebellum, NSP-4 staining outlined cell bodies of neuroblasts and migrating granule cells in the external granular layer. Post-migratory granule cells and Purkinje cells were negative. As in the adult, the labeled structures in the internal granular layer were probably astrocytes. Our results on the in vivo and in vitro localization of NSP-4 show its expression by subclasses of neurones and astrocytes in the cerebellum and by a subclass of neurones in cultures from the peripheral nervous system. The developmentally-regulated changes in the molecular weight forms of the NSP-4 antigen together with the shift in its cellular localization during cerebellar ontogeny suggest a functional significance for this antigen in developmental processes.