Molecular heterogeneity and structural evolution during cerebellar ontogeny detected by monoclonal antibody of the mouse cell surface antigen BSP-2.

The monoclonal antibody anti-BSP-2 defines a set of glycoproteins present on the neuronal cell surface in dissociated mouse cerebellar cultures and on neurons and astrocytes in sections of the mouse cerebellum. This antibody was used in the present study to characterize the antigens recognized in cerebellar cultures and in the developing and adult mouse cerebellum in vivo. In extracts from cerebellar cultures and from late postnatal or adult cerebellum, the anti-BSP-2 antibody reacted with a triplet of glycosylated polypeptide chains of 180,000, 140,000 and 120,000 mol. wt. Early postnatal cerebellum contained a different form of BSP-2 antigen which migrated as one broad or several closely spaced diffuse bands in the 190,000-250,000 mol. wt. region of SDS polyacrylamide gels. During cerebellar ontogeny, the adult pattern emerged gradually between postnatal days 5 and 13. The cellular expression of the BSP-2 antigen was studied by immunohistochemistry on sections of the developing cerebellum. At postnatal day 3, the antigen was found mainly on cell bodies and fibers of the Bergmann glia and on astrocytes of the granular layer. Immature granule cells of the outer zone of the external granular layer lacked the antigen, but they appeared to acquire the antigen during their migration to the internal granular layer. At postnatal day 13, the immunofluorescence pattern was not different from the one seen in the adult. These results suggest that the neonatal 190,000-250,000 mol. wt. form of BSP-2 may at least in part be expressed by astroglial cells and they show a close correlation between the emergence of the adult forms of the antigen and the appearance of labeled granule cells in the internal granular layer. In vitro degradation implying cleavage of sialic acid residues, but probably also proteolysis and/or cleavage of different glycans converted the neonatal form of BSP-2 into the triplet pattern and ultimately into a p120 component. Neuraminidase digestion of the adult antigens produced small molecular weight shifts without converting one band into the other, but endogenous enzyme activities were capable of degrading the p180 and p140 bands by converting them into the p120 protein. Our findings support the idea that distinct, but structurally similar surface glycoproteins created by post-translational modifications from a common precursor molecule may be expressed by different cell types or during different developmental stages. As shown by sequential immunoprecipitation experiments, BSP-2 and the rat neuronal membrane protein D2 may belong to the same family of surface glycoproteins.