We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885-17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N-and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80 -110 nM range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80 -90% by heparin (2 g/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N-or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.In previous studies we biochemically characterized a major heparan sulfate proteoglycan of nervous tissue (1, 2) that we later cloned and identified as the rat homologue of glypican-1 (3), the initial member of a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans that is currently composed of six vertebrate proteins. High levels of glypican-1 mRNA are present in brain and skeletal muscle, and in situ hybridization histochemistry demonstrated that glypican-1 mRNA is especially prominent in cerebellar granule cells, large motor neurons in the brain stem, and CA3 pyramidal cells of the hippocampus (4). These results and parallel immunocytochemical studies indicate that glypican-1 is predominantly a neuronal product in the late embryonic and postnatal rat nervous system. Based on genetic studies, it would appear that the glypicans play a significant role in cell growth and development. Dally, the Drosophila homologue of glypican-1, is required for the control of cell division in the developing visual system and for morphogenesis of other tissues (5), and the human homologue of glypican-3/OCI-5 (GPC3) is found to be mutated in pat...

The localization of aggrecan and mRNA splice variants of versican in the developing rat central nervous system has been examined by using specific polyclonal antibodies to the nonhomologous glycosaminoglycan attachment regions of these hyaluronan-binding chondroitin sulfate proteoglycans. At embryonic day 16 (E16), aggrecan and versican splice variants containing either or both the ␣-and ␤-domains are present in the marginal zone and subplate of the cerebral cortex and in the amygdala, internal capsule, and the optic and lateral olfactory tracts. There is strong staining of versican but not of aggrecan in the hippocampus and dentate gyrus by E19, whereas both aggrecan and ␣-versican are present in the fimbria. At E19, aggrecan is seen throughout the cerebral cortex, whereas the distribution of versican is considerably more limited, being confined essentially to the marginal zone and subplate. At 1 week postnatal, both aggrecan and versican are present in the prospective white matter and in the molecular and granule cell layers of the cerebellum, but neither proteoglycan is seen in the external granule cell layer. ␣-but not ␤-versican staining is seen in Purkinje cells, and aggrecan staining of Purkinje cells is also rather minimal. In the spinal cord at E13, aggrecan is present in the dorsal root entry zone, ventral funiculus, mantle layer, and floor plate, as well as in the dorsal root ganglia and ventral roots. However, ␣-versican is confined to the dorsal root entry zone and the ependyma surrounding the spinal canal, and ␤-versican is not present in spinal cord parenchyma at this developmental stage, being limited to the surrounding connective tissue. By E19, there are significant amounts of all three proteoglycans in the spinal cord. Aggrecan staining is most intense in the lateral funiculus and the fasciculi gracilis and cuneatus, where ␣-versican staining is also strong. In contrast, ␤-versican is seen predominantly in the motor columns. Differences in the localization and temporal expression patterns of these chondroitin sulfate proteoglycans suggest that, like neurocan and phosphacan, they have partially complementary roles during central nervous system development.

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