Abstract.We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chow droitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of ~1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan.To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth, lmmunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.

We We have previously described a chondroitin sulfate proteoglycan isolated from a phosphate-buffered saline extract of rat brain by immunoaffinity chromatography with the 3F8 monoclonal antibody and which is developmentally regulated with respect to its sulfation, carbohydrate composition and oligosaccharide structure, and immunocytochemical localization (1). A chondroitin/keratan sulfate proteoglycan (designated 3H1) was also isolated from rat brain by using monoclonal antibodies to the keratan sulfate chains (1). 3F8 and neurocan, another chondroitin sulfate proteoglycan of brain, for which the primary structure has been described (2), interact with neurons and the neural cell-adhesion molecules (CAMs), Ng-CAM and N-CAM (3). From radioligandbinding studies it was found that the brain proteoglycans bind with high affinity (Kd "O.5 nM) to Ng-CAM and N-CAM but not to other cell-surface and extracellular matrix proteins such as laminin, fibronectin, several collagens, epidermal growth factor and fibroblast growth factor receptors, or the myelin-associated glycoprotein (4). The 3F8 proteoglycan and neurocan inhibited neurite outgrowth and binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes, and direct binding ofneurons to the proteoglycan core glycoproteins was demonstrated with an assay in which cell-substrate contact was initiated by centrifugation (3,4). Recent studies have also shown that embryonic chicken brain neurons bind the 3H1 proteoglycan and contain cell-surface keratan sulfate chains (M. Flad, R.U.M., and R.K.M., unpublished results). These results indicate that brain proteoglycans can bind to neurons and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 and 3H1 proteoglycans.The primary structure of the 3F8 proteoglycan has now been determined by cDNA cloning, and we have identified this major chondroitin sulfate proteoglycan of brain as a possible mRNA splice variant of a receptor-type protein tyrosine phosphatase (PTP) named PTPC (5) and RPTFP. (6) (RPTPC/P). The tyrosine phosphatases act in concert with tyrosine kinases to regulate the phosphorylation state of proteins, and their structural features and potential physiological roles in signal transduction and cell-cycle regulation have recently been reviewed (7-9). With probes based on conserved sequences in their phosphatase domains, over 30 PTPs have now been cloned, but a major question concerning this family of key regulatory enzymes is the identification of their ligands and substrates. Our fidings indicate that neural CAMs and the exellular matrix protein tenascin may serve as ligands for RPIPC/P. Amino acid sequencing of the 3H1 chondroitin/keratan sulfate proteoglycan of brain demonstrated that it is a glycosylation variant of the 3F8 proteoglycan. We have named the 3F8/3H1 proteoglycan phosphacant and suggest that it may modulate cell interactions and other developmental processes in nervous tissue.

Abstract. Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTPg'/B). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble ~25I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of ,~0.1 nM. Binding was reduced by "~15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity.Immunocytochemical studies of embryonic rat spinal cord and early postnatal cerebellum demonstrated that phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system.