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.

No abstract

We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2-7 nM range. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by ϳ60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by ϳ75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (K d ‫؍‬ 0.3-8 nM), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.

Proteoglycans appear to play an important role in modulating cell-cell and cell-matrix interactions during nervous tissue histogenesis. The nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta were found to be high-affinity ligands of the neural cell adhesion molecule TAG-1/axonin-1, with dissociation constants of 0.3 nM and 0.04 nM, respectively. Phosphacan binding was decreased by approximately 70% following chondroitinase treatment, whereas binding of neurocan was not affected. The contribution of chondroitin sulfate chains to the binding of neurocan and phosphacan to TAG-1/axonin-1 is therefore the opposite of that previously observed for their binding to two other Ig-superfamily neural cell adhesion molecules, Ng-CAM/L1 and N-CAM. Moreover, whereas phosphacan interactions with certain proteins are mediated at least in part by N-linked oligosaccharides on the proteoglycan, N-deglycosylation of phosphacan had no effect on its binding to TAG-1/axonin-1. In addition to the chondroitin sulfate proteoglycans described above, we have demonstrated that N-CAM is a high-affinity ligand of TAG-1/axonin-1 (Kd approximately 1 nM), and specific binding of TAG-1/axonin-1 to tenascin-C was also observed (Kd approximately 9 nM). Immunocytochemical studies of embryonic and early postnatal nervous tissue showed an overlapping localization of TAG-1/axonin-1 with all four of these ligands, further supporting the biological significance of their ability to interact in vitro.

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.

This review focuses primarily on studies concerning the potential roles of two nervous-tissue-specific chondroitin sulfate proteoglycans, viz., neurocan and phosphacan, in cell interactions and neurite growth in the developing central nervous system. The multiple ligands of these proteoglycans and the modulatory effects of various types of glycosylation are also considered. Other chondroitin sulfate proteoglycans, such as NG2, DSD-1, Cat-301, versican, and biglycan, are briefly discussed in relation to the functional properties that have been ascribed to them.