High affinity binding and overlapping localization of neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta with tenascin-R, amphoterin, and the heparin-binding growth-associated molecule

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 approximately 60% 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 approximately 75% 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 (Kd = 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.