High Affinity Binding and Overlapping Localization of Neurocan and Phosphacan/Protein-tyrosine Phosphatase-ζ/β 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 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.

Versican V2 Assembles the Extracellular Matrix Surrounding the Nodes of Ranvier in the CNS

“…The conjoint loss of versican V2, tenascin-R, and phosphacan from the nodal matrix in our mutants now confirms in vivo macromolecular interactions formerly postulated on the basis of in vitro findings. This includes interactions between versican V2 and tenascin-R (Aspberg et al, 1997) and between tenascin-R and phosphacan (Xiao et al, 1997;Milev et al, 1998). Since phosphacan is also absent from the nodes of Ranvier in TnR-null mice (Weber et al, 1999), we conclude that versican V2 first recruits tenascin-R, which in turn integrates phosphacan into the matrix.…”

Extracellular matrix of the central nervous system: from neglect to challenge

“…The transient deposition of neurocan and versican V0/V1, peaks in rats shortly after birth (Fig. 3) (MeyerPuttlitz et al 1995;Milev et al 1998a). Preferential accumulations are the marginal zone and the subplate of the neocortex, parts of the hypothalamus, the amygdala and the developing hippocampus and dentate gyrus (Meyer-Puttlitz et al 1996;Miller et al 1995;Popp et al 2003).…”

Identification of the Core Protein Carrying the Tn Antigen in Mouse Brain: Specific Expression on Syndecan-3.

“…In addition to glycosaminoglycans, many proteoglycans have other types of carbohydrate chains such as N-and O-glycans. Not only glycosaminoglycans but also N-and O-glycans and core proteins seem to be involved in a variety of biological interactions (Gould et al, 1992;Milev et al, 1998;Herndon et al, 1999;Oohira et al, 2000). Syndecan-3 is known to be a heparan sulfate proteoglycan present in the developing brain.…”