The Fibrinogen-like Globe of Tenascin-C Mediates Its Interactions with Neurocan and Phosphacan/Protein-tyrosine Phosphatase-ζ/β

Abstract

Two nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan (the extracellular domain of protein-tyrosine phosphatase-/␤), are highaffinity ligands of tenascin-C. Using portions of tenascin-C expressed as recombinant proteins in human fibrosarcoma cells, we have demonstrated both by direct radioligand binding assays and inhibition studies that phosphacan binding is retained in all deletion variants except those lacking the fibrinogen-like globe and that phosphacan binds to this single domain with nearly the same affinity (K d ϳ12 nM) as to native or recombinant tenascin-C. However, maximum binding of neurocan requires both the fibrinogen globe and some of the adjacent fibronectin type III repeats. Binding of phosphacan and neurocan to intact tenascin-C, and of phosphacan to the fibrinogen globe, is significantly increased in the presence of calcium. Chondroitinase treatment of the proteoglycans did not affect their binding to either native tenascin-C or to any of the recombinant proteins, demonstrating that these interactions are mediated by the proteoglycan core proteins rather than through the glycosaminoglycan chains. These results are also consistent with rotary shadowing electron micrographs that show phosphacan as a rod terminated at one end by a globular domain that is frequently seen apposed to the fibrinogen globe in mixtures of phosphacan and tenascin-C. C6 glioma cells adhere to and spread on deletion variants of tenascin-C containing only the epidermal growth factor-like domains or the fibronectin type III repeats and the fibrinogen globe. In both cases cell adhesion was inhibited by similar concentrations of phosphacan, demonstrating that the fibrinogen globe is not necessary for this effect, which is apparently mediated by a direct action of phosphacan on the cells rather than by its interaction with the proteoglycan binding site on tenascin-C.We have previously reported that neurocan and phosphacan/ protein-tyrosine phosphatase-/␤, two major nervous tissuespecific chondroitin sulfate proteoglycans, are high affinity ligands of tenascin-C (apparent K d ϳ3 nM) and that phosphacan inhibits the adhesion of C6 glioma cells to tenascin-C (1). Neurocan is a multidomain proteoglycan with a 136-kDa core protein (2) and together with versican and brevican is a member of the aggrecan family of hyaluronan-binding proteoglycans (3). It contains N-terminal immunoglobulin-like and hyaluronanbinding domains, a C-terminal domain consisting of EGF-, lectin-, and complement regulatory protein-like sequences, and a nonhomologous central domain of 593 amino acids, which contains the attachment sites for the three chondroitin sulfate chains and a large number of O-glycosidic oligosaccharides. In contrast, phosphacan (4, 5), which contains a 173-kDa core protein and three chondroitin sulfate chains, is an mRNA splicing product that represents the entire extracellular domain of a receptor-type protein-tyrosine phosphatase that also occurs as a chondroitin sulfate proteoglycan in brain (3). Phos...

Generation of an environmental niche for neural stem cell development by the extracellular matrix molecule tenascin C

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CALEB Binds via Its Acidic Stretch to the Fibrinogen-like Domain of Tenascin-C or Tenascin-R and Its Expression Is Dynamically Regulated after Optic Nerve Lesion

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

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