MAPPING OF A DEFINED NEUROCAN BINDING-SITE TO DISTINCT DOMAINS OF TENASCIN-C

Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-te rminal domains containing C type lectin like modules, To detect and en hance the affinity for complementary ligands of neurocan, the COOH ter minal neurocan domain was fused with the NH2-terminal region of tenasc in-C, which contains the hexamerization domain of this extracellular m atrix glycoprotein, The fusion protein was designed to contain the las t downstream glycosaminoglycan attachment site and was expressed as a proteoglycan, In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the additi on of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment co mprising the fourth and fifth fibronectin type III module, The fusion protein directly reacted with this fragment in ligand blot and enzyme linked immunosorbent assay procedures, Both tenascin-C and TNfn4,5 wer e retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded a K-D value of 17 nM for puri fied tenascin-C, We conclude that a divalent cation-dependent interact ion between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.