Mapping of a Defined Neurocan Binding Site to Distinct Domains of Tenascin-C

doi:10.1074/jbc.272.43.26905

Mapping of a Defined Neurocan Binding Site to Distinct Domains of Tenascin-C*

From the Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried and the ^§Department of Neurobiology, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany

Abstract

Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH₂-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH₂-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last 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 addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising 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 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded aK _D value of 17 nm for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.

Footnotes

↵* This work was supported in part by German Research Council Grants DFG, Ra 544/3-1 (to U. R.) and SFB 317/A2 (to A. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany. Tel.: 49-89-8578-2215; Fax: 49-89-8578-2422; E-mail:rauch{at}biochem.mpg.de.
↵¶ Supported by graduate training stipends of the Studienstiftung des Deutschen Volkes and the Graduiertenkollegs Zelluläre und Molekulare Neurobiologie.
↵‖ Supported by a Schilling Professorship for Neuroscience.
↵** Supported by a Schilling Professorship for Neuroscience.
↵1 The abbreviations used are: EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; FNIII, fibronectin type III; BSA, bovine serum albumin; mAb, monoclonal antibody; TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module; ELISA, enzyme-linked immunosorbent assay; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
↵2 U. Rauch, unpublished observation.
↵3 A. Clement and A. Faissner, unpublished observation.
- Received July 22, 1997.