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-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 a KD 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.
Mesh Terms:
Amino Acid Sequence, Animals, Antibodies, Monoclonal, Binding Sites, Binding, Competitive, Brain, Cell Line, Chickens, Chromatography, Affinity, Edetic Acid, Humans, Immunoblotting, Lectins, C-Type, Ligands, Mice, Models, Structural, Molecular Sequence Data, Nerve Tissue Proteins, Peptide Fragments, Protein Conformation, Proteochondroitin Sulfates, Rats, Recombinant Fusion Proteins, Recombinant Proteins, Tenascin
J. Biol. Chem.
Date: Oct. 24, 1997
Download Curated Data For This Publication
7600
Switch View:
  • Interactions 1