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.
Rauch
U
U
Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany. rauch@biochem.mpg.de
Clement
A
A
Retzler
C
C
Fröhlich
L
L
Fässler
R
R
Göhring
W
W
Faissner
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry* / Q000378:metabolism*; D002846:Chromatography, Affinity; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D037181:Lectins, C-Type; D008024:Ligands; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D058580:Neurocan; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019063:Tenascin / Q000737:chemistry* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011508:Chondroitin Sulfate Proteoglycans; D037181:Lectins, C-Type; D008024:Ligands; D009419:Nerve Tissue Proteins; D058580:Neurocan; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019063:Tenascin; C076817:NCAN protein, human; D004492:Edetic Acid