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

Abstract

Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH 2 -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 NH 2 -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 K 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.

Alternative Splicing in the Aggrecan G3 Domain Influences Binding Interactions with Tenascin-C and Other Extracellular Matrix Proteins

“…5 and Table I). This is in agreement with the neurocan binding to FnIII-(4 -5) reported previously (20). Interestingly, exactly the same region (FnIII-(3-5)) was identified as the lectin interaction site on tenascin-R (15).…”

“…We were, however, unable to detect versican interaction with tenascin-C in the same assays, even though immunoblotting revealed its presence. It has since been demonstrated that the neurocan C-type lectin domain can bind tenascin-C in similar assays (20). We now confirm the neurocan interaction with tenascin-C and demonstrate that aggrecan, versican, and brevican also bind tenascin-C.…”

“…Alternative splicing of the EGF and SCR modules in the G3 domain is well established (22)(23)(24)(25)(26) and could have an effect on the interactions of the C-type lectin repeat. Although there are binding studies using full-length proteoglycan (17) or recombinant G3 domains (14,15,20), no thorough investigation of the effects of alternative splicing in the G3 domain has been published. To this end we produced a set of recombinant aggrecan G3 modules containing different combinations of the EGF1, EGF2, C-type lectin, and SCR modules.…”

“…The G3 domain also binds sulfated glycolipids on the cell surface (19). In addition, neurocan has been reported to bind to tenascin-C (20). The ECM protein ligands for the G3 domains are all dimeric or multimeric proteins, and we have shown that they can crosslink proteoglycans from different hyaluronan/proteoglycan aggregates (17).…”

Two-Tiered Inhibition of Axon Regeneration at the Dorsal Root Entry Zone

“…Direct lesions to the CNS induce CSPG deposition at the lesion site, which halts the axonal progress of transplanted DRG neurons (Davies et al, 1997(Davies et al, , 1999. The extent to which the astrocytic reaction at the DREZ mimics that after CNS lesions is unknown, but in addition to proliferation of astrocytes, oligodendrocyte precursors, and microglia (Liu et al, 1998;Fawcett and Asher 1999), common features include increased NG2 expression by glial cells (Dou and Levine, 1994;Levine, 1994;Zhang et al, 1999) and upregulation of tenascin-C and tenascin-R (Zhang et al, 1997Fawcett and Asher, 1999), glycoproteins that bind many CSPGs and myelin-associated glycoprotein (MAG) in the extracellular matrix (Rauch et al, 1997;Yang et al, 1999).…”