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.

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Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan of the aggrecan family which has been shown to interact with neural cell adhesion molecules and tenascin, could be visualized by rotary shadowing electron microscopy as two globular domains interconnected by an extended flexible filament of 60 -90 nm. Several recombinant neurocan fragments generated in the human embryonic kidney cell line 293 represent as observed by electron microscopy the expected parts of this structure, which indicates a correct folding of these molecules. Biological activity of the recombinant N-terminal globular domain could be demonstrated by its coelution with hyaluronan in gel permeation chromatography. In addition, the modification of the recombinant fragments with certain carbohydrate structures was analyzed. High mannose oligosaccharides could be mapped to the N-terminal globular domain of the brainderived molecule. Only recombinant fragments containing parts of the central region of the molecule were modified with chondroitin sulfate chains and with the HNK-1 epitope, and could be considerably altered in their migratory behavior on SDS-polyacrylamide gel electrophoresis by neuraminidase treatment. These findings and the electron microscopic shape indicate a mucin-like character for the central neurocan region.

Neurocan is a brain-specific chondroitin sulfate proteoglycan, which has been shown to bind to the neural cell adhesion molecule N-CAM and to inhibit its homophilic interaction. To study in more detail the structures of neurocan responsible for this interaction, various recombinant neurocan fragments were generated. The ability of these fragments to interact with N-CAM was investigated in several different in vitro assay systems, enzyme-linked immunosorbent assaytype binding assays, Covasphere-aggregation assays, and assays based on an optical biosensor (BIAcore™) system. The analysis of the homophilic N-CAM interaction in the BIAcore system revealed a K D of 64 nM. This homophilic interaction could be reduced by preincubation of soluble N-CAM with neurocan. Direct binding of N-CAM to immobilized neurocan core protein and recombinant neurocan fragments could also be demonstrated, and K D values between 25 and 100 nM were obtained. In addition, direct binding of N-CAM to chondroitin sulfate could be demonstrated.Binding of N-CAM to the immobilized neurocan core protein could be inhibited with all recombinant fragments containing chondroitin sulfate or major parts of the mucin-like central region of neurocan. For the inhibition of homophilic N-CAM interactions, however, a combination of globular and extended structures was required.