Analysis of Neurocan Structures Interacting with the Neural Cell Adhesion Molecule N-CAM

Abstract

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.

Neurocan Is Dispensable for Brain Development

“…Neurocan message and protein were found in many parts of the brain, but not ubiquitously (6,22). Neurocan can bind to N-CAM, Ng-CAM, and axonin and interfere with the cell adhesive function of Ng-CAM (10,27,42). In vitro experiments showed a direct repulsive effect of neurocan on neurons and an inhibition of neurite outgrowth (8), suggesting that developmentally regulated neurocan expression in certain regions of the brain will have a significant impact on axon guidance and fasciculation.…”

“…Furthermore, it binds with high affinity to the neural cell adhesion molecules Ng-CAM/L1 (10), axonin (27), and N-CAM (42), which show an overlapping expression with neurocan. The N-terminal domain of neurocan mediates the binding to hyaluronan (43).…”

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

“…Experiments were performed and affinities were calculated in essentially the same way as described earlier (36), in part, with the identical covalently immobilized ligands.…”

Proteoglycans in Perineuronal Nets.

“…Retzler et al have shown that N-CAM binds to all major components of neurocan using various recombinant neurocan fragments [41]. Their results suggest that both neurocan-C and the N-terminal fragments are able to bind to N-CAM.…”