ANALYSIS OF NEUROCAN STRUCTURES INTERACTING WITH THE NEURAL CELL-ADHESION MOLECULE N-CAM

Neurocan is a brain-specific chondroitin sulfate proteoglycan, which h as been shown to bind to the neural cell adhesion molecule N-CAM and t o inhibit its hemophilic interaction, To study in more detail the stru ctures of neurocan responsible for this interaction, various recombina nt neurocan fragments were generated, The ability of these fragments t o interact with N-CAM was investigated in several different in vitro a ssay systems, enzyme-linked immunosorbent assay-type binding assays, C ovasphere-aggregation assays, and assays based on an optical biosensor (BIA-core(TM)) system, The analysis of the hamophilic N-CAM interacti on in the BLAcore system revealed a K-D of 64 nM. This hemophilic inte raction could be reduced by preincubation of soluble N-CAM with neuroc an. Direct binding of N-CAM to immobilized neurocan care protein and r ecombinant neurocan fragments could also be demonstrated, and K-D valu es behveen 25 and 100 nM were obtained. in addition, direct binding of N-CAM to chondroitin sulfate could be demonstrated Binding of N-CAM t o the immobilized neurocan core protein could be inhibited with all re combinant fragments containing chondroitin sulfate or major parts of t he mucin-like central region of neurocan. For the inhibition of hemoph ilic N-CAM interactions, however, a combination of globular and extend ed structures was required.