Retzler C, Göhring W, Rauch U
Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany.
J Biol Chem. 1996 Nov 1;271(44):27304-10. doi: 10.1074/jbc.271.44.27304.
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 assay-type binding assays, Covasphere-aggregation assays, and assays based on an optical biosensor (BIAcoreTM) system. The analysis of the homophilic N-CAM interaction in the BIAcore system revealed a KD 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 KD 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.
神经黏蛋白是一种脑特异性硫酸软骨素蛋白聚糖，已证明它能与神经细胞黏附分子N-CAM结合并抑制其同源相互作用。为了更详细地研究神经黏蛋白中负责这种相互作用的结构，制备了各种重组神经黏蛋白片段。在几种不同的体外检测系统中研究了这些片段与N-CAM相互作用的能力，包括酶联免疫吸附测定型结合检测、Covasphere聚集检测以及基于光学生物传感器（BIAcoreTM）系统的检测。在BIAcore系统中对N-CAM同源相互作用的分析显示解离常数（KD）为64 nM。可溶性N-CAM与神经黏蛋白预孵育可降低这种同源相互作用。还证实了N-CAM与固定化神经黏蛋白核心蛋白及重组神经黏蛋白片段的直接结合，获得的KD值在25至100 nM之间。此外，还证实了N-CAM与硫酸软骨素的直接结合。含有硫酸软骨素或神经黏蛋白类黏蛋白样中心区域主要部分的所有重组片段均可抑制N-CAM与固定化神经黏蛋白核心蛋白的结合。然而，为了抑制N-CAM同源相互作用，需要球状结构和伸展结构的组合。
