Friedlander D R, Milev P, Karthikeyan L, Margolis R K, Margolis R U, Grumet M
Department of Pharmacology, New York University Medical Center, New York 10016.
J Cell Biol. 1994 May;125(3):669-80. doi: 10.1083/jcb.125.3.669.
We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.
我们之前已经表明，用可溶性神经黏蛋白（一种脑硫酸软骨素蛋白聚糖）孵育可抑制包被有神经细胞黏附分子（N-CAM）和神经元胶质细胞黏附分子（Ng-CAM）的微珠聚集，这表明神经黏蛋白可与这些细胞黏附分子结合（格鲁梅特，M.，A. 弗拉克斯，和 R. U. 马戈利斯。1993年。《细胞生物学杂志》120:815）。为了更直接地研究这些相互作用，我们测试了可溶性125I-神经黏蛋白与包被有不同糖蛋白的微孔板的结合情况。神经黏蛋白与Ng-CAM和N-CAM有高水平结合，但与髓鞘相关糖蛋白、表皮生长因子受体、纤连蛋白、层粘连蛋白和IV型胶原几乎没有或没有检测到结合。与Ng-CAM和N-CAM的结合是可饱和的，并且在每种情况下，Scatchard图都表明存在一个高亲和力结合位点，解离常数约为1 nM。用软骨素酶处理神经黏蛋白后，结合显著减少，游离硫酸软骨素抑制神经黏蛋白与Ng-CAM和N-CAM的结合。这些结果表明硫酸软骨素在这一过程中起作用，尽管核心糖蛋白也具有结合活性。已表明神经黏蛋白的COOH末端一半具有与全长蛋白聚糖基本相同的结合特性。为了研究神经黏蛋白的潜在生物学功能，分析了其对神经元黏附和神经突生长的影响。当神经元在包被有不同组合的神经黏蛋白和Ng-CAM的培养皿上孵育时，神经元黏附和神经突延伸受到抑制。使用抗Ng-CAM抗体作为底物的实验也表明，神经黏蛋白对神经元黏附和神经突生长有直接抑制作用。组织切片的免疫过氧化物酶染色显示，神经黏蛋白、Ng-CAM和N-CAM在发育中小脑的分子层和纤维束中浓度最高。体内的重叠定位、分子结合研究以及对神经元黏附和神经突生长的显著影响支持了这样一种观点，即神经黏蛋白可能在发育过程中通过与神经细胞黏附分子结合来调节神经元黏附和神经突生长。
