Force Probe Investigations of NCAM Adhesion

描述(由申请人提供)：我们的目标是通过一个 直接力测量和遗传操作的结合 蛋白质结构--亲和性黏附的基本物理机制 神经细胞黏附分子与其调控之间的关系 翻译后修饰的粘附性。NCAM是一个大型的多域 通过亲和性结合来调节细胞间相互作用的蛋白质。它是 一种含量最丰富、分布最广的细胞黏附蛋白 免疫球蛋白超家族，分子的黏附功能已被证明 来影响一些重要的发展过程。这种蛋白质是 不同寻常的是，它展示了两种结构形式：其中一种展示了 一种是具有粘合活性，另一种是具有防粘功能。虽然 广泛的研究解决了这些功能的生物学作用，两者都没有 其潜在的机制已经在分子细节上得到了定义。这个 抗粘连性能与蛋白质的糖基化有关，糖基化可产生 聚唾液酸(PSA)。这一修改不仅影响NCAM的粘附性，而且 还有由其他黏附分子介导的细胞相互作用。这些建议 研究将量化膜间作用力的距离依赖关系 显示不同形式的NCAM，以确定分子 聚唾液酸改变NCAM与AS之间相互作用的机制 以及另外两个黏附蛋白N-钙粘蛋白和LI的相互作用。这个 测量将直接检验PSA在空间上阻碍 细胞的紧密结合，从而对 细胞间的相互作用。关于粘接功能，还增加了 NCAM和NCAM碎片均缺乏特异性的力/距离测量 域将进一步测试当前关于特定的几个假设 结构域相互作用，调节同嗜性结合。这些建议 力-距离测量将产生独特的分子水平信息 关于NCAM黏附的机制，不能从 对整个细胞、溶液结合测量或晶体结构的研究。

DESCRIPTION (provided by applicant): Our objectives are to identify, by a combination of direct force measurements and the genetic manipulation of protein structure, the fundamental physical mechanisms of homophilic adhesion between neural cell adhesion molecules (NCAM) and the regulation of NCAM adhesion by post-translational modification. NCAM is a large, multidomain protein that mediates cell-cell interactions through homophilic binding. It is one of the most abundant and widespread cell adhesion proteins of the immunoglobulin superfamily, and the molecule's adhesive function has been shown to influence a number of important developmental processes. The protein is unusual in that it exhibits two structural forms: one of which exhibits adhesive activity and the other which displays anti-adhesive function. Although extensive research addressed the biological role of these functions, neither of the underlying mechanisms has been defined in molecular detail. The anti-adhesive property is linked to the protein glycosylation, which produces polysialylic acid (PSA). This modification affects not only NCAM adhesion but also cell interactions mediated by other adhesion molecules. These proposed studies will quantify the distance dependence of the forces between membranes displaying the different forms of NCAM, in order to determine the molecular mechanism by which polysialic acid alters both interactions between NCAM as well as the interactions of two other adhesion proteins N-cadherin and Li. The measurements will directly test the hypothesis that PSA sterically impedes the close apposition of cells and thereby exerts a general regulatory influence on cell interactions. With regard to the adhesive function, additional force/distance measurements with both NCAM and NCAM fragments lacking specific domains will further test several current hypotheses regarding the specific domain interactions that mediate homophilic binding. These proposed force-distance measurements will generate unique, molecular level information concerning the mechanisms of NCAM adhesion that cannot be determined from studies with whole cells, solution binding measurements, or crystal structures.