MYELIN PROTEIN ZERO--MECHANISMS OF MEMBRANE ADHESION

蛋白0(P-0，MW=28,500)是哺乳动物周围神经系统髓鞘的主要粘附性糖蛋白。人类P0基因的突变是影响周围神经系统的某些髓鞘疾病(夏科氏-玛丽-牙病和德杰林-索塔斯病)的基础。P0也是免疫球蛋白基因超家族中最简单的跨膜成员，因此该分子作为免疫球蛋白超家族相互作用的典型代表引起了人们极大的兴趣。明确地建立P0：P0膜粘附性相互作用的结构基础将直接关系到理解其他Ig超家族分子发挥作用的结合元件。我们唯一的具体目标是对P0：P0分子相互作用的建议模型进行实验评估，该相互作用会导致强大的膜粘附性。在这些研究的第一阶段，我们将设计编码多肽的全长P0cDNA，这些多肽通过细胞外域的点突变而突变。战略突变将被放置，以便我们可以测试首先通过分析P0胞外区揭示的几个特定接触点的氨基酸残基的作用。每个诱变的cdna将被用作mRNA合成的模板，这反过来将对我们设计的青蛙卵母细胞耦合系统进行编程，以测试膜间黏附。在平行研究中，突变的P0表达基因的永久表达的纯克隆细胞系将用于细胞分析，用于正常非黏附、非聚集细胞的黏附和聚集，如HeLa或L细胞。

Protein zero (P-0, mw = 28,500) is the major adhesive glycoprotein of the myelin sheath in the mammalian peripheral nervous system. Mutations in the P0 gene in humans underlie certain myelin diseases that affect the peripheral nervous system (Charcot-Marie-Tooth and Dejerine-Sottas Diseases). P0 is also the "simplest" transmembrane member of the immunoglobulin gene (Ig) superfamily, and so this molecule has attracted great interest as a prototypic representative of Ig superfamily interactions. Definitively establishing the structural basis for P0:P0 membrane adhesive interactions will have direct bearing on understanding the binding elements by which other Ig superfamily molecules exert their' actions. Our single Specific Aim is to experimentally evaluate a proposed model for P0:P0 molecular interactions which lead to strong membrane adhesion. In the first phase of these studies, we will engineer full length P0 cDNAs encoding polypeptides mutagenized by point mutation in the extracellular domain. Strategic mutations will be placed such that we may test the roles for amino acid residues at several specific contact points first revealed by analysis of the P0 extracellular domain. Each mutagenized cDNA will be used as template for mRNA synthesis, which in turn will program a coupled frog oocyte system we devised to test for intermembrane adhesion. In parallel studies, permanent expressing pure clonal cell lines of mutagenized P0 expressors will be used in cellular assays for adhesion and aggregation in normally non-adherent, non-aggregating cells such as HeLa or L-cells.