MYELIN PROTEIN ZERO--MECHANISMS OF MEMBRANE ADHESION

蛋白零（P-0, mw = 28,500）是哺乳动物周围神经系统髓鞘的主要黏附糖蛋白。人类P0基因的突变是影响周围神经系统的某些髓鞘疾病（Charcot-Marie-Tooth病和Dejerine-Sottas病）的基础。P0也是免疫球蛋白基因（Ig）超家族中“最简单”的跨膜成员，因此该分子作为Ig超家族相互作用的原型代表引起了极大的兴趣。明确建立P0:P0膜粘附相互作用的结构基础，将直接关系到了解其他Ig超家族分子发挥作用的结合元件。我们的单一特定目标是通过实验评估P0:P0分子相互作用的拟议模型，该模型导致强膜粘附。在这些研究的第一阶段，我们将设计全长P0 cdna编码在细胞外区域通过点突变诱变的多肽。策略突变将被放置，这样我们就可以测试氨基酸残基在几个特定接触点的作用，这些接触点首先是通过分析P0细胞外结构域揭示的。每个突变的cDNA将被用作mRNA合成的模板，这反过来将编程一个偶联的青蛙卵母细胞系统，我们设计用于测试膜间粘附。在平行研究中，永久表达突变P0表达物的纯克隆细胞系将用于通常不粘附、不聚集的细胞（如HeLa或l细胞）的粘附和聚集的细胞试验。

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.