MCP--MV/C3B/C4B BINDING SITES AND ISOFORM VARIATION

描述(改编自申请人的摘要)：超过 二十年来，申请人对补充性制度进行了一次 努力了解其在健康和疾病方面的工作原理。早些时候 研究集中在豚鼠、小鼠和人类C4的生物学上。自.以来 20世纪80年代的S，注意力一直集中在补体受体及其调控上 蛋白质。一种这样的调节因子，膜辅因子蛋白(MCP；CD46)， 通过与因子I一起充当辅因子来保护宿主组织 灭活沉积在自体细胞上的C3b和C4b。自.以来 申请人在1984年发现MCP是一种C3结合蛋白，它的作用是 逐渐扩大到只有补体抑制剂的范围。MCP 最近被确认为麻疹病毒(MV)受体，目前正在 用作相关病毒受体(牛瘟和犬病)的探针 瘟热)。此外，MCP对生殖生物学也很感兴趣 在精子和母胎界面上的表达。作为一种 补体激活的抑制剂，MCP正在被工程改造成 异种移植，并作为一种可溶性的治疗剂生产。因为. MCP的这些和其他值得注意的交互作用，有三个具体目标 建议。首先，确定MV、C3b和MCP上的结合位点 C4b、删除/替换构建体将被表达和评估 用于配基相互作用。二、MCP的细胞保护作用 通过使用稳定转染的克隆来比较异构体 配基功能分析中异构体的等效拷贝数 结合、辅因子活性和细胞保护。第三，功能界别 将分析MCP的两个细胞质尾巴的意义 涉及高甘露糖前体的加工(PRO-MCP)和 磷酸化。尾部介导的四重作用机制 使用MCP确定PRO-MCP的加工差异 表达每个尾部、尾部嵌合体和尾部突变体的转染体 确定内质网(ER)运输加速因子， ER或其他过程中的寡聚反应解释了这一点 不同。MCP的细胞质尾部有几个推测 磷酸化位点。以确定MCP是否在 将对Tail、MCP转染体、人类细胞和细胞系进行评估 补体激活(或MCP上调)后的挑战 策略。完成本提案的具体目标将 不仅为补充制度提供相关信息，而且 也用于其他领域，如生殖免疫学、异种移植 和传染病。

DESCRIPTION (Adapted from the applicant's abstract): For more than twenty years, the applicant has examined the complement system in an effort to understand its workings in health and disease. Earlier studies focused on the biology of guinea pig, mouse and human C4. Since the 1980's, attention has centered on complement receptors and regulatory proteins. One such regulator, Membrane Cofactor Protein (MCP; CD46), protects host tissue by acting as a cofactor, along with factor I, to inactivate C3b and C4b that deposit on autologous cells. Since the applicant identified MCP as a C3-binding protein in 1984, its role has progressively expanded beyond that of only a complement inhibitor. MCP was recently identified as the measles virus (MV) receptor and is being used as a probe for related virus receptors (rinderpest and canine distemper). Additionally, MCP is of interest in reproductive biology due to its expression on sperm and at the maternal-fetal interface. As an inhibitor of complement activation, MCP is being engineered into xenografts and produced as a soluble therapeutic agent. Because of these and other remarkable interactions of MCP, three specific aims are proposed. First, to identify the binding sites on MCP of MV, C3b and C4b, deletion/substitution constructs will be expressed and evaluated for ligand interaction. Second, the cytoprotective properties of MCP isoforms will be compared by using stably transfected clones bearing equivalent copy numbers of isoforms in functional analyses of ligand binding, cofactor activity and cytoprotection. Third, the functional significance of the two cytoplasmic tails of MCP will be analyzed as they relate to processing of high mannose precursors (pro-MCP) and to phosphorylation. The mechanism governing the tail-mediated fourfold difference in processing of pro-MCP will be determined by using MCP transfectants expressing each tail, tail chimeras and tail mutants to determine if an endoplasmic reticulum (ER) transport accelerating factor, oligomerization in the ER or another process accounts for this difference. The cytoplasmic tails of MCP possess several putative phosphorylation sites. To determine if MCP is phosphorylated on the tail, MCP transfectants, human cells and cell lines will be evaluated following challenge by a complement activating (or MCP upregulating) strategy. Completion of the specific goals of this proposal will provide relevant information not only for the complement system, but also for other areas such as reproductive immunology, xenotransplantation and infectious diseases.