CALNEXIN AND CLASS I MHC FUNCTION

Calnexin 和 I 类 MHC 功能

• 批准号: 2004608
• 负责人: RUSSELL D. SALTER
• 金额: $ 19.33万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2004608
• 关键词: MHC class I antigen; antigen presentation; antigenic peptide transporter; cell line; endoplasmic reticulum; intermolecular interaction; membrane proteins; molecular chaperones; molecular cloning; protein biosynthesis; protein transport; site directed mutagenesis; transfection

DESCRIPTION (Adapted from the investigator's abstract): Class I major histocompatibility molecules play a key role in recognition of antigens by CD8+ T lymphoctyes. By binding peptides in the endoplasmic reticulum and transporting them to the cell surface, class I molecules allow T lymphocytes to scan for abnormal protein expression. This allows elimination of cancerous cells or cells infected with intracellular pathogens. The peptide binding clefts of individual class I molecules differ drastically in shape and peptide content. Such diversity allows T lymphocytes to survey a wide variety of antigens, and suggests that specificity of peptide binding is controlled largely by the shape of the class I binding cleft. How peptides bind to class I molecules in vivo is presently not well understood, and interactions with accessory proteins influence the process. How several accessory proteins influence the biogenesis and peptide binding properties of class I molecules within cells, with the eventual goal of manipulating antigen presentation will be investigated. 1) The effect of polymorphism in class I molecules on binding to calnexin and calreticulin will be examined. The investigators previously showed that two human class I proteins, encoded by A*0201 and B*0702, bind weakly and strongly to calnexin respectively. A panel of sixteen additional HLA-A, -B and -C heavy chains will be studied to determine if patterns of binding can be discerned, and whether strong and weak binders have different kinetics of transport. 2) The position dependence of the glycan on class I heavy chains for binding calnexin and calreticulin will be tested. Novel glycan acceptor sites will be introduced by site directed mutagenesis into class I heavy chains carrying a mutation which prevents their glycosylation and binding to calnexin. 3) How class I molecules bind to TAP/tapasin involved in transporting peptides into the endoplasmic reticulum will be determined. Potential sites of interaction in A*0201 and B*0702 include parts of the a2 and a3 domains, as well as the peptide binding cleft. 4) Regions of calnexin important for ligand binding will be identified by mutagenesis followed by transfection of mutant clones into the calnexin negative human cell, NKR. Several assays have been established to determine whether calnexin mutants are functional.

描述（改编自研究者摘要）：I类专业 组织相容性分子在抗原识别中起关键作用， CD 8 + T淋巴细胞。 通过结合内质网中的肽， I类分子将它们运送到细胞表面，使T淋巴细胞 扫描异常蛋白质表达 这允许消除 癌细胞或被细胞内病原体感染的细胞。 所述肽 单个I类分子的结合裂缝在形状上有很大差异 和肽含量。 这种多样性使T淋巴细胞能够在广泛的 多种抗原，并表明肽结合的特异性是 主要由I类结合裂缝的形状控制。 肽如何 与I类分子在体内的结合目前还不清楚， 与辅助蛋白的相互作用影响该过程。 如何几 辅助蛋白影响生物发生和肽结合特性 细胞内I类分子的最终目标是操纵 将研究抗原呈递。 1)多态性的影响 将检测I类分子与钙连接蛋白和钙网蛋白的结合。 研究人员先前表明，两种人类I类蛋白质，编码 A*0201和B*0702分别与钙连接蛋白弱结合和强结合。 一 将研究一组16个额外的HLA-A、-B和-C重链， 确定是否可以识别绑定模式，以及是否强大， 弱粘合剂具有不同的传输动力学。 2)的位置 聚糖对I类重链结合钙连接蛋白的依赖性， 将检测钙网蛋白。 将引入新的聚糖受体位点 通过定点诱变进入携带突变的I类重链 其阻止它们的糖基化和与钙连接蛋白结合。 3)I类如何 分子与TAP/tapasin结合，参与将肽转运到 将测定内质网。 潜在的相互作用位点 A*0201和B*0702包括a2和a3结构域的部分，以及 肽结合裂缝 4)钙连接蛋白对配体结合重要的区域 将通过诱变，然后转染突变体克隆来鉴定 钙连接蛋白阴性的人类细胞NKR。 已经进行了几次测定， 以确定钙连接蛋白突变体是否有功能。