MOLECULAR BASIS OF CD8 CO-RECEPTOR FUNCTION

CD8已被证明在I类mhc限制性T细胞发育和抗原识别功能中起关键作用。CD8共受体通过亮氨酸拉链（LC）序列以cd8α / α同二聚体或cd8α α分子的形式存在，允许cd8α / α同二聚体的可溶性igg样结构域的分泌和形成，导致mcd8α / α /Kb复合物的结晶，并衍射到2.8 a的分辨率。cd8 α -的精确分子相互作用仍不明确。在本提案中，将利用血管基质炎病毒八肽（VSV8）与Kb分子结合的良好特征复合物，详细分析cd8 - α肽/MHC相互作用。首先，CD8alphabeta将被设计用于真核细胞（Lec3.2.8.1 CHO）的分泌，真核细胞可以合成均质聚糖，其糖蛋白可以很容易地使用Endo-H去糖基化。材料将单独提供用于x射线晶体学，以及与均匀负载vsv8的Kb分子络合。CD8alpha -LZ也会与各种针对LZ、CD8alpha或CD8beta的单克隆抗体的Fag片段络合。CD8-VSV8/Kb相互作用的生物物理分析将使用表面等离子体共振进行。cd8 α - β外结构域和Ig片段对VSV8/Kb、tcr和TCR-VSV8/kappaB和beta2M的亲和力将通过PCR方法建立，并用于功能研究，以探索cd8 -肽/MHC相互作用的基础。通过共受体分子之间的结构交换，cd8α - β的茎区功能将被解剖，cd8α - β与Kb之间的精确相互作用将通过诱变被定义。第三，将通过诱变实验剖析cd8 α或cd8 β细胞质结构域在共受体功能中的作用。参与cd8α或cd8β共受体功能的信号转导分子将通过生化分析进行检查。与此同时，参与cd8 β信号转导的潜在分子将在酵母双杂交系统中克隆。

CD8 has been shown to be critically involved in class I MHC-restricted T cell development and antigen recognition function. CD8 co-receptors exist as either CD8alpha/alpha homodimers or CD8alphabeta molecules through the use of leucine zipper (LC) sequences that permit the secretion and formation of soluble Ig-like domains of the CD8alpha/alpha homodimer which led to crystallization of the mCD8alpha/alpha/Kb complex and diffracted to a 2.8 A resolution. The precise molecular interaction of CD8alphabeta is still ill-defined. In the present proposal a detailed analysis of CD8alphabeta peptide/MHC interaction will be performed utilizing a well-characterized complex of a vascular stromatitis virus octapeptide (VSV8) bound tot he Kb molecule. First, CD8alphabeta will be engineered for secretion in eukaryotic cells (Lec3.2.8.1 CHO) which synthesize homogeneous glycans and whose glycoproteins can be readily deglycosylated using Endo-H. Material will be provided for X-ray crystallography by itself as well as complexed with homogeneously VSV8-loaded Kb molecules. CD8alphabeta-LZ will also be complexed with various Fag fragments of mAbs directed against the LZ, CD8alpha or CD8beta. Biophysical analysis of the CD8-VSV8/Kb interaction will be performing using surface plasmon resonance. The affinity of the CD8alphabeta ectodomains and Ig segments for VSV8/Kb, TCRs and TCR-VSV8/kappaB and beta2M will created by PCR method and used in functional studies to probe the basis of CD8-peptide/MHC interaction. The function of the stalk region of CD8alphabeta will be dissected by structural swap between co-receptor molecules and the precise interaction between CD8alpha-beta and Kb will be defined by mutagenesis. Third, the function of the cytoplasmic domain of CD8alpha or CD8beta in the co-receptor function will be dissected by mutagenesis experiments. The signal transduction molecules involved in co-receptor function of CD8alpha or CD8beta will be examined by biochemical analysis. In parallel, the potential molecules involved in signal transduction of CD8beta will be cloned in the yeast two hybrid system.