MOLECULAR BASIS OF CD8 CO-RECEPTOR FUNCTION

CD 8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。CD 8共受体通过使用亮氨酸拉链（LC）序列以CD 8 α/α同源二聚体或CD 8 α分子的形式存在，所述亮氨酸拉链（LC）序列允许分泌和形成CD 8 α/α同源二聚体的可溶性Ig样结构域，这导致mCD 8 α/α/Kb复合物结晶并衍射至2.8 A分辨率。 CD 8 α的精确分子相互作用仍然不清楚。在本提案中，将利用与Kb分子结合的血管瘤病毒八肽（VSV 8）的良好表征的复合物进行CD 8 α肽/MHC相互作用的详细分析。首先，将对CD 8 α进行工程改造，使其在真核细胞（Lec3.2.8.1 CHO）中分泌，这些细胞合成均质聚糖，其糖蛋白可以使用Endo-H容易地去糖基化。材料本身将被提供用于X射线晶体学，以及与均匀VSV 8负载的Kb分子复合。CD 8 α-LZ还将与针对LZ、CD 8 α或CD 8 β的mAb的各种Fag片段复合。将使用表面等离子体共振对CD 8-VSV 8/Kb相互作用进行生物物理分析。通过PCR方法建立CD 8 α胞外结构域和IG片段对VSV 8/Kb、TCR和TCR-VSV 8/kappaB和β 2 M的亲和力，并用于功能研究，以探索CD 8-肽/MHC相互作用的基础。将通过共受体分子之间的结构交换来剖析CD 8 α-β的茎区的功能，并且将通过诱变来定义CD 8 α-β和Kb之间的精确相互作用。第三，将通过诱变实验剖析CD 8 α或CD 8 β的胞质结构域在共受体功能中的功能。将通过生化分析检查参与CD 8 α或CD 8 β共受体功能的信号转导分子。与此同时，在酵母双杂交系统中克隆了CD 8 β信号转导的潜在分子。

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.