MOLECULAR BASIS OF CD8 CO-RECEPTOR FUNCTION

CD8已被证明在I类MHC限制性T细胞的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同源二聚体或CD8αβ分子的形式存在，该序列允许CD8α/α同源二聚体的可溶性Ig样结构域的分泌和形成，从而导致mCD8α/α/KB复合体的结晶并衍射到2.8A的分辨率。CD8Alphabeta的确切分子相互作用仍不清楚。在本提案中，将利用与KB分子结合的血管间质炎症病毒八肽(VSV8)的特征良好的复合体来详细分析CD8αβ肽/MHC的相互作用。首先，CD8Alphabeta将在真核细胞(Lec3.2.8.1CHO)中被设计用于分泌，这些真核细胞合成均一的多糖，其糖蛋白可以很容易地用内切-H去糖基化。材料本身将为X射线结晶学提供材料，也将与均匀负载VSV8的KB分子络合。CD8αβ-LZ还将与针对LZ、CD8α或CD8β的单抗的各种FAG片段形成复合体。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。CD8αβ胞外区和Ig片段与VSV8/Kb、TCRs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。CD8α-β的茎区域的功能将通过共受体分子之间的结构交换而被解剖，CD8α-β与KB之间的精确相互作用将通过突变来确定。第三，CD8α或CD8β胞浆结构域在共受体功能中的功能将通过诱变实验进行剖析。参与CD8α或CD8β共同受体功能的信号转导分子将通过生化分析进行检测。同时，CD8β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.