BASIS OF TCR RECOGNITION OF PEPTIDE/MHC COMPLEXES

T细胞受体（TCR）是一种多亚基复合物，介导 识别与MHC分子复合的肽抗原。由于 事实上，受体复合物的各个组分是 跨膜分子，因此，不适合在溶液中研究， 这种认识的结构基础是不明确的。最近我 设计了一种通用的方法来促进可溶性TCR α的结合， 和β亚基，通过使用亮氨酸拉链序列 只有异二聚体形成。 在本提案中，详细分析了 TCR-肽/MHC相互作用将利用TCR特异性 对于一个充分表征的水泡性口炎病毒（VSV）八肽， Kb MHC I类分子的背景。第一个可溶性TCR将是 工程化用于在真核细胞（Lec 3281 CHO）中分泌， 合成均质聚糖且其糖蛋白可容易地 使用Endo-H进行去糖基化。将提供材料进行X射线检查 晶体学以“脱辅基蛋白”的形式存在， 与均匀VSV负载的Kb分子复合， 以前通过X射线晶体学显示出高分辨率的衍射。 sTCR还将与抗TCR的各种Fab片段复合 针对可变区、恒定区和克隆型的mAb 决定因素第二，TCR残基以及Kb α螺旋的突变， 将通过定点诱变产生残基， 在VSV八肽的p1、p4和p6位置具有肽变体 被认为是TCR接触，用于功能研究，以探索基础 TCR-肽MHC识别。不同TCR识别的比较 将制造相同的VSV-8/Kb复合物。此外，改变的肽 这些TCR中的几种TCR的配体（APL）将通过功能性免疫印迹来鉴定。 标准和复杂的Kb结构研究。后者将 对肽/MHC复合物提供了相当深入的了解， 刺激或拮抗T细胞活化。第三，生物物理 TCR/VSV-Kb相互作用的分析将使用等离子体激元进行 共振 几种TCR对VSV/Kb以及APL/Kb的亲和力将 被定性。单体受体对Kb亲和力的相关性 以及使用TCR的胸腺选择过程 将研究Rag-2-/-背景中的转基因动物。

The T cell receptor (TCR) is a multi-subunit complex that mediates recognition of peptide antigens complexed to MHC molecules. Owing to the fact that the individual components of the receptor complex are transmembrane molecules and therefore, not amenable to study in solution, the structural basis of this recognition is ill-defined. Recently I have devised a general method to facilitate association of soluble TCR alpha and beta subunits through the use of leucine zipper sequences that permit only heterodimer formation. In the present proposal, a detailed analysis of TCR-peptide/MHC interaction will be performed utilizing TCRs specific for a well-characterized vesicular stomatitis virus (VSV) octapeptide in the context of the Kb MHC class I molecule. First soluble TCRs will be engineered for secretion in eukaryotic cells (Lec328l CHO) which synthesize homogeneous glycans and whose glycoproteins can be readily deglycosylated using Endo-H. Material will be provided for x-ray crystallography in the form of the "apoprotein" by itself as well as complexed with homogeneously VSV-loaded Kb molecules that have been previously shown to defract to high resolution by x-ray crystallography. The sTCRs will also be complexed with various Fab fragments of anti-TCR mAbs directed against variable region, constant region and clonotypic determinants. Second, mutations in TCR residues as well as Kb a helical residues will be created by site-directed mutagenesis and, in conjunction with peptide variants at the p1, p4 and p6 position of the VSV octapeptide thought to be TCR contacts, used in functional studies to probe the basis of TCR-peptide MHC recognition. Comparison of different TCRs recognizing the same VSV-8/Kb complex will be made. In addition, altered peptide ligands (APL) of several of these TCRs will be identified by functional criteria and complexed with Kb for structural studies. The latter will offer considerable insight into peptide/MHC complexes which are stimulatory or antagonistic of T cell activation. Third, biophysical analysis of TCR/VSV-Kb interaction will be performed using plasmon resonance. The affinity of several TCRs for VSV/Kb as well as APL/Kb will be characterized. Correlations between monomeric receptor affinity for Kb with variant peptides and the processes of thymic selection using TCR transgenic animals in the Rag-2-/- background will be investigated.