BASIS OF TCR RECOGNITION OF PEPTIDE/MHC COMPLEXES

T细胞受体(TCR)是一种多亚单位复合体，介导 识别与MHC分子络合的多肽抗原。由于 事实上，受体复合体的各个成分是 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模糊的。最近我有 设计了一种促进可溶性TCRα结合的通用方法 和β亚基通过使用亮氨酸拉链序列 只有杂二聚体形成。在本提案中，详细分析了 TCR-肽/MHC的相互作用将利用TCR特异性的 对于一种特性良好的水疱性口炎病毒(VSV)八肽，在 Kb MHC I类分子的背景。第一批可溶解的TCR将是 在真核细胞(Lec328l CHO)中进行分泌工程 均一多聚糖的合成及其糖蛋白的研究 使用Endo-H脱糖基。将为x射线提供材料 以“载脂蛋白”的形式存在的结晶学 与均相负载的VSV Kb分子络合 先前通过x射线结晶学显示，它可以变形到高分辨率。 STCR还将与抗TCR的各种Fab片段复合 针对可变区、恒定区和克隆型的单抗 决定因素。第二，TCR残基和Kb a螺旋的突变 残基将通过定点突变产生，并结合 在VSV八肽的p1、p4和p6位置有多肽变体 被认为是TCR触点，用于功能研究，以探索基础 TCR多肽MHC的识别。不同TCR识别方法的比较 同样的VSV-8/KB复合体将被制造出来。此外，改变后的多肽 其中几个TCR的配体(APL)将通过官能团进行鉴定 标准，并与知识库复配，用于结构研究。后者将 提供了对多肽/MHC复合体的相当深入的见解 刺激或拮抗T细胞的激活。第三，生物物理 TCR/VSV-KB相互作用的分析将使用等离子激元 共鸣。几个TCR对VSV/Kb以及APL/Kb的亲和力将 被刻画出来。KB单体受体亲和力的相关性 与多肽和胸腺选择过程的TCR 将对RAG-2-/-背景中的转基因动物进行调查。

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.