Molecular Interactions Of Lymphoid Cell Receptors

在前一年，我们报道了MHC/CD 8 α β复合物的三维结构，这种结构多年来一直困扰着科学家。 这项研究的初步结论揭示了CD 8 β链相对于α的位置的拓扑结构，并提出了几个与CD 8依赖性信号传导相关的重要功能问题。 在过去的一年中，我们的计划是：1）开发用于表达人CD 8 α β的系统; 2）利用这种蛋白质进行结合和结构研究; 3）扩展我们对小鼠CD 8 α β分子的观察，以包括CD 8 α β异二聚体的结构可塑性茎区。 经过相当大的努力，我们现在已经在几种哺乳动物细胞表达系统中改造了人CD 8 α/β。 更多的努力来了解TCR/MHC相互作用的重要方面已经更加富有成效。 我们最近完成了MHC-I（H-2Dd/P18肽）/B4.2.3 TCR复合物的2.0 X射线晶体学结构，这使我们能够详细研究TCR是否对MHC具有固有反应性的问题，而不依赖于结合的肽。 为此，我们使用了高分辨率的三维结构的复合物作为基础的TCR的靶向诱变，以进一步定量评估的规则，在定量方面的TCR/MHC相互作用。 具体地，该结构表明TCR α和β链的CDR 3区主要参与与抗原肽的分子接触，并且两条链的CDR 1和CDR 2区以互补方式起作用以与MHC-I分子（在这种情况下为H-2Dd）相互作用。 已经制备了一组仔细考虑的TCR CDR 3区的缺失和取代突变体，以探索这些分子中的一些将保留与MHC-I的反应性的可能性，尽管消除或改变与结合肽的反应性。 旨在理解Treg表达分子GARP（也称为LRRC 32）的结构和功能的努力旨在：1）理解GARP的基本结构，预测为富含亮氨酸的重复蛋白; 2）评估GARP与潜在形式的TGF-β的相互作用; 3）开发针对人和小鼠形式的GARP的单克隆抗体，用于功能和进一步的结构研究。 为了这些目的，我们已经成功地在果蝇S2细胞中表达人和小鼠GARP蛋白。 人潜伏性TGF-β（LAT）也已在CH 0-Lec3.8.2.1（一种缺乏末端碳水化合物添加的细胞系）中表达。 使用表面等离子体共振，我们已经测量了GARP/潜在TGF β相互作用的结合常数。 与Shevach实验室合作，利用我们的重组人和小鼠GARP，我们正在开发针对人和小鼠GARP的单克隆抗体。 已经获得了针对小鼠和人分子的抗体，现在正在进行表征。 这些抗体应该允许表征在其细胞表面上表达GARP的T细胞，并且在理解GARP与潜伏TGF-β的相互作用中是必不可少的。 我们在通过SPR评估分子相互作用方面的专业知识允许直接比较一组小鼠抗SEB单克隆抗体与经工程改造具有人恒定区的嵌合抗体的结合亲和力。 我们的结果清楚地表明，嵌合抗体对SEB具有与原始小鼠抗体基本相同的亲和力。 在最近的一系列实验中，我们对MHC-I分子HLA-B*5701的特征作出了贡献，当与抗逆转录病毒药物阿巴卡韦结合时，该分子可引起严重的急性超敏反应。 我们已经成功地改造了HLA-B*5701，并获得了用于衍射研究的蛋白质晶体。

In the previous year, we had reported the three-dimensional structure of the MHC/CD8alpha beta complex, a structure that had eluded scientists for many years. The initial conclusion from this study revealed the topology of the location of the CD8beta chain with respect to alpha, and posed several important functional questions related to CD8 dependent signaling. Our plan in the past year was to: 1) develop systems for the expression of the human CD8 alpha beta; 2) to exploit such protein for binding and structural studies; 3) to extend our observations on the mouse CD8 alpha beta molecule to include the structurally plastic stalk region of the CD8 alpha beta heterodimer. With considerable effort, we now have engineered human CD8 alpha/beta in several mammalian cell expression systems. Additional efforts to understand important aspects of the TCR/MHC interaction have been more productive. We have recently completed the 2.0 X-ray crystallographic structure of an MHC-I (H-2Dd/P18 peptide)/B4.2.3 TCR complex, which allows us to examine in detail questions relating to whether or not the TCR has inherent reactivity toward the MHC, independent of the bound peptide. To this end, we have used the high resolution three-dimensional structure of the complex to serve as the basis for targeted mutagenesis of the TCR to further evaluate quantitatively the rules that govern the TCR/MHC interaction in quantitative terms. Specifically, the structure suggests that CDR3 regions of the TCR alpha and beta chains primarily are involved in molecular contacts with the antigenic peptide, and that CDR1 and CDR2 regions of both chains function in a complementary fashion to interact with the MHC-I molecule, in this case, H-2Dd. A set of carefully considered deletion and substitution mutants of the TCR CDR3 regions have been made to explore the possibility that some of these molecules will retain reactivity with MHC-I despite eliminating or changing reactivity with the bound peptide. Efforts to understand the structure and function of the Treg expressed molecule, GARP, also known as LRRC 32, are designed to; 1) understand the fundamental structure of GARP, predicted to be a leucine rich repeat protein; 2) evaluate the interact of GARP with the latent form of TGF-beta; 3) develop monoclonal antibodies to both human and mouse forms of GARP for functional and further structural studies. To these ends, we have successfully engineered the expression of both human and mouse GARP proteins in Drosophila S2 cells. Human latent TGF-beta (LAT) has also been expressed in CH0-Lec 3.8.2.1 cells, a cell line deficient in the addition of terminal carbohydrates. Using surface plasmon resonance, we have measured the binding constant for the GARP/Latent TGF beta interaction. In collaboration with the Shevach laboratory, utilizing our recombinant human and mouse GARP, we are developing monoclonal antibodies to both human and mouse GARP. Antibodies to both the mouse and human molecules have been obtained, and are now being characterized. These antibodies should permit the characterization of T cells that express GARP on their cell surface and will be essential in understanding the interactions of GARP with latent TGF-beta. Our expertise in evaluating molecular interactions by SPR has allowed a direct comparison of the binding affinity of a panel of mouse anti-SEB monoclonal antibodies with chimeric antibodies engineered to have human constant regions. Our results show clearly that the chimeric antibodies have essentially the same affinity for SEB that the original mouse antibodies have. In a recent series of experiments we have contributed to the characterization of the MHC-I molecule, HLA-B*5701, which, when bound to an anti=retroviral drug, abacavir, can cause severe acute hypersensitivity. We have successfully engineered HLA-B*5701, and have obtained protein crystals for diffraction studies.