Molecular Interactions Of Lymphoid Cell Receptors

在前一年，我们已经报道了MHC/CD8αβ复合体的三维结构，这是科学家们多年来一直未能理解的结构。这项研究的初步结论揭示了CD8β链相对于α的位置的拓扑结构，并提出了几个与CD8依赖信号有关的重要功能问题。我们在过去一年的计划是：1)开发表达人CD8αβ的系统；2)利用这种蛋白质进行结合和结构研究；3)扩大我们对小鼠CD8αβ分子的观察，包括CD8αβ异二聚体的结构可塑性茎区域。经过相当大的努力，我们现在已经在几个哺乳动物细胞表达系统中设计了人CD8α/β。 为了解TCR/MHC相互作用的重要方面所作的额外努力取得了更大的成效。我们最近完成了MHC-I(H-2DD/P18肽)/B4.2.3 TCR复合物的2.0X射线晶体结构，这使我们能够详细研究与TCR是否与MHC具有内在反应性有关的问题，而不依赖于结合的肽。为此，我们利用该复合体的高分辨率三维结构作为TCR靶向突变的基础，进一步从定量的角度评估控制TCR/MHC相互作用的规则。具体地说，该结构表明TCRα链和β链的CDR3区域主要参与与抗原肽的分子接触，两链的CDR1和CDR2区域以互补的方式与MHC-I分子相互作用，在这种情况下为H-2DD。一组仔细考虑的TCRCDR3区的缺失和替换突变体已经被用来探索其中一些分子在消除或改变与结合肽的反应性的情况下仍保持与MHC-I的反应性的可能性。 为了了解Treg表达分子的结构和功能，GARP，也被称为LRRC 32，旨在：1)了解GARP的基本结构，预计它是一种富含亮氨酸的重复蛋白；2)评估GARP与潜在形式的转化生长因子-β的相互作用；3)开发抗人和小鼠形式的GARP的单抗，以便进行功能和进一步的结构研究。为此，我们成功地在果蝇S2细胞中表达了人和小鼠GARP蛋白。人潜伏的转化生长因子-β(LAT)也在CH0-Lec 3.8.2.1细胞中表达，这是一种缺乏末端碳水化合物的细胞系。利用表面等离子体共振技术，测定了GARP与潜伏的转化生长因子β相互作用的结合常数。与舍瓦赫实验室合作，利用我们的重组人和小鼠GARP，我们正在开发抗人和小鼠GARP的单抗。针对老鼠和人类分子的抗体已经获得，并正在进行鉴定。这些抗体应该能够鉴定细胞表面表达GARP的T细胞，并对了解GARP与潜在的转化生长因子-β的相互作用至关重要。 我们在通过SPR评估分子相互作用方面的专业知识使我们能够直接比较一组小鼠抗SEB单抗与具有人类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的亲和力基本上与原始小鼠抗体相同。 在最近的一系列实验中，我们对MHC-I分子的特性做出了贡献，人类白细胞抗原-B*5701，当它与抗逆转录病毒药物阿巴卡韦结合时，可以引起严重的急性超敏反应。我们成功地设计了人类白细胞抗原-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.