Molecular Interactions Of Lymphoid Cell Receptors

我们之前的工作主要是基于小鼠模型系统，探索CD8αβ辅助受体与MHC-I的相互作用。由于CD8在细胞免疫中的重要作用，我们一直在为获得具有生物学活性的重组人CD8αβ而努力工作，有几个目标：1)完成CD8αβMHC-I相互作用的详细表征；2)详细了解CD8αβ分子的结构方面。我们的进展是：1)开发表达人CD8αβ的系统；2)利用这种蛋白进行结合和结构研究。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，并正在探索这些重组制剂的结合特性。在这个项目的第二个主要方面，我们扩大了我们对NK细胞激活受体和T细胞辅助受体NKG2D与其应激诱导配体的相互作用的理解。虽然NKG2D与人MICA和ULBP3相互作用的结构细节已知，但与小鼠RAE1的相互作用仅在低分辨率下已知，同一家族的其他几种小鼠应激诱导配体尚未被详细研究。为此，M.Hong博士在实验室设计了两个以前研究较少的小鼠NKG2D配体MULT1和H60，纯化了它们，通过表面等离子体共振定量确定了它们与NKG2D的相互作用，并确定了几个关键的X射线结构，使我们能够了解这些分子的演化。我们确定了未连接的MULT1的结构为2.8，而H60与NKG2D的络合物的分辨率为3.3。然后，通过生产突变的MULT1和H60分子电池并测量它们与NKG2D的亲和力，证实了这些结构。这些研究为NKG2D如何识别大量不同的配体(人类有8种不同的配体，老鼠有9种不同的配体)提供了新的见解，这些配体在结构上是相关的，但氨基酸序列的一致性(从10%到40%不等)是不同的。这里的第三个子项目是与John Coligan的实验室合作，在这个项目中，我们贡献了我们在测量蛋白质/蛋白质相互作用方面的专业知识，并已经开始解决CD300家族NK细胞/髓系细胞蛋白与建立在细胞表面的各种分子相互作用的特异性问题，这些分子可以被引入脂体中。有希望的初步数据表明，我们可以探索CD300家族成员与由不同组成的磷脂制成的脂质体的相互作用，以及在理解这些分子的配体特异性方面应该被证明有价值的区域。在最后一个正在进行的子项目中，我们正在与简胡丽和威廉·保罗合作，努力设计细胞因子IL-1β和各种突变体，通过与特定抗体的偶联来靶向T淋巴细胞学的独特亚群。初步结果表明，我们可以制备所需数量的纯化的IL1β及其突变体，以允许进行生物学实验和生化耦合实验。

Our previous work explored the interaction of the CD8 alpha beta coreceptor with MHC-I, based primarily on the mouse model system. Because of the importance of CD8 function in cellular immunity, we have continued to work diligently towards obtaining biologically active recombinant human CD8alpha beta, with several goals in mind: 1) to complete the detailed characterization of the CD8 alpha beta MHC-I interaction, and 2) to understand in detail the structural aspects of the the stalk of the CD8 alpha beta molecule. Our progress has been to: 1) develop systems for the expression of the human CD8 alpha beta; 2) to exploit such protein for binding and structural studies. With considerable effort, we now have engineered human CD8 alpha/beta in several mammalian cell expression systems, and are exploring the binding characteristics of these recombinant preparations. In the second major aspect of this project, we have extended our understanding of the interactions of the NK cell activating receptor and T cell coreceptor, NKG2D, with its stress induced ligands. Although the structural details of the interaction of NKG2D with human MICA and ULBP3 are known, the interaction with the mouse RAE1 is only known at low resolution, and several other murine stress induced ligands of the same family have not been studied in detail. To this end, Dr. M. Hong in the laboratory has engineered two of the previously less studied murine NKG2D ligands, MULT1, and H60, purified them, determined quantitatively their interactions with NKG2D by surface plasmon resonance, and determined several critical X-ray structures that permit us to understand the evolution of these molecules. We have determined the structure of MULT1 unliganded to 2.8 resolution, and a complex of H60 with NKG2D to a resolution of 3.3 . These structures were then confirmed by the production of a battery of mutant MULT1 and H60 molecules and measurement of their affinities for NKG2D. These studies provide additional insight into the general problem of how NKG2D can recognize a large number of distinct ligands (8 different ligands in the human and 9 in the mouse) that are structurally related but are varied in amino acid sequence idnetity (ranging from 10 to 40%). The third sub-project here is a collaboration with the laboratory of John Coligan in which we have contributed our expertise in measurement of protein/protein interactions and have begun to address the question of the specificity of the interaction of the CD300 family of NK cell/ myeloid cell proteins with a variety of molecules founds on cell surfacesmolecules that can be introduced into lipsomes. Promising preliminary data suggest that we can explore the interaction of CD300 family members with liposomes made with various compositions of phospholipids, and area that should prove valuable in understand the ligand specificity of these molecules. In the last ongoing subproject, we are collaborating with Jane Hu-Li and William Paul in efforts to engineer the cytokine IL-1beta and various mutants for targetting, via coupling to particular antibodies, to unique subsets of T lymphoctyes. Preliminary results suggest that we can prepare the needed amounts of the purified IL1 beta and its mutants to allow both biological experiments and biochemical coupling experiments.