Biochemical Characterization of the Regulatory T-cell Protein LAG-3.

调节性 T 细胞蛋白 LAG-3 的生化表征。

基本信息

批准号：
7667932
负责人：
Zarixia Zavala-Ruiz
金额：
$ 11.25万
依托单位：
UNIVERSITY OF PUERTO RICO RIO PIEDRAS
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-05 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7667932
关键词：
Activated Lymphocyte Activated Natural Killer Cell Adjuvant Affect Affinity Antigen-Presenting Cells Antigens B-Lymphocytes Binding Biochemical CD8-Positive T-Lymphocytes CD8B1 gene Cancer Patient Cell surface Cells Characteristics Chemicals Cleaved cell Complex Crystallography Dendritic Cells Diagnosis Diffusion Disease Escherichia coli Escherichia coli Proteins Estrogens Gel Chromatography Generations Genes Goals Histocompatibility Antigens Class II Homeostasis Homologous Gene Image Immune response In Vitro Inclusion Bodies Influenza Insecta Kinetics Laboratories Ligands Lymphocyte Activation MART-1 Tumor Antigen MHC Class II Genes Malignant Neoplasms Melan-A protein Modification Nature Patients Peripheral Blood Mononuclear Cell Play Preparation Principal Investigator Progesterone Receptors Progress Reports Property Proteins Recombinant Proteins Recombinants Reporting Research Research Personnel Resistance Roentgen Rays Role Serum Signal Transduction Site Solutions Structure Surface Plasmon Resonance Survival Rate System T cell regulation T-Cell Activation T-Lymphocyte Tuberculosis X-Ray Crystallography crosslink experience in vivo light scattering malignant breast neoplasm molecular mass neoplastic cell novel receptor research study response survivin therapeutic target three dimensional structure tumor

项目摘要

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize the regulatory T-cell protein lymphocyte activation gene-3 (LAG- 3). The LAG-3 protein plays an important role in negatively regulating T-cell activation and proliferation; and in its soluble form has the power to activate antigen presenting cells. This protein is expressed in activated natural killer cells, CD4+ and CD8+ T-cells. B-cells also express LAG-3 in a T-cell dependent manner. LAG- 3 is a CD4-homolog that, like CD4, binds to class II MHC proteins. A dimeric state of LAG-3 is observed on the cell surface of activated T-cells, but very little is known about the protein after it is cleaved from the cell surface, which is known as soluble LAG-3. We propose here experiments to: a) complete ongoing efforts to obtain refolded and functional LAG-3 protein, b) determine the biophysical properties of the oligomeric state of sLAG-3 as well as that of the interaction of LAG-3 with class II MHC proteins, c) determine the interaction site of possible LAG-3 oligomers and that of LAG-3 with MHC II molecules. We will continue our efforts in producing soluble functional LAG-3 by purifying it from inclusion bodies and refolding it in vitro as well as by expressing it in Drosphila Schneider-2 cells. Characterization of LAG-3 oligomers and the LAG-3:MHC II complex will be done by gel filtration to determine the size and molecular mass under native conditions, dynamic light scattering to determine the size distribution and the translational diffusion coefficient D, as well as cross linking studies to characterize the oligomeric state of sLAG-3. Affinity constants of the binding of LAG-3 with class II MHC proteins will be examined by gel filtration and dynamic light scattering while kinetic and affinity parameters of the interaction will be studied by surface plasmon resonance. The three-dimensional structure of LAG-3 by itself and in complex with a class II MHC will be obtained by x-ray crystallography to determine the oligomeric state of LAG-3, and the interaction site between these two molecules; any potential conformational changes on LAG-3 upon MHC II binding will be examined as well. Differential chemical modifications of a class II MHC protein in the presence and absence of LAG-3 of the complex will be performed. Understanding the biochemical characteristics of LAG-3 will facilitate the identification of the mechanism by which this protein regulates T-cell activation and is capable of activating the most potent antigen presenting cells, dendritic cells.

描述（由申请人提供）：本提案的目标是表征调节性 T 细胞蛋白淋巴细胞激活基因 3 (LAG-3)。 LAG-3蛋白在负调节T细胞活化和增殖中发挥重要作用；其可溶形式具有激活抗原呈递细胞的能力。该蛋白在激活的自然杀伤细胞、CD4+ 和 CD8+ T 细胞中表达。 B 细胞还以 T 细胞依赖性方式表达 LAG-3。 LAG-3 是一种 CD4 同源物，与 CD4 一样，与 II 类 MHC 蛋白结合。在活化的 T 细胞的细胞表面观察到 LAG-3 的二聚体状态，但人们对这种蛋白质从细胞表面裂解后的情况知之甚少，这种蛋白质被称为可溶性 LAG-3。我们在此建议进行实验：a) 完成获得重折叠和功能性 LAG-3 蛋白的持续努力，b) 确定 sLAG-3 寡聚状态的生物物理特性以及 LAG-3 与 II 类 MHC 相互作用的生物物理特性蛋白质，c) 确定可能的 LAG-3 寡聚体以及 LAG-3 与 MHC II 分子的相互作用位点。我们将继续努力生产可溶性功能性 LAG-3，方法是从包涵体中纯化 LAG-3，并在体外重新折叠，以及在果蝇 Schneider-2 细胞中表达。 LAG-3 寡聚物和 LAG-3:MHC II 复合物的表征将通过凝胶过滤来确定天然条件下的尺寸和分子质量、动态光散射来确定尺寸分布和平移扩散系数 D，以及交联研究来表征 sLAG-3 的寡聚状态。 LAG-3 与 II 类 MHC 蛋白结合的亲和常数将通过凝胶过滤和动态光散射进行检查，而相互作用的动力学和亲和参数将通过表面等离子共振进行研究。通过X射线晶体学获得LAG-3本身以及与II类MHC复合物的三维结构，以确定LAG-3的寡聚状态，以及这两个分子之间的相互作用位点；还将检查 MHC II 结合后 LAG-3 上任何潜在的构象变化。将在复合物存在和不存在 LAG-3 的情况下对 II 类 MHC 蛋白进行差异化学修饰。了解 LAG-3 的生化特征将有助于识别该蛋白调节 T 细胞激活的机制，并能够激活最有效的抗原呈递细胞——树突状细胞。