Determining the Origins of Nonclassical Class I molecules through Molecular and Functional Approaches

通过分子和功能方法确定非经典 I 类分子的起源

基本信息

批准号：
10501472
负责人：
Erin June Adams
金额：
$ 71.04万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-14 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10501472
关键词：
Adaptive Immune System Address Affinity Amphibia Animals Antibodies Antigen Receptors Area Autoimmunity Back Binding Binding Sites Biochemical Biochemistry Biology Biophysics Birds CD1 Antigens Cells Characteristics Chimera organism Chondrichthyes Communicable Diseases Complement Complex Cytomegalovirus Data Docking Drug or chemical Tissue Distribution Elasmobranchii Evolution Explosion Family Fire - disasters Fishes Gene Family Genes Genetic Genetic Polymorphism Genetic study Genome HIV Head and neck structure Human Human Characteristics Humoral Immunities Hydrophobicity Immune Immune Sera Immune system Immunity Immunoglobulin Somatic Hypermutation Immunoglobulins Immunology In Situ Hybridization Jaw Knowledge Ligands Link Lipid Binding Lymphocyte MHC Class I Genes Major Histocompatibility Complex Malignant Neoplasms Modeling Modification Molecular Monoclonal Antibodies Multigene Family Natural Killer Cells Osteichthyes Pathway interactions Peptide Leader Sequences Peptides Phylogenetic Analysis Preparation Property Proteins Publishing Qa-1 Antigen Reagent Recombinant Proteins Recombinants Regulation Reptiles Research Research Personnel Shark Stains Stress Structural Biologist Structural Models Structure System T-Cell Receptor Technology Tissues Up-Regulation Vertebrates Work X-Ray Crystallography adaptive immune response adaptive immunity analog antigen binding base biophysical analysis combat comparative experience follow-up in silico insight member nanobodies public health relevance receptor response single cell analysis transcriptome

项目摘要

SUMMARY Sharks are members of the oldest class of living Vertebrates with adaptive immunity based on immunoglobulin, T cell receptors, and the major histocompatibility complex. Eight class I lineages have recently been uncovered by the Flajnik lab, the classical class I (UAA) and seven nonclassical lineages (UBA to UHA). Unexpectedly, two of these MHC-linked shark nonclassical class I (Class Ib) molecules show striking similarities to the human nonclassical class I molecules CD1 and HLA-E. Preliminary data on genetics, expression, and sequence similarity strongly in the Flajnik lab suggest that UFA is the orthologue of CD1. Structural modeling and in silico biophysical analyses in the Adams lab are consistent with UFA’s clear similarity to CD1, revealing a strongly hydrophobic binding site. The second shark class Ib molecule, UDA, is predicted to bind peptides like classical class I, yet it is monomorphic, single-copy, and has properties similar to mammalian HLA-E/Qa-1. We will biochemically define UFA and UDA ligands and elucidate their structures. We will express and purify recombinant versions of UDA and UFA for biochemical analysis including ligand elution and preparation of tetramers to identify the shark cells recognizing these nonclassical class I molecules. Highly purified forms of recombinant proteins will be used for structural analysis through X-ray crystallography. Using in-house technologies, we will generate nanobody binding reagents to these proteins for structural analysis and also complement the antisera and monoclonal antibodies already generated for tissue-expression analysis. By in situ hybridization and antibody/nanobody staining we will define UDA/UFA cellular expression and examine their upregulation in cells under stress. UDA and UFA tetramers will reveal which lymphocytes interact with UDA/UFA and, with single-cell analysis, the adaptive and/or innate receptors that interact with them. We will perform biochemical analysis of shark primary cells to characterize UDA/UFA biosynthetic pathways. In this proposal, therefore, we will combine the expertise and experience of the two investigators, Martin Flajnik, who has studied the genetics, biochemistry, and function of the shark immune system for 35 years and generally the evolution of adaptive immunity, and Erin Adams, a structural biologist with deep knowledge of the human class I system and evolutionary biology. In summary, we hypothesize that CD1 and HLA-E analogues arose at the Big Bang of adaptive immunity 500 million years ago, suggesting that peptide-binding by the classical class I, lipid-binding by “CD1,” and a peptide-binding by a jack-of-all-trades “HLA-E-like” nonclassical class I were significant features in the earliest adaptive immune system.

概括鲨鱼是现存脊椎动物中最古老的一类成员，具有基于免疫球蛋白的适应性免疫，最近发现了 T 细胞受体和主要的组织相容性复合体。由 Flajnik 实验室、经典 I 类 (UAA) 和七个非经典谱系（UBA 到 UHA）组成。其中两个与 MHC 相关的鲨鱼非经典 I 类（Ib 类）分子与人类显示出惊人的相似性非经典 I 类分子 CD1 和 HLA-E 的遗传学、表达和序列的初步数据。 Flajnik 实验室的强烈相似性表明 UFA 是结构建模和计算机模拟的同源物。 Adams 实验室的生物物理分析与 UFA 与 CD1 的明显相似性一致，揭示了强烈的第二个鲨鱼 Ib 类分子 UDA 预计会像经典的肽一样结合肽。 I 类，但它是单态、单拷贝，并且具有与哺乳动物 HLA-E/Qa-1 相似的特性。我们将通过生物化学方法定义 UFA 和 UDA 配体并阐明它们的结构。我们将表达和纯化它们。用于生化分析的 UDA 和 UFA 重组版本，包括配体洗脱和制备四聚体来识别识别这些非经典 I 类分子的高度纯化形式的鲨鱼细胞。重组蛋白将用于通过内部 X 射线晶体学进行结构分析。技术，我们将生成这些蛋白质的纳米抗体结合试剂，用于结构分析补充已生成的用于组织表达分析的抗血清和单克隆抗体。原位杂交和抗体/纳米抗体染色我们将定义 UDA/UFA 细胞表达并检查 UDA 和 UFA 四聚体在细胞中的上调将揭示哪些淋巴细胞与它们相互作用。 UDA/UFA 以及通过单细胞分析，我们将了解与它们相互作用的适应性和/或先天受体。对鲨鱼原代细胞进行生化分析，以表征 UDA/UFA 生物合成途径。因此，在这项提案中，我们将结合两位研究人员 Martin 的专业知识和经验 Flajnik 对鲨鱼免疫系统的遗传学、生物化学和功能进行了 35 年的研究一般来说，适应性免疫的进化，以及艾琳·亚当斯（Erin Adams），一位对适应性免疫有深入了解的结构生物学家人类 I 类系统和进化生物学总之，我们追求 CD1 和 HLA-E 类似物。出现在 5 亿年前的适应性免疫大爆炸中，这表明肽结合经典的 I 类，通过“CD1”进行脂质结合，以及通过多才多艺的“HLA-E 样”非经典结合进行肽结合 I 类是最早的适应性免疫系统的显着特征。