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：
10645114
负责人：
Erin June Adams
金额：
$ 69.68万
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/10645114
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 TikTok Tissues Up-Regulation Vertebrates Work X-Ray Crystallography adaptive immune response adaptive immunity analog antigen binding 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.

概括鲨鱼是具有自适应免疫植物蛋白的最古老的活脊椎动物的成员， T细胞接收器和主要的组织相容性复合物。最近发现了八个I类血统由Flajnik实验室，经典的I类（UAA）和七个非经典血统（UBA至UHA）。不料，其中两个与人类的MHC链接鲨鱼I类（IB类）分子显示出与人类的惊人相似之处非经典I类CD1和HLA-E。遗传，表达和序列的初步数据 Flajnik实验室中强烈的相似性表明UFA是CD1的直系同源物。结构建模和计算机亚当斯实验室中的生物物理分析与UFA与CD1的明显相似性一致疏水结合位点。预计第二鲨鱼类IB分子UDA被预测会结合经典的肽 I类是单态的单拷贝，具有类似于哺乳动物HLA-E/QA-1的特性。我们将在生化上定义UFA和UDA配体并阐明其结构。我们将表达和净化 UDA和UFA的重组版本用于生化分析，包括配体洗脱和制备四聚体鉴定识别这些非经典I类分子的鲨鱼细胞。高度纯化的形式重组蛋白将通过X射线晶体学用于结构分析。使用内部技术，我们将生成与这些蛋白质的纳米病结合试剂进行结构分析以及补充已经生成用于组织表达分析的抗血清和单克隆抗体。由In 原位杂交和抗体/纳米染色我们将定义UDA/UFA细胞表达和检查它们在压力下的细胞上的上调。 UDA和UFA四聚体将揭示哪些淋巴细胞与 UDA/UFA以及单细胞分析，与它们相互作用的自适应和/或先天接收器。我们将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，在此提案中，我们将结合两个调查人员马丁的专业知识和经验 Flajnik，研究了鲨鱼免疫系统的遗传学，生物化学和功能已有35年了，通常是自适应免疫的演变，以及对艾琳·亚当斯（Erin Adams）人类I类系统和进化生物学。总之，我们假设CD1和HLA-E类似物在5亿年前的自适应免疫的大爆炸中出现，这表明辣椒结合经典的I类，“ CD1”的脂质结合，以及一个全部交易的肽结合。第一类是最早的自适应免疫系统中的重要特征。