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

总结 鲨鱼是现存最古老的脊椎动物，具有基于免疫球蛋白的适应性免疫力， T细胞受体和主要组织相容性复合体。最近发现了八个I类血统 由Flajnik实验室，经典I类（UAA）和七个非经典谱系（乌巴到UHA）。没想到， 其中两个与MHC相关的鲨鱼非经典I类（Ib类）分子与人类的有着惊人的相似性， 非经典I类分子CD 1和HLA-E。遗传学、表达和序列的初步数据 Flajnik实验室中的相似性强烈表明乌法是CD 1的直向同源物。结构建模和计算机模拟 亚当斯实验室的生物物理分析与乌法与CD 1的明显相似性一致，揭示了一种强烈的 疏水结合位点。第二种鲨鱼Ib类分子UDA被预测可以像经典的 I类，但它是单态的、单拷贝的，并且具有与哺乳动物HLA-E/Qa-1相似的性质。 我们将生化定义乌法和UDA配体，并阐明其结构。我们将表达和净化 用于生化分析的重组形式的UDA和乌法，包括配体洗脱和制备 四聚体，以确定鲨鱼细胞识别这些非经典的I类分子。高度纯化的 重组蛋白将通过X射线晶体学用于结构分析。利用内部 技术，我们将产生纳米抗体结合试剂，这些蛋白质的结构分析， 补充已经产生的用于组织表达分析的抗血清和单克隆抗体。采用 原位杂交和抗体/纳米抗体染色，我们将定义UDA/乌法细胞表达并检查 它们在压力下的细胞中的上调。UDA和乌法四聚体将揭示哪些淋巴细胞与 UDA/乌法，以及与它们相互作用的适应性和/或先天性受体。我们将 进行鲨鱼原代细胞的生化分析，以表征UDA/乌法生物合成途径。 因此，在本提案中，我们将联合收割机结合两位调查员的专业知识和经验， Flajnik研究鲨鱼免疫系统的遗传学、生物化学和功能已有35年， 一般来说，适应性免疫的进化，和艾琳亚当斯，一个结构生物学家与深入的知识， 人类I类系统和进化生物学。总之，我们假设CD 1和HLA-E类似物 产生于5亿年前的适应性免疫大爆炸，这表明， 经典的I类，通过“CD 1”结合脂质，通过“HLA-E样”非经典的多面手结合肽 I类是最早的适应性免疫系统中的重要特征。

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.