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

摘要 鲨鱼是现存最古老的脊椎动物之一，具有基于免疫球蛋白的适应性免疫， T细胞受体和主要组织相容性复合体。最近发现了8个I类血统 由Flajnik实验室、经典I类(UAA)和七个非经典谱系(UBA到UHA)。出乎意料的是， 其中两个与MHC相连的鲨鱼非经典I类(Ib)分子显示出与人类惊人的相似之处 非经典I类分子CD1和人类白细胞抗原-E关于遗传学、表达和序列的初步数据 Flajnik实验室的相似性很强，表明UFA是CD1的同源基因。结构建模和硅胶技术 亚当斯实验室的生物物理分析与UFA与CD1的明显相似性一致，揭示了一种强烈的 疏水结合部位。第二个鲨鱼类Ib分子UDA被预测能与经典的多肽结合 第一类，但是它是单态、单拷贝的，并且具有与哺乳动物的人类白细胞抗原-E/Qa-1相似的特性。 我们将用生物化学方法定义UFA和UDA配体，并阐明它们的结构。我们将表达和提纯 用于包括配体洗脱和制备的生化分析的UDA和UFA的重组版本 四聚体来识别识别这些非经典I类分子的鲨鱼细胞。高度纯化的形式 重组蛋白将通过X射线结晶学进行结构分析。在内部使用 技术，我们将产生与这些蛋白质的纳米体结合试剂用于结构分析，还 补充已经为组织表达分析产生的抗血清和单抗。由In 原位杂交和抗体/纳米体染色我们将确定UDA/UFA细胞的表达并检测 它们在应激状态下在细胞中的表达上调。UDA和UFA四聚体将揭示与哪些淋巴细胞相互作用 UDA/UFA，以及通过单细胞分析，与其相互作用的适应性和/或先天受体。我们会 对鲨鱼原代细胞进行生化分析，以确定UDA/UFA的生物合成途径。 因此，在这项提案中，我们将结合马丁这两位调查员的专业知识和经验。 弗拉伊尼克研究鲨鱼免疫系统的遗传学、生化和功能已有35年， 一般来说，获得性免疫的进化，以及结构生物学家艾琳·亚当斯对 人类第一类系统与进化生物学。综上所述，我们假设CD1和人类白细胞抗原E类似物 在5亿年前的获得性免疫大爆炸中出现，表明由 经典的I类，由“CD1”的脂类结合，以及由万能的“类人类白细胞抗原-E”非经典的多肽结合 I类是最早的获得性免疫系统的重要特征。

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.