Human complement C4 isotypes in Lupus

 描述（由申请人提供）：发现携带C1 q或C4遗传或获得性缺陷的个体几乎总是发展狼疮，被称为“狼疮性瘫痪”。与人类白细胞抗原基因座（HLA）内的许多基因座一样，位于III类区域的补体C4基因座是高度多态性的。存在两种高度保守的同种型C4 A和C4 B，其编码同种型区域（4个氨基酸）中的结构变异性，所述同种型区域调节内部硫酯的共价结合。虽然最常见的单倍型是C4 A和C4 B，但C4基因座上的等位基因数量可以从0到4不等。例如，C4 A或C4 B中的等位基因缺陷分别以约4%和1%的频率发生。与人类相反，小鼠表达单个功能性C4等位基因;同种型区域中的鼠C4基因的DNA序列是人类C4 A和C4 B的杂交体。C4的一个关键功能是在酶促裂解后与其靶标共价结合。C4一旦被激活就具有高度反应性，并且使用小分子配体的体外研究确定C4 A保持其活性更长时间并且主要形成酰胺键;而C4 B与抗原表面形成酯键。预计体外化学差异将对体内C4功能产生显著影响。动物模型的可用性来测试体内两种亚型的重要性对于推动该领域的发展至关重要。当前提案的目标是表征两种新型小鼠品系，其中C4基因座在同型区域中被“编辑”以表达C4 A或C4 B蛋白。目的1：表征携带hu C4 A或C4 B同种型序列的基因“编辑”小鼠品系。我们希望从这个目标中了解C4 A和C4 B在保护小鼠免受病毒和细菌病原体侵害方面是否存在差异。目的2：检验C4同种型区域的结构差异影响狼疮小鼠模型的保护作用的假设。我们期望从这个目标中了解C4 A蛋白在清除核仁抗原和保护小鼠狼疮模型中是否比C4 B更有效。总结：这项研究的成功完成不仅将提供有价值的试剂和新的工具，推动该领域的发展，而且可能导致蛋白质替代疗法的发展。

 DESCRIPTION (provided by applicant): The finding that individuals bearing genetic or acquired deficiency in C1q or C4 almost always develop lupus has been referred to as the "Lupus Paradox". Like many loci within the Human Leukocyte Antigen locus (HLA), the complement C4 locus, which is located in the class III region, is highly polymorphic. There are two highly conserved isotypes, C4A and C4B, which encode structural variability in the isotypic region (4 amino acids) which regulate covalent binding of the internal thioester. While the most common haplotype is C4A and C4B, the number of alleles at the C4 locus can vary from 0 to 4. For example, allelic deficiency in C4A or C4B occurs at a frequency of about 4% and 1%, respectively. In contrast to humans, mice express a single functional C4 allele; the DNA sequence of the murine C4 gene in the isotypic region is a hybrid of human C4A and C4B. A key function of C4 is to bind covalently to its target following enzymatic cleavage. C4 is highly reactive once it is activated and studies using small molecule ligands in vitro determined that C4A retains its activity longer and primarily forms amide bonds; whereas, C4B forms ester bonds with the antigen surface. It is predicted that the in vitro chemical difference will have significant effects on C4 function in vivo. The availability of animal models to test the importanc of the two isoforms in vivo would be important to move the field forward. The goal of the current proposal is characterize two novel strains of mice in which the C4 locus was "edited" in the isotypic region to express either C4A or C4B protein. Aim 1: Characterize gene "edited" strains of mice bearing hu C4A or C4B isotypic sequences. We expect to learn from this aim if C4A and C4B differ in their protection of mice against viral and bacterial pathogens. Aim 2: Test the hypothesis that structural differences in the isotypic region of C4 influence protection in a murin model of lupus. We expect to learn from this aim if the C4A protein is more efficient than C4B in clearance of nucleolar antigens and in protection in a murine lupus model. Summary: The successful completion of this study will not only provide valuable reagents and novel tools to push the field forward but could lead to development of protein replacement therapy.