Glycation inactivation of human CD59 and diabetic complications

描述（由申请人提供）：慢性高血糖水平（高血糖症）是糖尿病并发症的原因，包括肾病、视网膜病变、神经病变和加速动脉粥样硬化，这些都是成年人群发病率和死亡率的主要原因。然而，高血糖如何导致糖尿病并发症仍然知之甚少。我们和其他人的广泛临床研究表明，补体系统，免疫系统的主要效应子和炎症介质，可能在这些并发症的发病机制中发挥重要作用。特别是，我们已经确定，人CD 59，一个关键的补体调节蛋白，保护细胞免受补体介导的损伤，是抑制糖化，非酶的葡萄糖附着到蛋白质。这是因为人CD 59含有糖基化基序，其由蛋白质活性位点中的K41残基在H5 A0处的H44残基形成。有趣的是，这种糖基化基序不存在于其他物种的CD 59中。我们的假设是，由于其独特的H44残基的hCD 59的糖基化失活可能代表高血糖症和人类糖尿病的血管增殖并发症之间的难以捉摸的联系。然而，实际和伦理方面的考虑排除了进行临床研究，以全面测试这一假设在人类。因此，我们确定补体损害糖尿病并发症靶器官的细胞和分子机制的策略集中于开发我们实验室中现有的分子工程小鼠。我们将通过STZ注射使这些小鼠患糖尿病（1型糖尿病模型），并利用我们的前一代mCD 59 KO小鼠回交到Apoe-/-背景中来研究hCD 59的糖基化失活在糖尿病心血管疾病中的作用。具体地，我们将比较糖尿病hCD 59野生型（糖基化失活敏感性）转基因小鼠与糖尿病hCD 59 Gln 44突变体（糖基化失活抗性）转基因小鼠，两者都在mCd 59 KO和Apoe-/-背景中。我们还将使用可用的抗mC 5抗体来阻断末端补体级联反应，并为补体对糖尿病血管疾病的贡献提供直接的机制证据。这项工作的成功完成将提供：1）明确的证据证明补体和hCD 59的糖基化失活在糖尿病心血管并发症的发病机制中的作用; 2）研究糖尿病并发症的机制、治疗和预防所需的动物模型; 3）强有力的证据支持未来人类补体和糖尿病的研究。 公共卫生相关性：本提案的主要目标是验证我们的假设，即补体调节蛋白CD 59的糖基化失活在糖尿病并发症的发病机制中起关键作用，特别关注糖尿病患者常见的心血管疾病。为此，我们将使用STZ诱导的糖尿病小鼠，这些小鼠缺乏mCD 59（mCD 59 KO），回交到Apoe缺陷背景中，并转基因表达糖基化敏感野生型形式或糖基化不敏感突变体形式的人CD 59，所有这些都是在我们的实验室中产生和获得的。拟议的研究是高度翻译;其相关性突出了以下事实：1）糖尿病及其并发症是成年人群发病率和死亡率的主要原因，2）引起缺血性心脏病、中风和外周血管疾病的血管病理占糖尿病人群死亡率的50%以上，3）糖尿病心血管疾病的治疗占美国每年用于治疗慢性糖尿病并发症的超过1000亿美元的近65%（H1占美国总卫生预算的10美元），以及4）我们目标的成功实现将为发病机制的临床前研究提供急需的动物模型，治疗和预防糖尿病并发症。

DESCRIPTION (provided by applicant): Chronic high blood sugar levels (hyperglycemia) are responsible for diabetic complications including nephropathy, retinopathy, neuropathy and accelerated atherosclerosis, all major causes of morbidity and mortality in the adult population. However, how hyperglycemia causes diabetic complications is still poorly understood. Extensive clinical studies by us and others indicate that the complement system, a major effector of the immune system and mediator of inflammation, may play a significant role in the pathogenesis of these complications. In particular, we have established that human CD59, a key complement regulatory protein that protects cells from complement-mediated damage, is inhibited by glycation, the non-enzymatic attachment of glucose to proteins. This is because human CD59 contains a glycation motif, formed by its H44 residue at H 5A0 from its K41 residue in the active site of the protein. Interestingly, this glycation motif is not present in CD59 from other species. Our hypothesis is that glycation-inactivation of hCD59 due to its unique H44 residue could represent the elusive link between hyperglycemia and the vascular proliferative complications of human diabetes. However, practical and ethical considerations preclude conducting clinical studies to test this hypothesis comprehensively in humans. Therefore, our strategy to determine the cellular and molecular mechanism by which complement damages the target organs of diabetic complications has focused on developing molecular engineered mice that are now available in our laboratory. We will make these mice diabetic by STZ injection (type 1 diabetes model), and investigate the role of glycation-inactivation of hCD59 in diabetic cardiovascular disease taking advantage of our previous generation of mCD59KO mice backcrossed into the Apoe-/- background. Specifically, we will compare diabetic hCD59 wild type (glycation-inactivation sensitive) transgenic mice with diabetic hCD59Gln44 mutant (glycation- inactivation resistant) transgenic mice, both in a mCd59KO and Apoe-/-background. We will also use an available anti-mC5 antibody to block the terminal complement cascade and provide direct mechanistic evidence for the contribution of complement to diabetic vascular diseases. Successful accomplishment of this work will provide: 1) clear evidence for the role of complement and glycation-inactivation of hCD59 in the pathogenesis of the cardiovascular complications of diabetes; 2) needed animal models to study mechanism, therapy and prevention of diabetic complications; and 3) strong evidence to support future studies on complement and diabetes in humans. PUBLIC HEALTH RELEVANCE: The major goal of this proposal is to test our hypothesis that glycation-inactivation of the complement regulatory protein CD59 plays a critical role in the pathogenesis of diabetic complications with particular focus on the cardiovascular disease commonly seen in diabetic individuals. To this end, we will use STZ-induced diabetic mice that lack mCD59 (mCD59KO), are backcrossed into an Apoe deficient background, and transgenetically express human CD59 in either the glycation-senstive wild type form or as a glycation-insensitive mutant, all generated and available in our laboratory. The proposed studies are highly translational; their relevance is highlighted by the following facts: 1) Diabetes and its complications are a leading cause of morbidity and mortality in the adult population, 2) vascular pathology causing ischemic heart disease, stroke, and peripheral vascular disease account for more than 50% of the mortality rate in the diabetic population, 3) treatment of diabetic cardiovascular disease accounts for close to 65% of the more than 100 billion dollars annually spent in the US to treat chronic diabetic complications (H 1 out 10 dollars of the overall US health budget), and 4) successful accomplishment of our aims will provide a much needed animal model for pre-clinical studies on the pathogenesis, treatment and prevention of diabetic complications.