ENZYMATIC MECHANISM TO CONTROL NONENZYMATIC GLYCATION

葡萄糖是大多数哺乳动物细胞的必需宏量营养素，在其中它作为能量和碳的主要来源发挥作用。然而，在高浓度下，葡萄糖可对细胞产生有害影响。这在糖尿病中最显著地表现出来，其中慢性高血糖导致许多糖尿病并发症，包括：视网膜病变、血管疾病、神经病变和肾病。大量证据表明，高血糖症的一些有害作用是由蛋白质的非酶促葡糖基化引起的，其中葡萄糖与赖氨酸残基反应形成果糖赖氨酸（FL）。这种早期可逆的糖基化产物随后经历一系列进一步的反应，最终导致形成不可逆的晚期糖基化终产物（AGE）。早期和晚期的糖基化产物对细胞功能（特别是肾脏）都有不良影响，并且在衰老和糖尿病的许多组织中已经记录了AGE的积累。最近我们发现了一种明显普遍存在的酶（果糖赖氨酸磷酸转移酶，FLPT），其将蛋白质上的果糖赖氨酸磷酸化为果糖赖氨酸-3-磷酸（FL 3 P）。FL的这种修饰随后导致其自发分解和未修饰的赖氨酸残基的再生。我们推测，FLPT在哺乳动物细胞中的功能是分解FL，从而在最早可能的干预点逆转非酶葡萄糖基化过程。此外，我们提出FLPT介导的去糖基化的可变效率可能是个体对糖尿病并发症的易感性和正常衰老率的决定因素。我们打算通过两个特定的目的来探索这些假设：1）果糖赖氨酸磷酸转移酶（来自人红细胞）的纯化及其完整的分子表征2）评估FLPT途径在糖尿病肾病发展速度加快和减慢的糖尿病患者人群红细胞中的活性。

Glucose is an essential macronutrient of most mammalian cells where it functions as the main source of energy and carbon. However, at high concentration glucose can have detrimental effects on cells. This is most dramatically manifested in diabetes mellitus where chronic hyperglycemia leads to many diabetic complications including: retinopathy, vascular disease, neuropathy and nephropathy. There is substantial evidence to suggest that some of the deleterious effects of hyperglycemia are caused by the non- enzymatic glucosylation of proteins wherein glucose reacts with lysine residues to form fructoselysine (FL). This early, reversible, glucosylation product subsequently undergoes a series of further reactions leading ultimately to the formation of the irreversible Advanced Glycation Endproducts (AGE's). Both the early and advanced glucosylation products have adverse effects on cell function (especially in the kidney) and AGE accumulation has been documented in many tissues in aging and diabetes. Recently we discovered an apparently ubiquitous enzyme (Fructoselysine Phosphotransferase, FLPT) which phosphorylates fructoselysine on proteins to fructolysine-3-phosphate (FL 3P). This modification of FL subsequently leads to its spontaneous decomposition and the regeneration of an unmodified lysine residue. We postulate that the function of FLPT in mammalian cells is to breakdown FL thus reversing the nonenzymatic glucosylation process at the earliest possible point of intervention. Furthermore we propose that variable efficiency of the FLPT-mediated deglucosylation may be a determinant of the susceptibility of individuals to diabetic complications and the rate of normal aging. We intend to explore these hypotheses through two specific aims: 1) Purification of the fructoselysine phosphotransferase enzyme (from human erythrocytes) and its complete molecular characterization 2) Assessment of the activity of FLPT pathway in erythrocytes from populations of diabetic patients with accelerated and retarded rates of development of diabetic nephropathy.