LENS PROTEIN GLYCATION AND CATARACT DEVELOPMENT

眼晶状体的晶状体蛋白是长寿蛋白质，因为它们几乎不 翻个身。这样的蛋白质不断地受到翻译后翻译的影响 非酶糖基化(糖基化)等修饰。糖尿病患者 患者在年轻时患白内障的可能性要高出四到六倍。 晶状体蛋白的年龄和糖基化所起的作用 在糖尿病白内障的发生中起着重要作用。糖基化产物 主要分两个阶段，席夫碱和Amadori产物的形成或 早期糖基化和晚期糖基化终末产物的形成 具有荧光性和蛋白质交联性。长距离的 该提案的目标是研究糖基化在糖尿病中的作用 白内障的发生。目前的目标是确定氨基酸的位置。 不同的晶状体蛋白糖化并确定其速率 或糖尿病进展过程中这些部位的糖基化程度 以及链脲佐菌素糖尿病大鼠的白内障，衰老和糖尿病人的白内障， 在大鼠和人晶体蛋白的体外糖基化过程中 糖和糖磷酸盐。这样的信息对于理解 为什么糖基化会导致蛋白质构象变化，增加 硫醇的反应性和增强的硫醇氧化。为了实现这些目标 将应用蛋白质化学的现代技术，这些技术包括： 分子筛液相色谱分离[~3H]NaBH4反应的天然晶体蛋白 包括HMW聚集体、反相高效液相色谱分离结晶蛋白 亚基多肽、胰蛋白酶和胰凝乳素，Affigel 601 亲和层析法纯化糖化肽， 高效液相色谱/柱后联用法测定氨基酸组成 人工微测序的衍生化方法和氨基酸测序 方法。用于体外和体内晶体蛋白的鉴定 除了经典的化学物质外，还会产生可溶或不溶的部分 鉴定，免疫学鉴定将与 将产生针对各种大鼠和人的单抗 晶体蛋白。在早期糖基化和晚期糖基化抑制剂的帮助下 糖基化，如乙酰水杨酸(阿司匹林)和氨基胍 表明早期糖基化和晚期糖基化是否在 糖尿病白内障的发生。此外，通过阿司匹林治疗，它将是 显示晶体蛋白的糖基化在体外(在大鼠中)被抑制 和人晶体蛋白)和体内(仅在大鼠晶体蛋白中)以及 其机制涉及特定氨基酸残基的乙酰化。

Crystallins of the eye lens are long-lived proteins because they hardly turn over. Such proteins are constantly subjected to posttranslational modifications such as nonenzymatic glycosylation (glycation). Diabetic patients are four to six times more likely to develop cataract at a younger age than the normal population and glycation of lens crystallins could play a significant role in diabetic cataractogenesis. Glycation proceeds primarily in two stages, formation of Schiff-base and Amadori products or early glycation and formation of advanced glycation end products that are fluorescent and having protein cross-linking properties. The long-range goal of the proposal is to study the role of glycation in diabetic cataractogenesis. The immediate goals are to identify the amino acid sites of different crystallins that are being glycated and to determine the rates or extents of glycation of such sites during the progression of diabetes and cataract in streptozotocin-diabetic rats, in aging and diabetic humans, and during in vitro glycation of rat and human crystallins with various sugars and sugar phosphates. Such information is essential to understand why glycation would induce protein conformational changes, increased reactivity of thiols and enhanced thiol oxidation. To achieve these goals modern techniques in protein chemistry will be applied which are: molecular sieve HPLC for separation of [3H]NaBH4 reacted native crystallins including HMW aggregates, reverse-phase HPLC for separation of crystallin subunit polypeptides and tryptic and chymotryptic peptides, Affigel 601 affinity chromatography for purification of glycated peptides, determination of amino acid composition by a HPLC/post-column derivatization method and amino acid sequencing by a manual microsequencing method. For identification of the crystallins in the in vitro and in vivo generated soluble or insoluble fractions, in addition to classical chemical characterization, immunologic identification will be carried out with monoclonal antibodies that will be produced against various rat and human crystallins. With the aid of inhibitors of early glycation and advanced glycation such as acetylsalicylic acid (aspirin) and aminoguanidine it will be shown whether early glycation and advanced glycation play any role in diabetic cataractogenesis. Moreover, with aspirin treatment it will be shown that glycation of which crystallins are inhibited in vitro (in rat and human crystallins) and in vivo (only in rat crystallins) and whether the mechanism involves acetylation of specific amino acid residues.