LENS PROTEIN GLYCATION & CATARACT DEVELOPMENT

眼透镜的晶体蛋白是长寿的蛋白质，因为它们几乎不 转过来 这些蛋白质不断进行翻译后 修饰，如非酶糖基化（糖化）。 糖尿病 年轻人患白内障的可能性是年轻人的四到六倍。 年龄比正常人群和糖基化的透镜晶体蛋白可以发挥作用 在糖尿病性白内障发生中起重要作用。 糖化进行 主要分为两个阶段，形成希夫基地和阿马多里产品或 早期糖基化和晚期糖基化终产物的形成 发荧光并具有蛋白质交联性质。 远程 该提案的目的是研究糖化在糖尿病中的作用， 白内障形成 目前的目标是确定氨基酸位点 不同的晶体蛋白被糖化， 或在糖尿病进展过程中这些位点的糖化程度 链脲佐菌素糖尿病大鼠、老年人和糖尿病患者的白内障， 在大鼠和人晶状体蛋白的体外糖基化过程中， 糖和糖磷酸盐。 这些信息对于理解 为什么糖基化会引起蛋白质构象的变化， 硫醇的反应性和增强的硫醇氧化。 实现这些目标 将应用蛋白质化学的现代技术，这些技术是： 分子筛HPLC法分离[3 H] NaBH 4反应的天然晶状体蛋白 包括HMW聚集体，用于分离晶状体蛋白的反相HPLC 亚基多肽和胰蛋白酶和胰凝乳蛋白酶肽，Affigel 601 用于纯化糖化肽的亲和层析， HPLC/柱后法测定氨基酸组成 衍生化方法和通过手动微测序进行的氨基酸测序 法 用于体外和体内晶体蛋白的鉴定 产生的可溶性或不溶性部分，除了经典的化学 表征，免疫学鉴定将进行， 单克隆抗体，将产生针对各种大鼠和人类 晶体蛋白。 借助早期糖化和晚期糖化抑制剂， 糖基化如乙酰水杨酸（阿司匹林）和氨基胍， 显示早期糖基化和晚期糖基化是否在 糖尿病性白内障 此外，使用阿司匹林治疗， 显示其晶体蛋白糖基化在体外（大鼠中）被抑制 和人晶体蛋白）和体内（仅在大鼠晶体蛋白中）以及是否 该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.