ASPARTIC ACID RACEMIZATION IN RELATION TO HUMAN CATARACTOGENESIS

天冬氨酸外消旋与人类白内障发生的关系

• 批准号: 6349104
• 负责人: FELIX I IFEANYL
• 金额: --
• 依托单位: GRAMBLING STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349104
• 关键词: aging; aminoacid analyzer; aspartate; cataract; conformation; high performance liquid chromatography; human tissue; hydrolysis; lens proteins; matrix assisted laser desorption ionization; protein structure function; racemization

Older citizens are fast becoming a greater percentage of the American population. With this trend, there is increasing concern over functional disabilities amongst this segment of the population. Visual disorders like cataract and macular degeneration, are the leading causes of blindness and constitute a major part of functional disabilities amongst older-persons. Age-related cataract is due, at least in part, to chemical modifications in lens proteins during the aging process. Amino acid racemization is one of the age-related changes that have been reported. It involves the stereoinversion of the L-form of a protein amino-acid, to its naturally uncommon D-form. This conformational change may lead to a structural change that may ultimately alter the function(s) of the modified protein. Past studies have documented increased D/L rations of specific aspartate residues of alpha-A crystallins from aged, cataractous human lenses and suggested a possible involvement of racemization in the pathogenesis of age-related cataract will aid in developing strategies for treatment, or at least, delaying the onset of this disabling disease. The long term objective of this project is to determine if the extent of aspartic acid racemization in human eye lens proteins, correlates with incidence of age-related cataract. The specific objectives are to utilize more reliable, sensitize methods, to quantitatively compare racemization of Asp-58, Asp-151 in alpha-A crystalline, and Asp-36, Asp-62 of alpha- B crystallins, isolated from cataractous and age-matched normal human lenses. This will be done by HPLC analysis of peptide fragments (containing the specified aspartate residues), derivatized with Marfey's reagent. Aspartic acid racemization will also be quantitated in water insoluble, urea-soluble protein fractions from cataractous and age- matched normal lenses, using a method that minimizes background racemization and estimates D-aspartic acid by oxidation with D-aspartate oxidase.

老年公民在美国人口中所占的比例正在迅速增加。随着这一趋势的发展，人们越来越关注这部分人口中的功能性残疾。视力障碍，如白内障和黄斑变性，是失明的主要原因，也是老年人功能性残疾的主要部分。晶状体相关性白内障至少部分是由于老化过程中透镜蛋白质的化学修饰。氨基酸外消旋化是已报道的与年龄相关的变化之一。它涉及蛋白质氨基酸的L-形式立体转化为其天然不常见的D-形式。这种构象变化可导致结构变化，其可最终改变经修饰的蛋白质的功能。过去的研究已经证明了老年白内障患者晶状体中α-A晶体蛋白的特定天冬氨酸残基的D/L比率增加，并表明年龄相关性白内障发病机制中可能涉及外消旋化，这将有助于制定治疗策略，或至少延迟这种致残性疾病的发作。本项目的长期目标是确定人眼透镜蛋白中天冬氨酸外消旋化的程度是否与年龄相关性白内障的发病率相关。具体目的是利用更可靠的增敏方法，定量比较从白内障和年龄匹配的正常人晶状体中分离的α-A晶体蛋白中的Asp-58、Asp-151和α- B晶体蛋白中的Asp-36、Asp-62的外消旋化。这将通过对肽片段（含有特定天冬氨酸残基）进行HPLC分析来完成，肽片段用Marveland试剂衍生化。还将使用一种最大限度地减少背景外消旋化并通过D-天冬氨酸氧化酶氧化来估计D-天冬氨酸的方法，对来自白内障和年龄匹配的正常晶状体的水不溶性、尿素溶性蛋白质组分中的天冬氨酸外消旋化进行定量。