Staniszewska Magdalena M, Nagaraj Ram H
Departments of Ophthalmology and Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
J Biol Chem. 2005 Jun 10;280(23):22154-64. doi: 10.1074/jbc.M501419200. Epub 2005 Apr 6.
Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.
色氨酸在眼球晶状体中可通过酶促和非酶促机制被氧化。氧化产物，如犬尿氨酸，会与蛋白质反应形成黄棕色色素并导致共价交联。我们制备了一种针对3-羟基犬尿氨酸（3OHKYN）修饰的钥孔血蓝蛋白的单克隆抗体，并使用3OHKYN修饰的氨基酸和蛋白质对其进行了表征。这种单克隆抗体与3OHKYN修饰的N（α）-乙酰赖氨酸、N（α）-乙酰组氨酸、N（α）-乙酰精氨酸和N（α）-乙酰半胱氨酸发生反应。在所测试的几种色氨酸氧化产物中，3OHKYN与人晶状体蛋白反应时产生的抗原浓度最高。通过反相高压液相色谱法纯化了3OHKYN与N（α）-乙酰赖氨酸反应的主要抗原，通过光谱对其进行了表征，并鉴定为2-氨基-3-羟基-α-（（5S）-5-乙酰氨基-5-羧基戊基氨基）-γ-氧代苯丁酸。酶消化的白内障晶状体蛋白显示出3OHKYN衍生的修饰。免疫组织化学显示在与晶状体纤维细胞质膜相关的蛋白质中有3OHKYN修饰。与葡糖苷酶反应后从正常晶状体中分离出的低分子产物（<10,000 Da），再与蛋白质孵育后产生了3OHKYN衍生的产物。用3OHKYN孵育的人晶状体上皮细胞显示出强烈的免疫反应性。我们还研究了糖基化对色氨酸氧化和犬尿氨酸介导的晶状体蛋白修饰的影响。结果表明，糖基化产物不能氧化色氨酸或在蛋白质中产生犬尿氨酸修饰。我们的研究表明，3OHKYN独立于糖基化修饰晶状体蛋白，形成可能导致白内障形成过程中蛋白质聚集和褐变的产物。
