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J. Biol. Chem., Vol. 280, Issue 23, 22154-22164, June 10, 2005

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3-Hydroxykynurenine-mediated Modification of Human Lens Proteins

STRUCTURE DETERMINATION OF A MAJOR MODIFICATION USING A MONOCLONAL ANTIBODY^*

Magdalena M. Staniszewska and Ram H. Nagaraj¶||

From the Departments of Ophthalmology and ^¶Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

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-acetyl lysine, N-acetyl histidine, N-acetyl arginine, and N-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-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl--((5S)-5-acetamino-5-carboxypentyl amino)--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.

Received for publication, February 7, 2005 , and in revised form, March 18, 2005.

^* This study was supported in part by National Institutes of Health Grants R01EY-09912 and P30EY-11373 and grants from Research to Prevent Blindness (RPB), the Ohio Lions Eye Research Foundation, and Fight for Sight (FFS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A recipient of a postdoctoral fellowship from FFS.

^|| A recipient of an RPB Lew R. Wasserman Merit Award. To whom correspondence should be addressed: Dept. of Ophthalmology, Wearn Bldg., Rm. 643, Case Western Reserve University, Cleveland, OH 44106. Tel.: 216-844-1132; Fax: 216-844-7962; E-mail: ram.nagaraj{at}case.edu.

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