Determination of dideoxyosone precursors of AGEs in human lens proteins

Abstract

Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation end products (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-{[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl) benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 AU/mg protein vs. 31.7±19.5 AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates.

Biochemical and Molecular Markers of Congenital and Senile Cataractous Lenses

“…Lenticular oxindolealanine (tryptophan oxidation product) and its byproduct kynurenines were observed in the lens nucleus of diabetic cataractous patients [23] and experimental diabetic rats [24]. Higher levels of dideoxyosones (DDOs); the intermediate product of advanced glycation end products (AGEs), such as pentosidine and glucosepane were detected in senile cataractous lenses [25]. Other findings mentioned deamidation of glutamine and asparagine residues in older lenses [26].…”

Non‐enzymatic glycation and glycoxidation protein products in foods and diseases: An interconnected, complex scenario fully open to innovative proteomic studies

“…In the human lens, in addition to sugars, ascorbate is a potential source of dideoxysones. Very recently, an elegant quasi‐proteomic approach was developed to detect dideoxysones in lens proteins (Linetsky et al, ). A specific dideoxysone‐trapping agent, namely biotinyl‐diaminobenzene (3,4‐diamino‐ N ‐(3‐{[5‐(2‐oxohexahydro‐1 H ‐thieno[3,4‐ d ]imidazol‐4‐yl)pentanoyl]aminopropyl)benzamide) (BDAB) (Fig.…”

“…The sequence of the corresponding peptide (115–125), with a monoisotopic mass of 1817.8 Da, and the theoretical fragmentation pattern are shown. Adapted with permission from Linetsky et al (). Copyright 2011, Elsevier.…”

Overview of Congenital, Senile and Metabolic Cataract

“…Lens proteins possessed the higher levels of dideoxyosones (DDOs): intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane [103]. Deamidation of glutamine and asparagine residues was observed in older lenses [104].…”