Age-related cataract is one of the major causes of blindness throughout the world. It is thought to develop as a result of extensive post- translational modifications of lens cystallins accumulated during a life span of an individual. Mostly, due to the lack of turnover within the lens, these modifications lead to the formation of the pigmented and covalently crosslinked high molecular weight protein aggregates able to scatter light impinging on the lens. Except disulfides, only a few of non- disulfides lens protein crosslinks have been identified so far, all of them are acid stable, and can be divided in two classes, dehydroalanine- or advanced glycation endproducts (AGE) crosslinks. To date neither the quantities of the particular crosslink nor the total quantities of the discovered crosslinks can explain the extensive crosslinking seen in cataract. Therefore, the long-term objective of this project is to determine and structurally characterize the crosslinks involved in the formation of protein lens crosslinks as well as to determine the role they play in cataractogenesis of human lens. Therefore, the long-term objective of this project is to determine and structurally characterize the crosslinks involved in the formation of protein lens crosslinks as well as to determine the role they play in cataractogenesis of human lens. We hypothesize that all the non-enzymatic and non-reducible crosslinks are limited to the two classes of crosslinks, dehydroalanine type crosslinks are limited to the two classes of crosslinks, dehydroalanine type crosslinks and glycation-mediated crosslinks. We think that since dehydroalanine chemistry is intrinsically dependent upon the presence of disulfides and Ca2+ within the lens proteins, the formation of lens protein disulfides and oxidized glutathione during aging and cataractogenesis facilitates their formation in the aged and, to a greater extent, in cataractous lenses. We also postulate that the decrease in concentration of GSH in cataractous lens will lead to a degradation of ascorbic aid and, probably, to glucose auto-oxidation, subsequently causing the AGEs crosslinks to occur during cataractogenesis. To study this hypothesis and to better understand the involvement of sugar- mediated crosslinking in cataractogenesis we have developed a methodology that allows a synthesis and purification of total fractions of AGE crosslinks generated by any sugar under conditions that prevent a degradation of any labile crosslinks. The method allows raising specific, broad specificity, high titer antibodies against AGE crosslinks and their detection by our newly developed ELISAs. These antibodies will be further used for the determination, isolation, and structural characterization of natural Lys-Lys and Lys-Arg crosslinks within the structure of the aged and cataractous lens proteins and studying the factors that lead to their formation in human during cataractogenesis. The anticipated results should provide a solid basis for understanding the impact the formation of non-disulfide crosslinks has on the development of cataract in humans.
| Linetsky, Mikhail; Shipova, Ekaterina V; Argirov, Ognyan K (2006) Influence of glutathione fructosylation on its properties. Arch Biochem Biophys 449:34-46 |
| Linetsky, Mikhail D; Shipova, Ekaterina V; Legrand, Roy D et al. (2005) Glucose-derived Amadori compounds of glutathione. Biochim Biophys Acta 1724:181-93 |
| Linetsky, Mikhail; LeGrand, Roy D (2005) Glutathionylation of lens proteins through the formation of thioether bond. Mol Cell Biochem 272:133-44 |
| Linetsky, Mikhail; Hill, J M W; LeGrand, Roy D et al. (2004) Dehydroalanine crosslinks in human lens. Exp Eye Res 79:499-512 |
| Linetsky, Mikhail; Hill, Jana M W; Chemoganskiy, Vitaliy G et al. (2003) Studies on the mechanism of the UVA light-dependent loss of glutathione reductase activity in human lenses. Invest Ophthalmol Vis Sci 44:3920-6 |
