The role of alpha- and epsilon-amino groups in the glycation-mediated cross-linking of gammaB-crystallin. Study of three site-directed mutants
| Autor: | H R, Zhao, R H, Nagaraj, E C, Abraham |
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| Rok vydání: | 1997 |
| Předmět: | |
| Zdroj: | The Journal of biological chemistry. 272(22) |
| ISSN: | 0021-9258 0781-2078 |
| Popis: | In the previous report we demonstrated that gammaB-crystallin is glycated predominantly at the N-terminal alpha-amino group (Casey, E. B., Zhao, H. R., and Abraham, E. C. (1995) J. Biol. Chem. 270, 20781-20786). To investigate the possible role of alpha- and epsilon-amino groups of gammaB-crystallin in glycation-mediated cross-linking, Lys-2 or Lys-163, or both, were mutated to threonine by site-directed mutagenesis in bovine gammaB-crystallin cDNA. Wild type and mutant gammaB-crystallins were expressed in Escherichia coli cells. Cross-linking studies were performed by incubating wild type and mutant gammaB-crystallins with glyceraldehyde, ribose, and galactose followed by SDS-polyacrylamide gel electrophoresis under reducing conditions. When both of the lysines of gammaB-crystallin were mutated to threonines (gammaB-K2T/K163T), the quantity of cross-linked products was greatly reduced, indicating that, despite the fact that the alpha-amino group is a major glycated site, epsilon-amino groups play a predominant role in cross-linking. Therefore, cross-linking ability depends not only upon the level of glycation but also upon which amino group is glycated. Steric hindrance may decrease the cross-linking ability of the alpha-amino group. Our results also show that Lys-2 and Lys-163 play almost equal roles in cross-linking of gammaB-crystallin. By incubating carbonic anhydrase, a protein with a blocked N terminus, and our novel "no lysine" gammaB (gammaB-K2T/K163T) with sugar, we were able to show for the first time that significant cross-linking occurs between lysines and non-lysine sites. The fact that pentosidine and imidazolysine, formed from ribose and methylglyoxal, respectively, were present in the cross-linked gammaB-crystallins revealed the existence of Lys-Arg and Lys-Lys cross-linking. |
| Databáze: | OpenAIRE |
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