THE ROLE OF ALPHA-AMINO AND EPSILON-AMINO GROUPS IN THE GLYCATION-MEDIATED CROSS-LINKING OF GAMMA-B-CRYSTALLIN - STUDY OF 3 SITE-DIRECTED MUTANTS

In the previous report we demonstrated that gamma B-crystallin is glyc ated 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 gamma B-crystallin in glycation-mediated cross-linking, Lys-2 or L ys-163, or both, were mutated to threonine by site-directed mutagenesi s in bovine gamma B-crystallin cDNA. Wild type and mutant gamma B-crys tallins were expressed in Escherichia coli cells, Cross-linking studie s were performed by incubating wild type and mutant gamma B-crystallin s with glyceraldehyde, ribose, and galactose followed by SDS-polyacryl amide gel electrophoresis under reducing conditions, When both of the lysines of gamma B-crystallin were mutated to threonines (gamma B-K2T/ K163T), the quantity of cross-linked products was greatly reduced, ind icating that, despite the fact that the alpha-amino group is a major g lycated site, epsilon-amino groups play a predominant role in cross-li nking, Therefore, cross-linking ability depends not only upon the leve l of glycation but also upon which amino group is glycated. Steric hin drance may decrease the cross-linking ability of the a-amino group. Ou r results also show that Lys-2 and Lys-163 play almost equal roles in cross-linking of gamma B-crystallin, By incubating carbonic anhydrase, a protein with a blocked N terminus, and our novel ''no lysine'' gamm a B (gamma B-K2T/K163T) with sugar, we were able to show for the first time that significant cross-linking occurs between lysines and non-ly sine sites, The fact that pentosidine and imidazolysine, formed from r ibose and methylglyoxal, respectively, were present in the cross-linke d gamma B-crystallins revealed the existence of Lys-Arg and Lys-Lys cr oss-linking.