In the previous report we demonstrated that ␥B-crystallin is glycated predominantly at the N-terminal ␣-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 ␣-and ⑀-amino groups of ␥B-crystallin in glycation-mediated cross-linking, Lys-2 or Lys-163, or both, were mutated to threonine by site-directed mutagenesis in bovine ␥B-crystallin cDNA. Wild type and mutant ␥B-crystallins were expressed in Escherichia coli cells. Cross-linking studies were performed by incubating wild type and mutant ␥B-crystallins with glyceraldehyde, ribose, and galactose followed by SDS-polyacrylamide gel electrophoresis under reducing conditions. When both of the lysines of ␥B-crystallin were mutated to threonines (␥B-K2T/K163T), the quantity of cross-linked products was greatly reduced, indicating that, despite the fact that the ␣-amino group is a major glycated site, ⑀-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 ␣-amino group. Our results also show that Lys-2 and Lys-163 play almost equal roles in cross-linking of ␥B-crystallin. By incubating carbonic anhydrase, a protein with a blocked N terminus, and our novel "no lysine" ␥B (␥B-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 ␥B-crystallins revealed the existence of Lys-Arg and Lys-Lys cross-linking.