To determine the role of Gly-1 and Lys-2 of bovine B-crystallin in glycation and cross-linking, Lys-2 was changed to Thr by site-directed mutagenesis. A polymerase chain reaction was used to perform site-directed mutagenesis on the third codon (AAG ACG) of bovine B-crystallin cDNA. The wild type and mutant cDNAs were cloned into pMON5743 and expressed in JM101 Escherichia coli cells, and the identity of B-crystallin was confirmed by Western blotting after purification by cation exchange high performance liquid chromatography. Glycation of B-crystallin by [C]glucose was reduced significantly due to the mutation of Lys-2, supporting the view that Lys-2 is a major glycation site. Peptide mapping showed the presence of two major labeled peptides containing N-terminal sequences, and in the mutant these peptides had longer retention times and reduced radioactivity. Amino acid analysis, after glycation with [C]glucose, revealed N-terminal glycine as the most predominant glycation site. Lys-2 was glycated slower than Gly-1 but faster than Lys-163. Glycation with DL-glyceraldehyde showed an important role for both Gly-1 and Lys-2 in the glycation-mediated B-crystallin cross-linking.

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