Abstract
To determine the role of Gly-1 and Lys-2 of bovine gamma 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 gamma B-crystallin cDNA. The wild type and mutant cDNAs were cloned into pMON5743 and expressed in JM101 Escherichia coli cells, and the identity of gamma B-crystallin was confirmed by Western blotting after purification by cation exchange high performance liquid chromatography. Glycation of gamma B-crystallin by [14C]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 [14C]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 gamma B-crystallin cross-linking.
Highlights
To determine the role of Gly-l and Lys-2 of bovine yB-crystallin in glycation and cross-linking, Lys-2 was changed to Thr by site-directed mutagenesis
Glycation alters the conformation of crystallins [10, 11], and this may increase their susceptibility of sulfhydryl oxidation, resulting in the formation of high molecular weight protein aggregates [12,13,14]
Cloning and Mutagenesis-After cloning the wild type and the mutant cDNA into pMON5743 the plasmids were double digested with Ncol and HindUI and run on 1% agarose gel to confirm that the cDNA insert is present
Summary
(Received for publication, February 21, 1995, and in revised form, June 14, 1995). From the Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100. To determine the role of Gly-l and Lys-2 of bovine yB-crystallin in glycation and cross-linking, Lys-2 was changed to Thr by site-directed mutagenesis. The presence of early stage glycation products (Schiff-base adducts and Amadori products) has been demonstrated to form on lens crystallins in vitro and in vivo [6, 7]. The complete sequence of calf yB-crystallin, the major species in the bovine lens, shows that there are two lysine residues. Glycation of yB-crystallin can occur at one or more of the following three sites: the a-amino group of the N-terminal glycine and the e-amino groups of the two lysines (Lys-2 and Lys-163). An earlier study has indicted that glycation of v-crystallin occurs primarily at the N-terminal residues [21] It remains unclear whether glycation of Gly-1, Lys-2, or both are involved and to what extent
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