Role of glycine 1 and lysine 2 in the glycation of bovine gamma B-crystallin. Site-directed mutagenesis of lysine to threonine.
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.
Casey
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EB
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Zhao
H R
HR
Abraham
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EC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D003459:Crystallins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D005947:Glucose / Q000378:metabolism; D005998:Glycine; D006031:Glycosylation; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013912:Threonine
D003459:Crystallins; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013912:Threonine; D005947:Glucose; D008239:Lysine; D005998:Glycine