Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of alpha A- and alpha B-crystallins.
Earlier studies have shown significant loss of chaperone activity in alpha-crystallin from diabetic lenses. In vitro glycation studies have suggested that glycation of alpha-crystallin could be the major cause of chaperone activity loss. The following lysine (K) residues in alpha-crystallin have been identified as the major glycation sites: K11, K78, and K166 in alpha A-crystallin and K90, K92, and K166 in alpha B-crystallin. The present study was aimed to assess the contribution of each of the above glycation site in the overall glycation and loss of chaperone activity by mutating them to threonine followed by in vitro glycation with fructose. Level of glycated protein (GP) was determined by phenylboronate affinity chromatography, advanced glycation end products (AGEs) by direct ELISA using anti-AGE polyclonal antibody, and chaperone activity by using alcohol dehydrogenase as the target protein. K11T, K78, and K166T mutants of alpha A showed 33, 17, and 27% decrease in GP and 32, 18, and 21% decrease in AGEs, respectively, as compared to alpha A-wt. Likewise, K90T, K92T, K90T/K92T, and K166T mutants of alpha B showed 18, 21, 29, and 12% decrease in GP and 22, 24, 32, and 16% decrease in AGEs, respectively. Chaperone activity also showed concomitant increase with decreasing glycation and AGEs formation. alpha A-K11T and alpha B-K90T/K92T mutants showed the largest decrease in glycation and increase in chaperone activity.
D000818:Animals; D005632:Fructose / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006031:Glycosylation; D008239:Lysine / Q000235:genetics*; D018832:Molecular Chaperones / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D051381:Rats; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism*
D017127:Glycation End Products, Advanced; D018832:Molecular Chaperones; D050505:Mutant Proteins; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D013912:Threonine; D005632:Fructose; D008239:Lysine