Reversal of chaperone activity loss of glycated alphaA-crystallin by a crosslink breaker.
The chaperone function of alpha-crystallin is significantly affected in diabetes. Increased formation of advanced glycation end products (AGEs) is the likely cause. This study was aimed to investigate the effect of AGE crosslinks on the chaperone activity of alpha-crystallin and to show the effect of an AGEs crosslink breaker, phenacyl-4,5-dimethylthiazolium bromide (DMPTB). Recombinant alphaA-crystallin was prepared by expressing it in Escherichia coli and purified by size exclusion chromatography. Glycation of alphaA-crystallin was performed with 1-100 mM glucose-6-phosphate (G6P) as the glycating agent for a period of 1-15 days. To break AGE crosslinks, pre-glycated alphaA-crystallin was treated with 0.1-20 mM DMPTB for 3 days. Excess G6P and DMPTB were removed by gel filtration before performing additional experiments. AGEs and crosslinked proteins were estimated by measuring non-tryptophan fluorescence and by SDS-PAGE. Chaperone activity was determined with alcohol dehydrogenase as the target protein. With increasing duration of glycation and G6P concentration, chaperone activity of alpha-crystallin decreased. When pre-glycated alphaA-crystallin was treated with 5-20 mM DMPTB, a DMPTB concentration-dependent recovery of chaperone activity was seen. Lower concentrations, 0.1, 0.5, and 1.0 mM, of DMPTB also showed significant recovery of the chaperone activity. SDS-PAGE analysis after DMPTB treatment showed 40% decrease in crosslinked proteins and fluorescence scan indicated 30% decrease in AGEs. DMPTB is expected to regain alpha-crystallin chaperone activity and provide structural stability to other eye lens proteins that are in aggregation mode which emphasizes the clinical importance of the present finding.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Poppy
P
Datta
Latha
L
Kallur
Edathara C
EC
Abraham
D003432:Cross-Linking Reagents / Q000494:pharmacology*; D019298:Glucose-6-Phosphate / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014364:Tryptophan / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000378:metabolism*
D003432:Cross-Linking Reagents; D017127:Glycation End Products, Advanced; C477945:N-phenacyl-4,5-dimethylthiazolium bromide; D013844:Thiazoles; D038202:alpha-Crystallin A Chain; D019298:Glucose-6-Phosphate; D014364:Tryptophan