Cleavage of in vitro and in vivo formed lens protein cross-links by a novel cross-link breaker.
The purpose of this study was to investigate the effect of N-phenacyl-4,5-dimethylthiazolium bromide (DMPTB), an advanced glycation end product (AGE) cross-link breaker, on lens protein cross-links formed in vitro and in vivo. DMPTB was synthesized and its structure confirmed by its NMR spectrum. To show whether DMPTB can inhibit AGE cross-linking, recombinant human alphaA-crystallin was glycated with glucose-6-phosphate (G6P) in the presence and absence of DMPTB. Reversal of the already formed cross-links was studied by treating pre-glycated alphaA-crystallin with DMPTB. The ability of DMPTB to cleave in vivo formed cross-links was ascertained by treating water-insoluble protein fractions from diabetic human lenses with this compound. Glycation of alphaA-crystallin with G6P showed several high molecular weight (HMW) protein bands on the SDS-PAGE gel; DMPTB inhibited the formation of these HMW proteins. Molecular sieve HPLC confirmed the inhibition of formation of larger aggregates not separated by SDS-PAGE. Treatment of pre-glycated alphaA-crystallin with DMPTB gave evidence for the degradation of the already formed cross-linked HMW aggregates. Both molecular sieve HPLC and reverse-phase HPLC of the water-insoluble protein fractions from two diabetic human lenses showed that DMPTB could degrade a major portion of the cross-linked HMW aggregates to lower molecular weight proteins. This suggests that the cross-linked proteins in human lenses are formed predominantly by the advanced glycation process and cross-link breakers like DMPTB may have application for the intervention of protein cross-linking in the eye lens.
Hollenbach
Seth
S
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Thampi
Prajitha
P
Viswanathan
Tito
T
Abraham
Edathara C
EC
D002851:Chromatography, High Pressure Liquid; D003720:Densitometry; D003920:Diabetes Mellitus / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019298:Glucose-6-Phosphate / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000187:drug effects*; D008875:Middle Aged; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology*; D013997:Time Factors; D038202:alpha-Crystallin A Chain / Q000737:chemistry
D017127:Glycation End Products, Advanced; C477945:N-phenacyl-4,5-dimethylthiazolium bromide; D011994:Recombinant Proteins; D013844:Thiazoles; D038202:alpha-Crystallin A Chain; D019298:Glucose-6-Phosphate