Carnosine inhibits modifications and decreased molecularchaperone activity of lens α-crystallin induced by ribose andfructose 6-phosphate

摘要

Purpose: α-Crystallin, a major structural protein in the lens,prevents heat- and oxidative stress-induced aggregation of proteins andinactivation of enzymes by acting as a molecular chaperone. Modificationof α-crystallin by some posttranslational modifications results inconformational changes and decreases in chaperone activity, which maycontribute to cataractogenesis in vivo. Carnosine(β-alanyl-L-histidine), an endogenous histidine dipeptide, preventsprotein modifications including glycation and oxidation. The purpose ofthis study was to further explore whether carnosine can protectα-crystallin against glycation by a sugar and a sugar phosphate,and in particular to find whether it can protect against its decreasedchaperone activity. Additionally, we investigated whether carnosinecould directly react with a sugar and a sugar phosphate.Methods: Bovine lens αL-crystallin was separated bysize-exclusion chromatography on a Sephacryl S-300 HR column. αL-crystallin was incubated with different concentrations of fructose6-phosphate (F6P) and ribose with or without carnosine for differenttimes. The chaperone activity of αL-crystallin was monitored usingthe prevention of thermal aggregation of βL-crystallin. Themodified αL-crystallin was examined by SDS-PAGE and fluorescencemeasurements. The absorbance spectra of solutions of carnosine andsugars were investigated.Results: Carnosine inhibited the crosslinking of αL-crystallininduced by F6P and ribose in a dose- and time-dependent manner. Itprotected αL-crystallin against its decreased chaperone activityinduced by 100 mM F6P during four days incubation, but not againstribose-induced change. Control αL-crystallin gave 96% protectionagainst aggregation of βL-crystallin after four days incubation,but only 85% protection was achieved in the presence of F6P, rising to96% (p=0.0004) in the presence of carnosine. After more extensivemodification by sugar and a sugar phosphate, there was no significantprotective effect of carnosine on αL-crystallin cross-linking orchaperone activity. The tryptophan fluorescence of modified αL-crystallin was remarkably decreased in the presence of F6P and ribose.However, the decrease was less when 50 mM carnosine was present duringeight days incubation with F6P. Carnosine did not maintain thefluorescence when ribose was used. The nontryptophan fluorescence wasincreased with a shift to longer wavelengths in a time-dependent manner.Carnosine readily reacted with F6P and ribose thereby inhibitingglycation-mediated protein modification as revealed electrophoretically.The increased absorbance was time-dependent, suggesting adducts may beformed between F6P, ribose, and carnosine.Conclusions: This is the first report showing that carnosine canprotect the chaperone activity of α-crystallin. This chaperone mayprotect against cataractous changes. In addition to demonstrating theeffects of carnosine on prevention crosslinking, our studies also bringout important evidence that carnosine reacts with F6P and ribose, whichsuggests carnosine's potential as a possible nontoxic modulator ofdiabetic complications.