Carnosine inhibits modifications and decreased molecular chaperone activity of lens alpha-crystallin induced by ribose and fructose 6-phosphate.
OBJECTIVE
Alpha-crystallin, a major structural protein in the lens, prevents heat- and oxidative stress-induced aggregation of proteins and inactivation of enzymes by acting as a molecular chaperone. Modification of alpha-crystallin by some posttranslational modifications results in conformational changes and decreases in chaperone activity, which may contribute to cataractogenesis in vivo. Carnosine (beta-alanyl-L-histidine), an endogenous histidine dipeptide, prevents protein modifications including glycation and oxidation. The purpose of this study was to further explore whether carnosine can protect alpha-crystallin against glycation by a sugar and a sugar phosphate, and in particular to find whether it can protect against its decreased chaperone activity. Additionally, we investigated whether carnosine could directly react with a sugar and a sugar phosphate.


METHODS
Bovine lens alphaL-crystallin was separated by size-exclusion chromatography on a Sephacryl S-300 HR column. alphaL-crystallin was incubated with different concentrations of fructose 6-phosphate (F6P) and ribose with or without carnosine for different times. The chaperone activity of alphaL-crystallin was monitored using the prevention of thermal aggregation of betaL-crystallin. The modified alphaL-crystallin was examined by SDS-PAGE and fluorescence measurements. The absorbance spectra of solutions of carnosine and sugars were investigated.


RESULTS
Carnosine inhibited the crosslinking of alphaL-crystallin induced by F6P and ribose in a dose- and time-dependent manner. It protected alphaL-crystallin against its decreased chaperone activity induced by 100 mM F6P during four days incubation, but not against ribose-induced change. Control alphaL-crystallin gave 96% protection against aggregation of betaL-crystallin after four days incubation, but only 85% protection was achieved in the presence of F6P, rising to 96% (p=0.0004) in the presence of carnosine. After more extensive modification by sugar and a sugar phosphate, there was no significant protective effect of carnosine on alphaL-crystallin cross-linking or chaperone activity. The tryptophan fluorescence of modified alphaL-crystallin was remarkably decreased in the presence of F6P and ribose. However, the decrease was less when 50 mM carnosine was present during eight days incubation with F6P. Carnosine did not maintain the fluorescence when ribose was used. The nontryptophan fluorescence was increased with a shift to longer wavelengths in a time-dependent manner. Carnosine readily reacted with F6P and ribose thereby inhibiting glycation-mediated protein modification as revealed electrophoretically. The increased absorbance was time-dependent, suggesting adducts may be formed between F6P, ribose, and carnosine.


CONCLUSIONS
This is the first report showing that carnosine can protect the chaperone activity of alpha-crystallin. This chaperone may protect against cataractous changes. In addition to demonstrating the effects of carnosine on prevention crosslinking, our studies also bring out important evidence that carnosine reacts with F6P and ribose, which suggests carnosine's potential as a possible nontoxic modulator of diabetic complications.
Yan
Hong
H
Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK. yhongb@fmmu.edu.cn
Harding
John J
JJ
D000818:Animals; D002336:Carnosine / Q000494:pharmacology*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005453:Fluorescence; D005636:Fructosephosphates / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000378:metabolism*; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012266:Ribose / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D038201:alpha-Crystallins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003432:Cross-Linking Reagents; D005636:Fructosephosphates; D018832:Molecular Chaperones; D038201:alpha-Crystallins; C027618:fructose-6-phosphate; D012266:Ribose; D002336:Carnosine