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EC Abraham, S Hollenbach, P Thampi, T Viswanathan; Reversal Of Lens Protein Crosslinking And Aggregation By A Novel Crosslink Breaker . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2387.
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Purpose: N-Phenacyl-4,5-dimethylthiazolium bromide (DMPTB), a known advanced glycation end product (AGE) crosslink breaker,has been shown to cleave tail tendon collagen crosslinks and reverse the diabetes induced stiffness of large artery. The purpose of this study is to investigate the effect of DMPTB on the formation of AGE crosslinks in vitro and on its ability to break or degrade the protein crosslinks and high molecular weight (HMW) aggrgates formed in vivo in senile/diabetic human lenses. Methods: DMPTB was synthesized and its structure confirmed by determining its NMR spectrum. To study the effect of DMPTB on the synthesis of AGE crosslinks, recombinant human alphaA-crystallin was glycated in vitro with glucose-6-phosphate in the presence and absence of DMPTB. To show whether DMPTB can break the crosslinks, pre-glycated alphaA-crystallin was treated with DMPTB. To show whether DMPTB can degrade crosslinks or HMW aggregates formed in vivo in human lenses, water-insoluble proteins from two diabetic human lenses were solubilized by sonication with 6 M guanidine hydrochloride and 1 mM DTT and then treated with DMPTB. Proteins were separated by SDS-PAGE followed by densitometry, by molecular sieve HPLC, and by reverse-phase HPLC. Results: In the absence of DMPTB, glycatyion with G6P showed the presence of several HMW bands on the SDS-PAGE gel; DMPTB significantly inhibited the formation of these HMW proteins. This was cofirmed by molecular sieve HPLC. Treatment of pre-glycated alphaA-crystallin with DMPTB showed evidence for breakdown of already formed crosslinks and HMW aggregates. Both molecular sieve HPLC and reverse-phase HPLC of human lens protein showed that DMPTB could degrade a major portion of the covalently linked (non-disulfide linked) HMW aggregastes to lower molecular weight proteins. Conclusions: The fact that a known AGE crosslink breaker could degrade a significant portion of the crosslinked HMW aggregates in human lenses suggest that they are formed predominantly by the AGE mechanism. Moreover, the data support the thinking that this or other AGE crosslink breakers may have therapautic application for the intervention of age and diabetes dependent crosslinking and agggregation of human lens protein.
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