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Cleavage of in vitro and in vivo formed lens protein cross-links by a novel cross-link breaker

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Abstract

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 αA-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 αA-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 αA-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 αA-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.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA

    Seth Hollenbach, Prajitha Thampi & Edathara C. Abraham

  2. Department of Chemistry, University of Arkansas at Little Rock, Little Rock, AR, 72204, USA

    Tito Viswanathan

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  1. Seth Hollenbach
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  2. Prajitha Thampi
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  3. Tito Viswanathan
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  4. Edathara C. Abraham
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Hollenbach, S., Thampi, P., Viswanathan, T. et al. Cleavage of in vitro and in vivo formed lens protein cross-links by a novel cross-link breaker. Mol Cell Biochem 243, 73–80 (2003). https://doi.org/10.1023/A:1021660105893

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