Reversal of chaperone activity loss of glycated alphaA-crystallin by a crosslink breaker.

The chaperone function of alpha-crystallin is significantly affected in diabetes. Increased formation of advanced glycation end products (AGEs) is the likely cause. This study was aimed to investigate the effect of AGE crosslinks on the chaperone activity of alpha-crystallin and to show the effect of an AGEs crosslink breaker, phenacyl-4,5-dimethylthiazolium bromide (DMPTB). Recombinant alphaA-crystallin was prepared by expressing it in Escherichia coli and purified by size exclusion chromatography. Glycation of alphaA-crystallin was performed with 1-100 mM glucose-6-phosphate (G6P) as the glycating agent for a period of 1-15 days. To break AGE crosslinks, pre-glycated alphaA-crystallin was treated with 0.1-20 mM DMPTB for 3 days. Excess G6P and DMPTB were removed by gel filtration before performing additional experiments. AGEs and crosslinked proteins were estimated by measuring non-tryptophan fluorescence and by SDS-PAGE. Chaperone activity was determined with alcohol dehydrogenase as the target protein. With increasing duration of glycation and G6P concentration, chaperone activity of alpha-crystallin decreased. When pre-glycated alphaA-crystallin was treated with 5-20 mM DMPTB, a DMPTB concentration-dependent recovery of chaperone activity was seen. Lower concentrations, 0.1, 0.5, and 1.0 mM, of DMPTB also showed significant recovery of the chaperone activity. SDS-PAGE analysis after DMPTB treatment showed 40% decrease in crosslinked proteins and fluorescence scan indicated 30% decrease in AGEs. DMPTB is expected to regain alpha-crystallin chaperone activity and provide structural stability to other eye lens proteins that are in aggregation mode which emphasizes the clinical importance of the present finding.

C-Terminal truncation affects subunit exchange of human alphaA-crystallin with alphaB-crystallin.

In human lenses, C-terminal cleavage of alphaA-crystallin at residues 172,168, and 162 have been reported. The effect of C-terminal truncation of alphaA-crystallin on subunit exchange and heterooligomer formation with alphaB-crystallin and homooligomer formation with native alphaA-crystallin is not known. We have conducted fluorescence resonance energy transfer studies which have shown that the rates of subunit exchange of alphaA(1-172 )and alphaA(1-168 )with alphaB-wt were two-fold lower than for alphaA-wt interacting with alphaB-wt. The subunit exchange rate between alphaA(1-162) and alphaB-wt was six-fold lower. These data suggest that cleavage of the C-terminal residues could significantly affect heterooligomerization. On the other hand, the subunit exchange rates between alphaA-wt and the truncated alphaA-crystallins were either unchanged or only slightly decreased, which suggest that homooligomerization may not be significantly influenced by C-terminal truncation. The main conclusion from this study is that cleavage of C-terminal residues of alphaA-crystallin including the nine residues of the flexible tail is expected to significantly affect the formation of heteroaggregates. Reconstitution experiments showed that the presence of an intact C-terminus is essential for the formation of fully integrated heteroaggregates with equal proportion of alphaA and alphaB subunits.