Construction Of A Model For Human Aldose Reductase Functional Expression In Saccharomyces Cerevisiae: Application For The Evaluation Of ARI On AR Activity

Aldose reductase (AR) is the rate-limiting enzyme in the polyol pathway (PP) which is widely expressed in vivo. PP has littile activity in the metabolism of glucose under normal conditions. However, AR and PP can be successively activated by hyperglycemia under diabetic conditions, and glucose were reduced into large amount of sorbitol. Osmotic stress resulted by sorbitol accumulation have been implicated to the development of diabetic chronic complication (DCC), while the mechanism still remains unclear. Since the PP hypothesis has been accepted generally, a great number of studies have been carried out in blocking PP by aldose reductase inhibitor (ARI) during the treatment of DCC. However, the clinical effects were unsatisfied. In the current study, a chimeric DNA fragment that encodes AR and green fluorescent protein (GFP) was introduced into the Saccharomyces cerevisia cells to construct an expression model. The functional expression of AR was demonstrated by the fluorescent expression of GFP. The inhibitory action of ARI was also preliminarily studied in this model.This study included three parts: (1) Construction of a yeast model of AR expression: The GFP gene was introduced at the 3' end of the AR cDNA to form recombination gene of AR::GFP. The fragments of AR and AR::GFP were then inserted respectively into the yeast expression vector pYEX-BX, transformed into the yeast strain INVSc1, and formed the strains of XAR and XAG The YEX strain formed by pYEX-BX was used as the normal control. (2) Analysis of the expression and activity of AR in yeast cells: There were no significantdifferences in the growth curves induced by CUSO4 within 10 hours among the three strains. The relative fluorescence (RF) of the XAG correlated with the time. Northern Blotting showed that the expressions of AR and AR..GFP mRNA were sustained and steady in the XAR and XAG strains. Western Blotting showed that proteins of AR and AR::GFP expressed in two strains respectively. Both of the strains were proved to have the AR activity checked by the fluorescent method. (3) The influences of ARI on the expression and activity of AR: Two classical ARIs, Sorbinil and Zopolrestat, were used in the present research. The total proteins were extracted from XAR and XAG strains after 6 hours' induction. AR activities were inhibited by both ARIs. When the ARIs were added into the culture medium of two strains directly, the GFP fluorescent signal and protein level of AR was not inhibited. However, a relatively lower activity of AR was observed in Zopolrestat-treated group but not in Sorbinil group.In conclusion, we transformed AR cDNA and ARr.GFP chimaera into yeast strain INVScl, constructed the expression model of aldose reductase, and observed GFP fluorescence and AR activity in the model. The inhibition effect of the ARIs on AR activity was also preliminarily studied. This model may be used in the research on the verification of the PP hypothesis and ARI preliminary screening.