Abstract
Abstract: :
Purpose: We have shown earlier that ablation of ALDH1A1 in the human lens epithelial cell line (HLEC) and rat lens resulted in increased susceptibility of these tissues to oxidation–induced toxicity. Furthermore, such ALDH1A1– inhibited tissues displayed a decrease in oxidation of the lipid peroxidation product, 4–hydroxynonenal (HNE), with no compensatory increase in the other HNE detoxification pathways suggesting a crucial role of ALDH1A1 in maintaining the lens transparency under oxidative stress. In order to better understand lens ALDH1A1, we have cloned, expressed and characterized this enzyme. Methods: Human lens cDNA library (UI–E–DWO) cloned in pT7T3–pac with a modified polylinked site was obtained from NCBI genebank. The cDNA library was amplified to subclone ALDH1A1, ALDH2A1, and ALDH3A1 isozymes by using standard PCR–strategy. The PCR product of ALDH1A1(1.5kb) was subcloned into E. coli expression system(pET28b). Expressed ALDH1A1 protein was purified by His–Tag Column and its His Tag were removed with thrombin. Human liver ALDH1A1 polyclonal antibodies (obtained from Dr. Henry Weiner, Purdue University) were used for Western blots. Various substrates such as acetaldehyde, HNE , benzaldehyde, propionaldehyde, malonaldehyde, etc. were tested for their Km and Vmax. Inhibitors such as citral , cyanamide, disulfiram, as well as activators such as diethylstilbestrol (DES) were tested for their efficacy to inhibit or activate ALDH1A1, respectively. Results: From human lens cDNA library, only ALDH1A1 was amplified; no transcripts were found for ALDH2A1 and ALDH3A1. Recombinant ALDH1A1 is composed of 1506 bp and 501 amino acids with a molecular weight of 54.8 KD and PI of 6.8. Human lens ALDH1A1 has 85 % homology with rat liver ALDH1A1 and 81% with mouse liver ALDH1A1. Purified ALDH1A1 has optimum activity at pH 8.0, and prefers NAD as its cofactor, with a low km for HNE ( 2.5 uM) and high Km for malonaldehyde (2459 uM). Km for propionaldehyde and acetaldehyde was found to be 50 and 490 uM , respectively. IC50 for citral, disualfiram and cyannamide were found to be 40 , 400 and 22610 uM, respectively, and AC50 for DES 11.4 uM . Conclusions: ALDH1A1 is the major ALDH isozyme in human lens and displays essentially similar substrate specificity as described for those obtained from other species and tissues. However, we speculate that due to the remarkably low Km of lens ALDH1A1 towards HNE, this enzyme has a crucial role to play in the detoxification of HNE and thus lens clarity.
Keywords: cataract • oxidation/oxidative or free radical damage • aging