Abstract
Purpose: :
Lipid peroxidation products such as 4–hydroxynonenal (HNE) have been shown by us to be deleterious to the lens if not detoxified. We have demonstrated that the NAD–dependent isozyme, aldehyde dehydrogenase 1 (ALDH1A1) is responsible for the oxidation of HNE in rat lens and human lens epithelial cells (HLEC). Ablation of ALDH1A1 by inhibitors, antisense or siRNA decreased HNE oxidation with a concomitant increase in oxidation–induced protein–HNE adducts, opacification of the lens and increased apoptosis of HLEC suggesting that activation of ALDH1A1 should ameliorate /prevent cataract formation. We have therefore cloned, expressed and purified lens ALDH1A1 and have studied its activation by an estrogen analogue, diethylstilbestrol (DES). Furthermore, we have attempted to crystallize the ALDH1A1–NAD–DES complex with the intention of designing potent, safe and specific activators of ALDH1 which will have a potential to prevent oxidation–induced cataractogenesis.
Methods: :
Human lens cDNA library was amplified to subclone ALDH1A1 . The PCR product of ALDH1A1 (1.5kb) was subcloned into E. Coli expression system (pET 28b). Expressed ALDH1A1 protein was purified by His–Tag column and its His–Tag was removed with thrombin. Purified enzyme was eluted for the kinetic parameters using various aldehyde substrates. Activation of the enzyme was studied using DES in the presence of NAD. For crystallization trials, ALDH1A1 was further purified using a superdex–75 size exclusion column. Concentrated enzyme solution (6–8mg/ml) was subjected to crystallization, in the presence of NAD and DES, using the hanging–drop vapor diffusion technique.
Results: :
Recombinant lens ALDH1A1 was found to be composed of 1506 bp and 501 amino acids with a molecular weight of 54.8 kD. Km for acetaldehyde, benzaldehyde, HNE, malonaldehyde ( MDA) and propionaldehyde was 256, 152 , 4.8, 3.5 and 119.5 uM , respectively. The catalytic efficiency of the enzyme towards HNE and MDA was very high. DES activated the enzyme 2–3 fold as evaluated by the enzyme activity using various substrates. Crystallization trials carried out using Hampton research crystal screen kits have resulted in small crystals. Optimization of the crystallization condition is in progress.
Conclusions: :
The enzyme has remarkably low Km for HNE and MDA supporting ALDH1A1 to be an essential enzyme in the maintenance of lens transparency under condition of oxidation stress. It is important that we design specific activators of ALDH1A1 which will have a potential to prevent oxidation–induced cataractogenesis.
Keywords: oxidation/oxidative or free radical damage • enzymes/enzyme inhibitors • cataract