Photo-Oxidative Damage to the Corneal Crystallin ALDH3A1: Structural and Functional Consequences of Direct UV Absorption

Abstract

Purpose: : Aldehyde dehydrogenase 3A1 (ALDH3A1), one of the most abundantly expressed proteins in the mammalian corneal epithelium, plays a critical and multifaceted roles in protecting the cornea from oxidative stress. For example, ALDH3A1 can protect other corneal enzymes such as glucse-6-phosphate dehydrogenase from UV-induced damage by directly absorbing the UV-light and by metabolizing toxic aldehydes produced by lipid peroxidation. The protective absorption of UV-light by ALDH3A1 is not without consequence, however, as ALDH3A1 becomes inactivated in the process. In the current study, we sought to characterize the effects of UV-light on the structure and function of ALDH3A1 with specific regard to the active site Cys residue.

Methods: : The impact of UV-exposure on the catalytic activity of ALDH3A1 was assessed by Michealis-Menten kinetics using benzaldehyde as the substrate. Aggregation of ALDH3A1 was monitored using SEC. Spectroscopic studies were performed using 2DUV, intrinsic fluorescence, bis-ANS binding fluorescence, and far UV CD. To detect and monitor UV-induced chemical modifications to the molecule, LysC peptide mapping was employed. .

Results: : The metabolism of benzaldehyde by ALDH3A1 followed Michaelis-Menten saturation kinetics. UV-inactivation led to a loss in the specific activity of the enzyme as well as a decrease in the V_max although the apparent K_m remained unchanged. UV-light caused soluble, non-native aggregation of ALDH3A1 due to both covalent and non-covalent interactions, which resulted in disruption of the secondary and tertiary structure of the protein. LysC peptide mapping revealed that UV-induced damage to ALDH3A1 also includes chemical modifications to Trp, Met, and Cys residues. Surprisingly, the conserved active site Cys of ALDH3A1 was not affected by UV-exposure.

Conclusions: : UVB-exposure leads to the covalent cross-linking and aggregation of ALDH3A1 molecules and partial unfolding of the protein. These structural changes, rather than the direct modification of the active site Cys residue, are the origin of UV-induced inactivation. The inactivation of ALDH3A1 by UV and other stresses may not render the cornea completely defenseless against toxic aldehydes, however, due to the abundant expression of this enzyme in the cornea as well as wide range in substrate specificity.

Keywords: crystallins • cornea: basic science • protein modifications-post translational