Molecular Cloning, Baculovirus Expression, and Tissue Distribution of the Zebrafish Aldehyde Dehydrogenase 2

Abstract

ABSTRACT:Ethanol is metabolized to acetaldehyde mainly by the alcohol dehydrogenase pathway and, to a lesser extent, through microsomal oxidation (CYP2E1) and the catalase-H 2 O 2 system. Acetaldehyde, which is responsible for some of the deleterious effects of ethanol, is further oxidized to acetic acid by aldehyde dehydrogenases (ALDHs), of which mitochondrial ALDH2 is the most efficient. The aim of this study was to evaluate zebrafish (Danio rerio) as a model for ethanol metabolism by cloning, expressing, and characterizing the zebrafish ALDH2. The zebrafish ALDH2 cDNA was cloned and found to be 1892 bp in length and encoding a protein of 516 amino acids (M r ‫؍‬ 56,562), approximately 75% identical to mammalian ALDH2 proteins. Recombinant zebrafish ALDH2 protein was expressed using the baculovirus expression system and purified to homogeneity by affinity chromatography. We found that zebrafish ALDH2 is catalytically active and efficiently oxidizes acetaldehyde (K m ‫؍‬ 11.5 M) and propionaldehyde (K m ‫؍‬ 6.1 M). Similar kinetic properties were observed with the recombinant human ALDH2 protein, which was expressed and purified using comparable experimental conditions. Western blot analysis revealed that ALDH2 is highly expressed in the heart, skeletal muscle, and brain with moderate expression in liver, eye, and swim bladder of the zebrafish. These results are the first reported on the cloning, expression, and characterization of a zebrafish ALDH, and indicate that zebrafish is a suitable model for studying ethanol metabolism and, therefore, toxicity.

Aldehyde Dehydrogenase 1B1: Molecular Cloning and Characterization of a Novel Mitochondrial Acetaldehyde-Metabolizing Enzyme

“…Our previous study demonstrated that recombinant mitochondrial ALDH2 enzyme is prone to inactivation, probably because of the oxidation of sulfhydryl residues that are essential for the catalytic activity of ALDHs (Lassen et al, 2005). Because ALDH1B1 shows Ϸ75% peptide sequence identity with ALDH2, we expected the same characteristic being shared by recombinant ALDH1B1.…”

“…To minimize the cross-reactivity toward ALDH2 protein, the antibody was preabsorbed by a ALDH2-bound column. Primary antibodies were used at dilutions of 1:5000 for anti-ALDH1B1, 1:1000 for polyclonal anti-ALDH2 (Lassen et al, 2005), 1:5000 for mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (Ambion, Austin, TX), and 1:10,000 for mouse monoclonal anti-␤-actin (Sigma-Aldrich, St. Louis, MO). Blocking reagents and corresponding secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE) or Calbiochem (San Diego, CA) and used according to the manufacturer's protocol.…”

Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (Danio rerio)

“…Furthermore, the basic layout of their brain is not fundamentally different from the typical vertebrate brain (Tropepe & Sive, 2003). Given the DNA sequence homologies found between zebrafish genes and other vertebrate genes including those of mammals (for ethanol metabolism related genes see, for example, Lassen et al, 2005;Reimers et al, 2004) and given the anatomical similarities among vertebrate brains, it has been argued that mechanisms of complex functional properties of the mammalian brain may be modeled and investigated using this species (e.g., Gerlai, 2003;Tropepe & Sive, 2003). Furthermore, it has also been argued (see e.g., Blaser & Gerlai, 2006) that behavioral analysis is perhaps the best and most objective method with which one can study the function of the brain and thus discover mutation or drug induced changes in this organ.…”

Molecular cloning and expression of a second zebrafish aldehyde dehydrogenase 2 gene (aldh2b)

“…Recently, Lassen et al (2005) reported molecular cloning of a zebrafish aldh2 gene and demonstrated that the zebrafish aldh2 had the similar activity to human aldh2 in metabolizing acetaldehyde and propionaldehyde. In this paper, we report that zebrafish has a second aldh2 gene (aldh2b).…”