Aldehyde Dehydrogenase 1B1 (ALDH1B1): Molecular Cloning and Characterization of A
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DMD Fast Forward. Published on July 8, 2010 as DOI: 10.1124/dmd.110.034678 DMDThis Fast article Forward. has not been Published copyedited and on formatted. July 8, The2010 final as version doi:10.1124/dmd.110.034678 may differ from this version. DMD #34678 Aldehyde dehydrogenase 1B1 (ALDH1B1): Molecular cloning and characterization of a novel mitochondrial acetaldehyde metabolizing enzyme Downloaded from Dimitrios Stagos †, Ying Chen †, Chad Brocker, Elizabeth Donald, Brian C. Jackson, David J. Orlicky, David C. Thompson, and Vasilis Vasiliou dmd.aspetjournals.org Department of Pharmaceutical Sciences (D.S., Y.C., C.B., E.D., B.J., V.V.), Department of at ASPET Journals on September 24, 2021 Pathology (D.O.), and Department of Clinical Pharmacy (D.T.), University of Colorado Denver, Aurora, CO 80045 1 Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on July 8, 2010 as DOI: 10.1124/dmd.110.034678 This article has not been copyedited and formatted. The final version may differ from this version. DMD #34678 RUNNING TITLE PAGE Running title: Initial characterization of human ALDH1B1 Corresponding author: Vasilis Vasiliou, PhD Department of Pharmaceutical Sciences University of Colorado Denver, Aurora, CO 80045, USA Downloaded from Phone: (303) 724-3520; fax: (303) 724-2666 e-mail: [email protected]. dmd.aspetjournals.org Number of text pages: 24 (including references) Number of tables: 1 Number of figures: 5 at ASPET Journals on September 24, 2021 Number of references: 40 Word count: Abstract: 243 Introduction: 701 (excluding citations) Di scussion: 1271 Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenases; RA, retinoic acid; 4-HNE, 4-Hydroxynonenal; M DA, Malondiadehyde; ORF, open reading frame; FPLC, fast protein liquid chromatography; Q- PCR, q uantitative r eal-time P CR; I HC, immunohistochemistry; IHF, immunoflurescence; DAPI, 4 ,6-diamidino-2-phenylindole; M OI, m ultiplicity o f infection; p-NPA, p-nitrophenyl acetate; GTN, glyceryl trinitrate; 2 DMD Fast Forward. Published on July 8, 2010 as DOI: 10.1124/dmd.110.034678 This article has not been copyedited and formatted. The final version may differ from this version. DMD #34678 ABSTRACT Ethanol-induced damage is largely attributed to its toxic metabolite, acetaldehyde. Clearance of acetaldehyde is achieved by its oxidation, primarily catalyzed by the mitochondrial class II aldehyde dehydrogenase (ALDH2). ALDH1B1 is another mitochondrial ALDH that shares 75% peptide sequence h omology w ith A LDH2. R ecent p opulation studies in C aucasians suggest a r ole f or ALDH1B1 in e thanol m etabolism. Ho wever, to da te, no f ormal Downloaded from documentation of t he biochemical pr operties of A LDH1B1 ha s been f orthcoming. I n th is current study, we cloned and expressed human recombinant ALDH1B1 in Sf9 insect cells. The resultant enzyme was purified by affinity chromatography to homogeneity. The kinetic dmd.aspetjournals.org properties o f pur ified h uman A LDH1B1 w ere a ssessed using a w ide range o f a ldehyde substrates. Human ALDH1B1 had an exclusive preference for NAD+ as the cofactor and was catalytically active towards short- and medium-chain aliphatic aldehydes, aromatic aldehydes at ASPET Journals on September 24, 2021 and the products of li pid pe roxidation, 4-hydroxy-nonenal ( 4-HNE) a nd malondialdehyde (MDA). M ost im portantly, hum an ALDH1B1 e xhibited an a pparent Km o f 55 μM f or acetaldehyde, making i t t he second low Km A LDH for m etabolism of t his su bstrate. The dehydrogenase activity of ALDH1B1 was sensitive to disulfiram inhibition, a feature a lso shared with ALDH2. The tissue distribution of ALDH1B1 in C57BL/6J mice and human was examined by Q- PCR, Western bl otting a nd im munohistochemical a nalysis. H ighest expression oc curred in th e li ver, f ollowed b y t he intestinal t ract, i mplicating a p otential physiological role of ALDH1B1 in these tissues. The current study is the first report on the expression, purification and biochemical characterization of human ALDH1B1 protein. 3 DMD Fast Forward. Published on July 8, 2010 as DOI: 10.1124/dmd.110.034678 This article has not been copyedited and formatted. The final version may differ from this version. DMD #34678 INTRODUCTION Excessive alcohol consumption leads to a variety of psychological and pathological consequences. Ch ronic alcohol i ntake i s often associated with damage t o mu ltiple o rgans, manifesting as hepatitis, liver cirrhosis, brain damage, and immune dysfunction. Many of the toxic effects of ethanol are attributable to ethanol metabolism. Ethanol is metabolized in the liver to acetaldehyde predominantly by class I alcohol dehydrogenase isoenzymes (ADH1A, Downloaded from ADH1B a nd ADH1C ) loc ated i n th e c ytosol o f h epatocytes. Ald ehyde d ehydrogenases (ALDHs) then oxidize acetaldehyde to acetate and water (Crabb et al., 2004). Acetaldehyde, the intermediate metabolite of ethanol, is toxic and its accumulation contributes significantly dmd.aspetjournals.org to ethanol-induced tissue damage (Eriksson, 2001; Meier and Seitz, 2008). The t oxicity o f acetaldehyde is caused by it covalently binding biological macromolecules, such as proteins and n ucleic acids, and f orming a dducts that impair t heir functions (N iemela, 2 007). at ASPET Journals on September 24, 2021 Acetaldehyde also induces lipid peroxidation, increased collagen synthesis and glutathione depletion (Esterbauer et al., 1991). Moreover, protein adducts with acetaldehyde and products of li pid peroxidation tr igger autoimmune responses th at a re associated wi th a lcoholic liver disease (Stewart et al ., 2 004; V iitala et al ., 2 000). Met abolism and el imination o f acetaldehyde is, therefore, of great importance for cellular d efense against ethanol-induced toxicities. Among the nineteen known human ALDHs, mitochondrial ALDH2 a nd, to a le sser extent, cytosolic ALDH1A1 have been shown to play a major role in acetaldehyde oxidation and elimination (Vasiliou and Pappa, 2000). Both enzymes, homotetramers with a 55 KDa subunit, exhibit low Km constants for acetaldehyde, viz. 3.2 and 180 μM for human ALDH2 and A LDH1A1, re spectively ( Klyosov et al ., 1 996). T he i mportance o f ALDH2 i n the clearance o f ac etaldehyde is exemplified i n humans car rying t he ALDH2*2 al lele, w hich 4 DMD Fast Forward. Published on July 8, 2010 as DOI: 10.1124/dmd.110.034678 This article has not been copyedited and formatted. The final version may differ from this version. DMD #34678 encodes a functional mutation (Glu487Lys) of ALDH2 and results in poor binding affinity to cofactor NAD+ and > 90% loss of enzyme activity (Larson et al., 2005; Yoshida et al., 1984). When compared t o w ild-type individuals, b lood acet aldehyde levels climb 2 0- an d 6-fold higher in homozygous a nd he terozygous in dividuals carrying t his p olymorphism, respectively, for equivalent levels of alcohol consumption (Crabb et al., 1989). This causes the “al cohol fl ushing” s yndrome c haracterized by the r eddening o f t he face, n eck, and in some cases the entire body due to dilation of capillaries (Eriksson, 2001). Similar elevations Downloaded from in acet aldehyde h ave been o bserved i n the Aldh2-null mice ( Yu e t al., 2009). The s econd enzyme involved in acetaldehyde metabolism is ALDH1A1. In addition to its crucial role in retinoic acid (RA) biosynthesis (Duester, 2000), cytosolic ALDH1A1 has been shown to be dmd.aspetjournals.org important in acetaldehyde metabolism and alcohol preference in rodent studies (Bond et al., 1991; B ond a nd S ingh, 1990; L ittle a nd P etersen, 1 983). H uman studies a lso r eport t he association of ALDH1A1 variants with enzyme deficiency in Caucasian “flushers” (Eriksson, at ASPET Journals on September 24, 2021 2001). Nevertheless, the contribution of ALDH1A1 to acetaldehyde metabolism appears to be less important in humans than rodent, likely because rodent ALDH1A1 has a much lower Km for acetaldehyde (15 μM) (Klyosov et al., 1996). ALDH1B1, p reviously known a s A LDHX a nd AL DH5, represents a nother mitochondrial AL DH i soenzyme. T he hum an ALDH1B1 ge ne loc ated on c hromosome 9 spans a 5,957 bp region and is the only known ALDH gene with an intronless coding region (Hsu and Chang, 1991). ALDH1B1 gene is composed of two exons and one intron and has only one known transcript variant (Black et al., 2009). ALDH1B1 enzyme, like ALDH2 and ALDH1A1, is predicted to be a homotetramer; the subunit contains 517 amino acids with a N-terminal 1 9-residue mi tochondrial l ead s ignal. Ba sed o n p eptide sequence al ignment, human A LDH1B1 shares 65% a nd 75 % ho mology with human A LDH1A1 a nd ALDH2, respectively. Northern blot analysis in human tissues have shown high levels of ALDH1B1 5 DMD Fast Forward. Published on July 8, 2010 as DOI: 10.1124/dmd.110.034678 This article has not been copyedited and formatted. The final version may differ from this version. DMD #34678 mRNA in the liver and testis and relatively low levels in other tissues (Hsu and Chang, 1991; Stewart et al., 1996). ALDH1B1 has also been found to be expressed abundantly in mouse and bovine corneas (Stagos et al., 2010), where cells are constantly exposed to UV-induced lipid p eroxidation. To date, li ttle is kn own r egarding th e biochemical properties a nd physiological roles of A LDH1B1. A s tudy u sing crude ly sate f rom HuH7 he patoma c ells reported that mitochondrial ALDH1B1 contributed to the oxidation of short chain aldehydes including acet aldehyde and propionaldehyde, i mplicating a ro le f or A LDH1B1 i n ethanol Downloaded from metabolism (Stewart et al., 1995).