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.
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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.
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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.
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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). In agreement with this report, two recent large population- based studies identified two ALDH1B1 variants that were associated with drinking behavior
(Ala69Val) and alcohol-induced hypersensitivity (Ala86Val) in Caucasians (Husemoen et al., dmd.aspetjournals.org
2008; Linneberg et al., 2010). These findings strongly suggest that ALDH1B1 enzyme may be i nvolved in ethanol de toxification a nd modifications i n t his enzyme may c ontribute to
alcohol-related diseases. To e xpand our c urrent kn owledge o n the c atalytic p roperties of at ASPET Journals on September 24, 2021
ALDH1B1, we c loned a nd pu rified hu man recombinant AL DH1B1. Dif ferent aldehydes, including acet aldehyde, were t ested as s ubstrates. In addition, w e surveyed the expression profile of ALDH1B1 mRNA and protein in multiple mouse tissues. This is the first report on the expression and biochemical characterization of human ALDH1B1 enzyme.
6 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.
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MATERIALS AND METHODS
Chemicals. 4-Hydroxynonenal ( 4-HNE) wa s obt ained f rom C ayman C hemical C ompany
(Ann Ha rbor, MI, US A). M alondiadehyde (MDA) was synthesized according to a method described previously (E sterbauer et al ., 1 991). A ll other chemicals a nd reagents w ere purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified,
Downloaded from Animals. Male C5 7BL/6J w ild-type mi ce ( ≈ 12-wk old ) were pu rchased f rom J ackson
Laboratory ( Bar H arbor, M aine). Aldh2(-/-) knockout m ice we re o btained f rom Dr .
Kawamoto’s laboratory (Yu et al., 2009) and maintained in the C57BL/6J background. Mice dmd.aspetjournals.org were euthanized by CO2 inhalation followed by cervical dislocation. All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) at the
University of Colorado Denver. at ASPET Journals on September 24, 2021
Construction, expression and purification of recombinant human ALDH1B1. The pCMV-
XL4-ALDH1B1 plasmid h arboring the full length o f hum an AL DH1B1 c DNA
(NM_000692.3) wa s pu rchased from Or iGene (Rockville, M D, US A). The O pen Reading
Frame ( ORF) of ALDH1B1 was amplified by polymerase chain reaction (PCR) using the primer pa ir 5’-CAAGGTACCTACAGGAAAGCCCACCATGCTGCGCTTCCTGGCA-3’
(forward) an d 5’–GTGAAGCTTTTACGAGTTCTTCTGAGGAACCTTGATGGTG–3’
(reverse). The forward primer was designed to contain a KpnI site (GGTACC) for subcloning and a sequence motif (CCACC) 5’-ward of the start codon (ATG) to ensure correct initiation of tr anslation in e ukaryotic cells ( Ding a nd Na m O ng, 2003). The r everse primer wa s designed to contain a HindIII site (AAGCTT) 3’-ward of the stop codon (TAA). The 1.6-kb
PCR product was then digested with KpnI and HindIII and subcloned into the pBlueBac4.5
7 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 expression ve ctor ( Invitrogen, Carlsbad, C A). T he correct sequence of ins erted OR F of
ALDH1B1 was confirmed by DNA sequencing analysis.
The pBlueBac4.5-ALDH1B1 plasmid was used to generate recombinant baculoviruse, which we re pl aque-purified and amplified i n S f9 in sect cells by the T issue Culture Core
Facility at the University of Colorado Denver as described previously (Manzer et al., 2003).
Approximately 6x 108 Sf9 c ells w ere h arvested 48 h r post-infection by c entrifugation at
1,000x g for 5 min and washed with phosphate-buffered saline (PBS), pH 7.4. Cell pellets Downloaded from were r esuspended in l ysis b uffer ( 100 mM p hosphate buffer, 1 mM E DTA, 0.1 m M 2- mercaptoethanol, 0.0 1% T riton X-100, 0. 5 μg/ml l eupeptin, 1 μg/ml pe pstatin, 0 .5 μg/ml
aprotinin a nd 100 μg/ml P MSF, pH 7.5) and cell s uspensions w ere h omogenized by dmd.aspetjournals.org sonication on ice. The resulting crude lysate was then centrifuged at 35,000x g for 1 hr at 40 C and the supernatant subjected to affinity purification by Fast Protein Liquid Chromatography
(FPLC) as previously described (Pappa et al., 2003). Briefly, 1-2 ml of Sf9 cell lysate was at ASPET Journals on September 24, 2021 applied to a 1. 6 X 6 c m 5’-AMP-Sepharose 4 B a ffinity c olumn ( Amersham Biosciences,
Piscataway, NJ) pre-equilibrated with the binding buffer (100 mM potassium phosphate, 1 mM E DTA, 0.1 mM 2-mercaptoethanol, 0. 01% T riton X -100, pH 7.4). The bo und
ALDH1B1 was eluted by applying a gradient of 0 to 0.25 mM NAD+ dissolved in the binding buffer ( 0.005 mM i ncrement per minute). Elution fractions ( 5 m l) were c ollected and examined for the presence of ALDH1B1 protein by Coomassie blue staining and Western blot analysis. F ractions containing A LDH1B1 were then p ooled and c oncentrated in concentrating b uffer ( 10 m M T ris-HCl, pH 7.4) a t 4ºC u sing a A micon® concentrator
(Millipore). The identity and purity of concentrated ALDH1B1 protein were confirmed by
Coomassie b lue staining, We stern b lot a nalysis and matrix-assisted la ser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis (see below).
8 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.
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Western immunoblot. Tissues from wi ld-type or Aldh2-/- kn ockout m ice we re c ollected, flash-frozen i n l iquid n itrogen a nd stored at -8 0oC un til us e. F rozen tiss ues we re homogenized in RIPA buffer (150 mM NaCl, 1% TritonX-100, 0.25% sodium deoxycholate,
0.1% SDS, 50 mM Tris, 1 mM EDTA, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin,
1 µg/ml pepstatin, pH 7.4) on ice with a tissue tearor (BioSpec Products, Bartlesville). Tissue homogenates we re c entrifuged a t 10, 000x g at 4 oC f or 20 m in a nd the s upernatant w as collected a nd us ed a s t issue ly sates. P roteins in t issue lysates (16-40 μg) o r purified Downloaded from recombinant human ALDH1B1 ( 25-200 ng ) w ere resolved by 10% S DS-PAGE and immunoblotted using primary antibodies as specified in the Results section. Rabbit polyclonal
α-ALDH1B1 was raised against human ALDH1B1 peptide sequence ELDTQQGPQVDKEQ dmd.aspetjournals.org
FERVLGYIQLGQKEGAKLLCGGERFGERGFFIKPTVFGGVQDDMR ( amino acids 3 53-
411) by the GenWay Biotech Inc. (San Diego, CA). To minimize the cross-reactivity towards
ALDH2 p rotein, t he antibody w as p re-absorbed b y a A LDH2-bound co lumn. Primary at ASPET Journals on September 24, 2021 antibodies were used at dilutions of 1:5000 for anti-ALDH1B1, 1:1000 for polyclonal anti-
ALDH2 ( Lassen e t a l., 200 5), 1:5000 f or mouse m onoclonal anti-GAPDH ( Ambion Inc.,
Austin, TX), a nd 1:10000 for m ouse m onoclonal a nti-β-ACTIN (Sigma, S t. L ouis, M O).
Blocking reag ents and co rresponding s econdary an tibodies w ere purchased f rom Li-Cor
Biosciences (L incoln, N ebraska) o r Cal biochem ( San Diego, CA) an d u sed according t o manufacturer’s pr otocol. Protein bands we re vis ualized us ing Li-Cor Odyssey I nfrared
Imaging System (Lincoln, Nebraska) or chemiluminescence (PerkinElmer, Boston, MA).
MALDI-TOF MS. Purified recombinant human ALDH1B1 p rotein (2 μg) wa s resolved by
15% S DS-PAGE. T he t wo p rotein bands, vis ualized b y C oomassie b lue staining, w ere excised and digested with trypsin (0.05 μg/μl in 25 mM ammonium bicarbonate) for 16 hr at
37ºC. Digested peptides were then analyzed b y MALDI-TOF MS on a 4800 p lus MALDI
9 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.
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TOF/TOF (ABI, Foster City, CA) equipped with a 200Hz solid-state laser using a matrix of
α-cyano-4-hydroxycinnaminic acid at 10 mg/ml in 5 0% ACN and 0.05% TFA. The sample was c alibrated externally usi ng a f ive po int p eptide calibration m ixture f rom Sigma
(ProteoMass Peptide Calibration Kit). Mass spectral data were collected in standard reflector mode over the mass range of 600 to 5000 m/z with a total of 400 shots collected at a laser intensity of 3300. MS/MS spectra were obtained using the standard 1kV acquisition method with CID t urned on , a ir a s th e collision ga s wit h a t hreshold of 1X10-6 t orr. T he laser Downloaded from intensity was set at 4300 and spectra were recorded for 2000 shots or until the accumulated spectra r eached an es timated S /N o f 7 5 (5 peaks an d 8 sub-spectra mi nimum). D atabase
searches w ere performed on M ASCOT s earch en gine ( www.matrixscience.com, Ma trix dmd.aspetjournals.org
Science, London, England) using ABI GPS software. Combined searches of both MS and
MS/MS data were performed using the MSDB database.
at ASPET Journals on September 24, 2021
Enzyme kinetic studies. The enzymatic activity of purified recombinant human ALDH1B1 was determined b y m onitoring t he formation of NADH a t 3 40 n m d uring the ox idation of aldehyde substrates using a Beckman D U-640 spectrophotometer. A molar ex tinction coefficient of 6.22 x 10-3 was used to c alculate the r ate of N ADH formation (Pappa e t al.,
2003). Prior to the enzyme assay, ALDH1B1 protein was first reactivated by incubation with
50 mM 2-mercaptoethanol at 25ºC in the dark for 1 hr . Aldehyde substrates were prepared either in ddH2O or 20% ethanol (the final concentration of ethanol being < 1%). Eight to ten concentrations of individual aldehyde substrate representing three to five below the apparent
Km and f ive a bove the a pparent Km were used. T he r eaction was initiated b y adding the substrate into the reaction mixture containing 0.1 M sodium pyrophosphate, 1 mM NAD+, 1 mM pyrazole, 1.0 mM 2-mercaptoethanol and 5 μg activated ALDH1B1 protein. Production of NADH w as m onitored f or 3 m in at 25ºC. F or t he Km s tudy fo r N AD+ a nd NADP +,
10 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 propionaldehyde (10 mM) was used a s the substrate t o d efine the a ctivity. The d isulfiram inhibition study wa s performed usin g p ropionaldehyde ( 100, 500 a nd 1000 μM) a s the substrate. In these studies, 100x stock solution of disulfiram dissolved in DMSO was added to the reaction mixture and pre-incubated with activated ALDH1B1 protein for 5 min prior to the addition of substrate.
The esterase activity of human A LDH1B1 w as de termined by monitoring t he formation o f p-nitrophenolate at 400 nm using a Beckman DU-640 spectrophotometer. A Downloaded from molar e xtinction c oefficient of 1 8.3x10-3 was us ed t o c alculate th e rate of p-nitrophenolate formation (Wang and Weiner, 1995). Briefly, activated ALDH1B1 protein (5 μg) was mixed with p-nitrophenyl acetate (p-NPA) at various concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 dmd.aspetjournals.org and 2.0 mM) in 0.1 M sodium phosphate buffer (pH 7.4); p-Nitrophenolate formation was then monitored for 3 min at 25ºC.
For all e nzyme ki netic analyses, apparent kin etic c onstants ( Vmax a nd Km) w ere at ASPET Journals on September 24, 2021 determined u sing Sigma Plot Enzyme Kinetic M odule (v ersion 7 .0, 2 001; SPSS Inc.,
Chicago, IL , U SA) b y mea ns o f t he M ichaelis-Menten mo del. T he e nzyme ac tivity w as reported as nmol/min/mg protein. Data were reported as mean ± S.E. from three independent experiments carried out in triplicate.
Reverse transcription and quantitative real-time PCR (Q-PCR). Total R NA wa s is olated from mo use tissues using RNeasy Mi ni k it (Qiagen, V alencia, CA) acco rding t o manufacturer’s protocol. c DNA was synthesized using a Superscript III R T kit (Invitrogen,
Carlsbad, CA) according to manufacturer’s instruction, using 5 µg of total RNA in a 20 µl reaction volume. Q-PCR reactions were carried out using 0.03 μg cDNA by the Taqman gene expression as say (A BI, Foster Ci ty, CA) f or m ouse Aldh1b1 ge ne ( primer ID:
Mm00728303_s1) a nd m ouse Aldh2 gen e (p rimer I D: M m00477463_m1) accor ding t o
11 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.
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β manufacturer’s pr otocol. Ex pression of -actin w as used f or no rmalization of C T da ta ΔΔ according t o the CT method ( Livak a nd Schmittgen, 2001). The m RNA le vel o f
ALDH1B1 in the liver was set as the control (=1) and relative mRNA levels were expressed as fold of control. Data were reported as mean ± S.E. of 3-4 animals.
Immunohistochemistry (IHC) and immunofluorescence (IHF). Formalin-fixed a nd paraffin-embedded normal h uman tissues w ere procured by IHCtech ( Aurora, CO) in Downloaded from accordance with a pprovals a nd guidelines t o use for I HC st aining. Tissues from C57BL/6J wild-type m ale m ice w ere f ixed in 4% paraformaldehyde, dehydrated in g raded e thanol
solutions and e mbedded in pa raffin. T issue sections ( 4-μm) w ere th en deparaffinized, dmd.aspetjournals.org rehydrated and processed f or hematoxylin and e osin ( HE) st aining or f or immunohistostaining. I HC staining of A LDH1B1 w as pe rformed us ing t he TS A B iotin
System Kit ( NEN Life S cience P roducts, B oston, MA) a ccording t o th e m anufacturer’s at ASPET Journals on September 24, 2021 protocol. P rimary a ntibodies we re used at a di lution of 1 :200 f or r abbit p olyclonal a nti-
ALDH1B1 (see above) or for rabbit pre-immune serum (serum control). Secondary antibody was used at a dilution of 1:250 for goat polyclonal anti-rabbit IgG (Sigma, St. Louis, MO).
Images were obtained using a Ni kon upright microscope linked t o a Ni kon digital camera.
For IHF s taining, t issue s ections w ere bl ocked wi th 3% BSA in PBS f or 1 hr a t r oom temperature, followed by incubation with rabbit polyclonal anti-ALDH1B1 (1:200) overnight at 4ºC . Samples we re the n washed a nd stained with F ITC-conjugated goa t anti-rabbit I gG
(Sigma, St. Louis, MO). Tissue sections were co-stained with 4,6-diamidino-2-phenylindole
(DAPI) to identify the nucleus. IHF staining was evaluated using a fluorescence microscope
(Nikon Eclipse E600).
12 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.
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RESULTS
Baculovirus-mediated expression and purification of recombinant human ALDH1B1
The OR F o f h uman ALDH1B1 was ins erted into the pBlueBac4.5 ve ctor f or recombination with baculovirus DNA and recombinant viruses were used to infect Sf9 insect cells. Eleven independently isolated plaques were amplified and screened for the expression of ALDH1B1 protein by Western blot analysis in crude extracts of Sf9 cells (data not shown). Downloaded from The r ecombinant v irus w ith the highest e xpression o f A LDH1B1 protein w as u sed f or subsequent infection of Sf9 cells at a multiplicity of infection (MOI) of 1. Cell lysates from
infected Sf9 cells were prepared a t 48 hr post-infection (as described in the Material and dmd.aspetjournals.org
Methods section) and used for the purification of recombinant human ALDH1B1.
Purification of ALDH1B1 protein was performed by affinity chromatography using a
5’-AMP-Sepharose 4B column a s d escribed i n th e Material and Methods se ction. This at ASPET Journals on September 24, 2021 single-step process yielded about 200-300 μg of purified pr otein from 500 m l of Sf9 c ell cultures. W hen v isualized b y Coomassie blue staining o n a SD S-PAGE gel ( Fig. 1A), the purified proteins revealed two peptide bands running s lightly above 50 kDa, in agreement with the predicted size of ALDH1B1 (≈55 kDa). B y W estern b lot analysis ( Fig. 1B ), both peptides im munoreacted wi th a r abbit polyclonal a ntibody r aised a gainst t he h uman
ALDH1B1 p eptide sequence (amino acids 3 53-411). T o f urther v erify the i dentity of the purified proteins, the two peptide bands were excised from a Coomassie blue-stained SDS-
PAGE gel and subjected to MALDI-TOF MS analysis following trypsin digestion (Fig. 2).
The M ALDI-TOF MS a nalysis s howed t hirteen p eaks f or both t he upper and l ower b and corresponding to predicted amino acid sequence of ALDH1B1 protein (Fig. 2A), with a total sequence c overage of 3 0% a nd 37% for t he hi gher and lowe r molecular we ight band, respectively (Fig. 2B).
13 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.
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Biochemical characterization of human ALDH1B1
Our previous study demonstrated that recombinant mitochondrial ALDH2 enzyme is prone to inactivation, likely due to the oxidation of sulfhydryl residues that are essential for the catalytic activity of ALDHs (Lassen et al., 2005). Since ALDH1B1 shows ≈75% peptide sequence i dentity with A LDH2, w e e xpected t he same characteristic be ing shared by recombinant A LDH1B1. I ndeed, following p urification, r ecombinant human ALDH1B1 Downloaded from exhibited negligible aldehyde oxidation activity even at saturating concentrations of cofactor and substrate ( data not sh own). Under these same r eaction conditions, r ecombinant human
ALDH1B1, like recombinant ALDH2, was reactivated by pre-incubation with the reducing dmd.aspetjournals.org agent 2-mercaptoethanol at an optimal concentration at 50 mM (data not shown). Thus, for all the enzyme kinetic studies, recombinant human ALDH1B1 was pre-activated by incubation
with 50 mM 2-mercaptoethanol at 25ºC for 1 hr. at ASPET Journals on September 24, 2021
The kinetic properties of human ALDH1B1 enzyme were studied using a wide range
+ + of aldehydes as substrates and using either NAD or NADP as the cofactor. The apparent Km and Vmax values derived from these studies are summarized in Table 1. ALDH1B1 showed an
+ absolute preference for NAD (Km = 3.6 μΜ) as the cofactor; no catalytic activity occurred when NADP + was u sed. T he re combinant human A LDH1B1 ex hibited a h igh aff inity f or aliphatic aldehydes, in cluding acetaldehyde, pr opionaldehyde, he xanal a nd n onanal, as reflected by apparent Km’s less than 100 μM; although there was a greater affinity towards medium-chain saturated a ldehydes ( Km = 0.4 a nd 0.8 μM for he xanal a nd nonanal, respectively) relative t o short-chain al dehydes ( Km = 5 5 a nd 14 μM f or acet aldehyde an d propionaldehyde, r espectively). Human ALDH1B1 ha d an apparent Km of 50 μM f or the aromatic aldehyde benzaldehyde. The order of the catalytic efficiency of human ALDH1B1 for these aldehydes, as ref lected b y Vmax/Km, was hexanal > nonanal > propionaldehyde >
14 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 acetaldehyde > benzaldehyde. ALDH1B1 was also capable of metabolizing products of lipid peroxidation, namely 4-HNE and MDA, albeit with low a ffinity for substrate binding (Km >
400 μM) and poor catalytic efficiency (Vmax/Km < 1). On the other hand, ALDH1B1 exhibited no catalytic activity for oxidizing unsaturated aliphatic aldehydes, i.e. trans-2-hexenal, trans-
2-nonenal and trans-2-dodecenal. Since disulfiram is a potent inhibitor of both ALDH1A1 and, to a l esser extend, ALDH2 in vitro, we also tested whether ALDH1B1 was a target of disulfiram i nhibition. As shown in Fig. 3 , A LDH1B1 w as l ess s ensitive to inhibition by Downloaded from disulfiram t han A LDH1A1. A t t he lowerst co ncentration o f s ubstrate t ested
(propionaldehyde = 1 00 μM), A LDH1B1 was not aff ected b y disulfiram. O nly at h igher concentrations of p ropionaldehyde ( 500 a nd 1 000 μM) w as A LDH1B1 a ctivity in hibited, dmd.aspetjournals.org with the extent of inhibition being < 40%. On the other hand, human ALDH1A1 activity was inhibited > 8 0% by th e same concentration o f di sulfiram a t a ll propionaldehyde concentrations. at ASPET Journals on September 24, 2021
Mitochondrial ALDH2 has be en reported to ha ve e sterase a ctivity (Sheikh and
Weiner, 1997). Gi ven the 75% p eptide homology sha red by AL DH2 a nd ALDH1B1, w e assessed w hether ALDH1B1 a lso ha d esterase a ctivity by m onitoring the formation of p- nitrophenolate (p-NPA). Human ALDH1B1 exhibited a maximal esterase activity of 1895 ±
91 nmol/min/mg and an apparent Km of 288 ± 46 μΜ for p-NPA. Compared with the reported
Km of human ALDH2 for p-NPA (Sheikh and Weiner, 1997), this Km of ALDH1B1 (current study) was 40-fold higher.
Expression of ALDH1B1 in mouse tissues
To understand the tissue distribution of ALDH1B1 expression, we first assessed the messenger level of Aldh1b1 gene in various tissues from male C57BL/6J mice by Q-PCR and compared i t with t he Aldh2 ge ne ( Fig. 4A ). F or e asy c omparison, the m RNA le vel of
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ALDH1B1 in the liver was set as the control (=1) and relative mRNA levels were expressed as fold of control. The highest ALDH1B1 mRNA expression was detected in liver and part of the s mall in testine ( ileum a nd jejunum); m oderate levels (≈ 0.5 fold) were observed in the testis, distal colon, lung, heart and duodenum, and lowest levels (≈ 0.03 fold) were found in the kidney and stomach. When compared to the Aldh1b1 gene, Aldh2 gene was comparably expressed in int estinal t issues ( < 3 -fold o f A LDH1B1 m RNA le vel) b ut m ore r obustly expressed i n other examined tissues, including the kidney, liver, t estes, stomach, lung an d Downloaded from heart, viz. 1135-, 145-, 64-, 63-, 16- and 11-fold of ALDH1B1 mRNA, respectively.
The p rotein l evels of AL DH1B1, ALDH2 and A LDH1A1 w ere determined by
Western blot analysis (Fig. 4B). To identify any potential compensatory interactions between dmd.aspetjournals.org these closely-related ALDHs, we also investigated ALDH1B1 and ALDH1A1 expression in tissues f rom Aldh2-/- kn ockout m ice ( Fig. 4B ). P artially in a greement with th e m RNA
expression profiles, ALDH1B1 protein was expressed abundantly in liver, small intestine and at ASPET Journals on September 24, 2021 testes. In these tissues, ALDH2 protein was also expressed at high levels. In large intestine and l ung, A LDH1B1 a nd A LDH2 were e xpressed m oderately a nd a t c omparable l evels.
Interestingly, when compared with ALDH1B1, ALDH2 seemed to be differentially expressed in certain t issues, i ncluding s tomach, k idney and h eart. In a s imilarly ma nner as the mitochondrial A LDHs, c ytosolic AL DH1A1 sh owed h igh expression i n li ver, t estes a nd small i ntestine; in terestingly, lu ng tiss ue had a r elatively h igh abundance of A LDH1A1 expression. As expected, ALDH2 protein was undetectable in examined tissues from Aldh2-/- knockout mice. In these tissues, however, ALDH1B1 and ALDH1A1 protein levels were no different from t hose obtained from wild-type mice, except t hat ALDH1A1 appeared t o b e upregulated in stomach from Aldh2-/- knockout mice.
The distribution of ALDH1B1 in s everal mouse tissues was further investigated by immunohistochemical a ssay ( Fig. 5 A). In the l iver, A LDH1B1 displayed a pe ri-central
16 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 distribution and w as detected i n t he cytoplasm o f he patocytes. I n the sm all inte stine,
ALDH1B1 was intensely stained in the cytoplasm of absorptive epithelial cells. In the large intestine (colon), mild staining of ALDH1B1 was observed mostly in locations where colon stem c ells reside. I n t he lung, A LDH1B1 a ppeared to be expressed pr edominantly in t he bronchiolar e pithelium a nd Clara cells of s mall a irways, e specially i n the te rminal bronchioles. In the testes, intense ALDH1B1 immunoreactivity was observed in the epithelial cells lin ing the seminiferous tubules. I n t he th ymus, AL DH1B1 seemed t o b e e xpressed Downloaded from specifically in the medulla, which contains fewer lymphocytes but more epithelial cells. To better v isualize t he c ellular l ocalization o f A LDH1B1 p rotein, we performed confocal
immunofluorescent staining f or A LDH1B1 in the two t issues tha t showed the hi ghest dmd.aspetjournals.org expression, i.e., li ver and small i ntestine ( Fig. 5B ). The staining of ALDH1B1 exhibited a cytoplasmic punctuate pattern in both tissues, supporting an organelle-associated localization
of ALDH1B1. at ASPET Journals on September 24, 2021
Expression of ALDH1B1 in human tissues
The e xpression of ALDH1B1 i n h uman ti ssues, i ncluding l iver, pa ncreas, small intestine, colon and lung, was investigated by immunohistochemical staining (Fig. 6 ). T he expression patterns of AL DH1B1, in terms o f the distribution and i ntensity of immunopositivity, in these human tissues were quite consistent with what was seen in mouse tissues ( Fig. 4A ). F urthermore, the punctuate positive-staining o f h uman A LDH1B1 w as clearly noted in the liver and panceas.
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DISCUSSION
The major enzyme th at metabolizes a cetaldehyde, the m ost t oxic metabolite of ethanol, is m itochondrial ALDH2. The inactive variant (Glu487Lys) of human ALDH2 is responsible f or e thanol-induced hypersensitivity w hich serves a s a deterrent against alcoholism in Asian pop ulations ( Yoshida et a l., 198 4). Whi le about 50% of As ians are carriers of this variant, it is nearly absent in Caucasians. Recent studies have identified the Downloaded from genetic determinants of drinking behavior and alcohol hypersensitivity among Caucasians to be po lymorphisms i n the h uman ALDH1B1 ge ne, whi ch encodes a nother m itochondrial
ALDH isoenzyme (Husemoen et al., 2008; Linneberg et al., 2010). These studies and others dmd.aspetjournals.org
(Stewart et al ., 1 995) are s uggestive o f a p otentially i mportant ro le o f A LDH1B1 in t he metabolism of acetaldehyde, but more direct confirmation remains to be established. Herein,
we have cloned, expressed and purified recombinant human ALDH1B1 using a baculovirus at ASPET Journals on September 24, 2021 expression system along with affinity c hromatography. H uman ALDH1B1 is catalytically- active towards a wide range of aldehyde substrates and utilizes NAD+, but not NADP+, as the cofactor. T he op timal substrates of h uman AL DH1B1 ( Km < 1 μM) are medium-chain aldehydes, i ncluding he xanal and nonanal. Short-chain p ropionaldehyde a nd aromatic benzaldehyde are also good substrates for ALDH1B1. Most importantly, human ALDH1B1 exhibits an intermediate Km (55 μM) for acetaldehyde when compared with human ALDH2
(Km = 3.2 μM) and human ALDH1A1 (Km = 180 μM) (Klyosov et al., 1996), suggesting that
ALDH1B1 indeed represents a low Km ALDH f or acetaldehyde. Thus, our st udy provides direct evidence to support the hypothesis that ALDH1B1 represents the second mitochondrial
ALDH, a side from ALDH2, that actively oxidizes a cetaldehyde and consequently m ay be important for the metabolism of ethanol.
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Disulfiram is a drug used to treat chronic a lcoholism by pr oducing a n undesirable acute sensitivity to alcohol. The effect of disulfiram is attributable to its action in elevating acetaldehyde l evels b y i nhibiting A LDH1A1 an d ALDH2 ( Petersen, 1 992). Recombinant human ALDH1A1 (Ki = 0.2 μM) is sixty-times more sensitive to inhibition by disulfiram than r ecombinant h uman AL DH2 (Ki > 12 μM) (L am et al ., 1997). I n t he present study, recombinant human A LDH1B1 w as in hibited by disulfiram bu t t o a l esser e xtent than
ALDH1A1 when propionaldehyde was used as substrate, a finding consistent with a previous Downloaded from study (Stewart et al., 1995). Such sensitivity of ALDH1B1 to disulfiram inhibition may have therapeutic ramifications on the use of disulfiram in vivo.
An interesting observation is that recombinant human ALDH1B1 e xpressed in S f9 dmd.aspetjournals.org insect cells migrates as double bands by SDS-PAGE. Based on their locations relative to the molecular we ight marker, the two p rotein ba nds correspond t o ≈55 and ≈58 KDa peptides.
MALDI-TOF MS and Western blot analysis confirmed the identity of both peptides to be at ASPET Journals on September 24, 2021
ALDH1B1. We propose th at th e 58 kDa pr otein band r epresents the 5 17-residue intact translated peptide and the 55 kDa protein represents the mature form after the mitochondrial lead signal is excised. Alternatively, the 58 KDa protein band could be a post-translationally modified form of ALDH1B1, e.g., one that has been glycosylated or phosphorylated or both.
Glycosylation has been documented previously for microsomal ALDH (Masaki et al., 1996).
Recently, phosphorylation has been identified to occur to ALDH2. One study reported that
ALDH2 is phosphorylated and activated by the survival kinase protein kinase C epsilon and that activation of ALDH2 is cardioprotective (Chen et al., 2008). On the other hand, it has also been reported that carbon tetrachloride (CCl4) exposure inhibits ALDH2 activity through
JNK-mediated p hosphorylation; i nactivation o f AL DH2 c ontributes to C Cl4-induced l iver damage ( Moon e t a l., 2010). Th ese s tudies suggest th at the e ffect o f p hosphorylation of
ALDH2, a nd likely of other A LDHs, is va riable, l eading to a ctivation or inactivation o f
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ALDH de pending on the specific s tress. F uther s tudies are n eeded to d etermine wh ether phosphorylation is a mechanism regulating ALDH1B1.
ALDH1B1 me ssenger is actively t ranscribed i n m ultiple human t issues (H su an d
Chang, 1991; S tewart et al ., 1 996). I n t he present study, w e o bserved a similar t issue expression profile in m ouse tiss ues. Liver h ad the highest le vel of ALDH1B1 and ALDH2 mRNAs r elative to oth er examined tis sues, supporting an im portant role of t he tw o mitochondrial ALDHs in th is or gan. In m ost t issues, e xcept in th e intestine, AL DH2 Downloaded from messenger is predominant by up to three orders of magnitude. Much higher levels of ALDH2 mRNA relative to ALDH1B1 mRNA has also been found in human liver (Hsu and Chang,
1991). T he pr edominance of he patic AL DH2 o ver ALDH1B1 could po ssibly e xplain the dmd.aspetjournals.org inability of A sian c arriers of ALD H2*2 a llele to efficiently metabolize a cetaldehyde produced after alcohol consumption in spite of the presence of ALDH1B1 enzyme. Along the
mouse gastrointestinal t ract, the re seems to be a gradient of A LDH1B1 m RNA expression at ASPET Journals on September 24, 2021 which starts at baseline in the duodenum, peaks in ileum and drops back to baseline in the colon. The levels of ALDH1B1 mRNA in these tissues are comparable to that of ALDH2 mRNA, suggesting a m ore i mportant r ole of ALDH1B1 in m ouse intestine. I t is well established t hat t he l iver plays a major role in drug a nd x enobiotic bio transformation.
However, metabolism d uring a bsorption across th e i ntestinal wall a lso i nfluences th e bioavailability of orally-administered compounds (van de Kerkhof et al., 2007). For instance, it has recently been reported that intestinal cytochrome P450 (CYP) 1A1, but not the hepatic
CYP1A1, is the key enzyme in oral benzo[a]pyrene detoxification (Shi et al., 2010). Given that ethanol is usually administered via the oral route, ALDH1B1 in the intestinal wall may play an important role in the initial metabolism of ethanol or of orally-ingested aldehydes, whereas ALDHs associated with the liver would be an ticipated t o process any a ldehydes in the hepatic portal vein or in the systemic circulation. Interestingly, ADH1, the major alcohol
20 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 metabolizing enzyme converting ethanol to acetaldehyde, is predominantly expressed in the small intestine ( Vaglenova e t a l., 2 003). It should be noted th at t he t issue dis tribution of
ALDH1B1 p rotein, i n both human and m ouse, a grees well wi th th e pa ttern of A LDH1B1 messenger, ind icating th at the t ranscriptional r egulation is li kely the ke y mechanism controlling the expression of ALDH1B1 protein.
Genome-wise, the t wo mitochondrial A LDHs, namely ALDH2 an d ALDH1B1, are conserved across taxa, including fungi, mammals and plants (Liu and Schnable, 2002). Why Downloaded from would na ture s elect to have two closely-related A LDHs present in the m itochondria? On e simple explanation could be that the two mitochondrial ALDHs have different biochemical
functions and/or differential expression in tissues at different developmental stages. Several dmd.aspetjournals.org studies ha ve a ssociated ALDH1B1 wi th f unctions oth er th an a cetaldehyde metabolism. A recent microarray s tudy sh owed tha t A LDH1B1 wa s up regulated in medulloblastoma c ell
lines resistant t o c yclophosphamide, suggesting a p ossible r ole of A LDH1B1 e nzyme i n at ASPET Journals on September 24, 2021 cancer drug r esistance ( Bacolod e t al., 2 008). A nother study r eported that the dynamic expression of A LDH1B1 c orrelated with gr anulocytic d evelopment of he matopoietic stem cells; in this r eport, the a uthor p roposed a possible r ole of A LDH1B1 in r etinoic acid metabolism mediating the effect (Luo et al., 2007). A transgenic mouse line having forced expression of human ALDH2*2 variant exhibited distinct phenotypes, including small body size, reduced muscle mass, diminished fat content and osteopenia (Endo et al., 2009), all of which are absent from ALDH2-null mice (Yu et al., 2009). One hypothesis to explain such a discrepancy is that the ALDH2*2 mutant subunit not only inactivates the ALDH2 wild-type subunit but also inactivates other ALDHs by forming heterotetramers, thereby blocking their functions. Gi ven >75% h omology in peptide se quence a nd t he s ame mitochondrial localization, A LDH1B1 subunit c ould be a p otential binding partner o f A LDH2 subunit.
Taken together, these observations i ndicate the in volvement of A LDH1B1 in multiple
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DMD #34678 physiological or p athological p rocesses, the biochemical m echanism(s) o f wh ich merits further investigation.
In summary, the present study describes, for the first time, the expression, purification and biochemical characterization of human ALDH1B1 enzyme. Our results demonstrate that mitochondrial AL DH1B1 r epresents a low Km A LDH for acet aldehyde me tabolism and therefore is consistent with ALDH1B1 being actively involved in the metabolism of ethanol.
The differential tissue distribution of ALDH1B1 argues in favor of a role for ALDH1B1 in Downloaded from intestinal tissu es. T he o bserved bi ochemical p roperties of ALDH1B1 are c onsistent with a physiological role(s) of this ALDH isoenzyme.
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at ASPET Journals on September 24, 2021
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FOOTNOTES
† These authors have contributed equally to this work.
Current address: Department of Biochemistry and Biotechnology, University of Thessaly,
Larissa, Greece 41221 (D.S.).
This work was supported by National Institutes of Health, USA [grants AA017754 and
EY17963]. Downloaded from
dmd.aspetjournals.org at ASPET Journals on September 24, 2021
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LEGENDS FOR FIGURES
Fig.1. Purified r ecombinant hu man A LDH1B1 revealed by SD S-PAGE. (A) P urified human ALDH1B1 protein was visualized by Coomassie blue staining following SDS-PAGE.
Purified ALDH1B1 appeared as double protein bands running around 55~58 kDa. Lane 1: molecular we ight m arker; la ne 2: recombinant human AL DH1B1 ( 2 μg). ( B) In creasing amounts of ALDH1B1 protein (25 – 200 ng) was loaded and detected by Western blot (WB) Downloaded from using a rabbit polyclonal antibody (anti-ALDH1B1) against human ALDH1B1 peptide (see
Materials and Methods). Both protein bands reacted with the antibody.
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Fig. 2. Con firmation of t he i dentity of purified AL DH1B1. (A) Th e tw o p rotein b ands visualized by Coomassie blue staining were excised and analyzed by MALDI–TOF MS. The
two bands each showed thirteen peaks corresponding to amino acid sequences of ALDH1B1 at ASPET Journals on September 24, 2021 protein predicted from the ALDH1B1 cDNA. (B) The predicted peptide sequence of human
ALDH1B1. T he N -terminal 19 re sidues (i n red) a re predicted m itochondrial l ead signal.
Distinct peptide sequences of upper and lower protein bands picked up by MALDI-TOF MS analysis are highlighted in yellow and blue, respectively. Peptide sequences positive for both protein bands are highlighted in orange. The peptide used to generate the polyclonal antibody against ALDH1B1 is underlined and highlighted in bold.
Fig. 3 . I nhibition o f t he dehydrogenase a ctivity of ALDH1B1 and ALDH1A1 by disulfiram. Disulfiram (10 μM) wa s p re-incubated with r ecombinant human ALDH1B1 or
ALDH1A1 f or 5 mi n prior t o t he i nitiation o f react ion. V arious concentrations o f propionaldehyde w ere u sed as substrate. T he A LDH act ivity o f en zymes i n presence o f disulfuram was expressed as a percentage of the activity observed at the same concentration
28 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 of s ubstrate in the absence of disulfiram (control). Data represent mean ± S.E. from three independent experiments.
Fig. 4. E xpression of AL DH1A1, ALDH1B1 and AL DH2 i n mouse t issues. (A) T he mRNA levels of A LDH1B1 and ALDH2 were m easured b y Q-PCR. Th e he patic le vel of
ALDH1B1 m RNA wa s set as the c ontrol (=1) after n ormalization wit h β-Actin. R elative levels of m RNA are r eported as f old of c ontrol. T he r atio of A LDH1B1/ALDH2 m RNA Downloaded from levels is indicated for each examined tissues. Data represent mean ± S.E. from 3-4 mice. (B)
Protein levels of A LDH1A1, AL DH1B1 a nd AL DH2 we re detected by We stern blot in
tissues from wild-type (+/+) and ALDH2-null (-/-) mice. dmd.aspetjournals.org
Fig. 5. Tissue distribution of ALDH1B1 protein by immunohistostaining. Mouse tissues
were pr ocessed f or im munohistochemical ( A) o r im munofluorescent ( B) an alysis of at ASPET Journals on September 24, 2021
ALDH1B1 a s d escribed in Materials and Methods. ( A) Representative i mages o f t issue sections st ained for g eneral his tology (H&E), A LDH1B1-immunoreactivity ( α-ALDH1B1) and serum c ontrol (Pre-immune). Liver displayed i ntense A LDH1B1 im munopositivity
(brown) with a pericentral distribution. The absorptive epithelial cells of small intestine also showed h igh staining. I mmunopositivity in t he l arge intestine ap peared t o colocalize with stem c ells. I n the l ung, ALDH1B1 was f ound to be expressed pr edominantly in the bronchiolar epithelium and clara cel ls o f small ai rways. I n t he testes, intense immunoreactivity was observed in the epithelial cells lining the seminiferous tubules. In the thymus, im munopositivity was de tected specifically i n the m edulla. ( B) C onfocal immunofluorescent staining for ALDH1B1 (green) in the liver and small intestine exhibited a cytoplasmic punctate p attern, in ag reement w ith an o rganelle-associated l ocalization o f
29 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.
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ALDH1B1. Sections were co-stained with DAPI for nuclear staining. Overlay represents the
ALDH1B1 and DAPI images combined. Magnification: A, 400x; B, 1000x.
Fig. 6. Distribution of ALDH1B1 in human tissues by immunohistochemistry. Formalin- fixed and p araffin-embedded normal hum an t issues w ere processed i n a ccordance w ith approvals a nd guid elines f or I HC st aining using polyclonal a nti-human A LDH1B1. T he absorptive epithelium of s mall intestine revealed the h ighest intensity o f A LDH1B1 Downloaded from immunopositivity ( brown). Hepatocytes and pa ncreatic acinar c ells a lso sh owed str ong positive-staining for ALDH1B1, whi ch displayed a c ytoplasmic p unctate p attern. The
immunopositivity of ALDH1B1 in colon appeared to colocalize with stem cells. In the lung, dmd.aspetjournals.org
ALDH1B1 seemed to be expressed predominantly in the bronchiolar epithelium and c lara cells o f s mall ai rways. Magnification: top panels, 4 00x; bottom panels, 100 0x. at ASPET Journals on September 24, 2021
30 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.
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TABLES
Table 1. Kinetic properties of recombinant human ALDH1B1.
Substrate Vmax Km Vmax/Km
(nmol/min/mg) (μΜ)
NAD+ 1277 ± 42 3.6 ± 0.5 359
+ NADP 0 0 0 Downloaded from
Acetaldehyde 665 ± 55 55 ± 10 12
Propionaldehyde 789 ± 155 14 ± 3 56
Hexanal 1007 ± 30 0.4 ± 0.002 2398 dmd.aspetjournals.org
Nonanal 686 ± 55 0.8 ± 0.1 879
Benzaldehyde 458 ± 36 50 ± 8 9.1
4-Hydroxynonenal (4-HNE) 2043 ± 163 3383 ± 304 0.6 at ASPET Journals on September 24, 2021
Malondialdehyde (MDA) 348 ± 21 466 ± 13 0.7
Unsaturated aldehydes (trans-2-hexenal, 0 0 0
Trans-2-nonenal, Trans-2-dodecenal)
p-Nitrophenol acetate (p-NPA) 1895 ± 91 288 ± 46 6.6
Apparent Km and Vmax va lues were de termined by f itting the data to th e Michaelis-Menten equation using Sigma Plot. Data represent mean ± S.E. from three independent experiments carried out in triplicate. The kinetic parameters for NAD+ and NADP+ were determined using propionaldehyde ( 10 m M) as the s ubstrate. NA D+ ( 1 m M) w as u sed f or aldehyde substrate experiments.
31 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. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021 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. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021 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. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021 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. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021 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. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021 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. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021