Aldehyde dehydrogenase 1B1: Molecular cloning and characterization of a novel mitochondrial acetaldehyde-metabolizing enzyme

Article abstract of DOI:10.1124/dmd.110.034678

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 aldehyde dehydrogenase (ALDH) that shares 75% peptide sequence homology with ALDH2. Recent population studies in whites suggest a role for ALDH1B1 in ethanol metabolism. However, to date, no formal documentation of the biochemical properties of ALDH1B1 has been forthcoming. In this 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 properties of purified human ALDH1B1 were assessed using a wide range of aldehyde substrates. Human ALDH1B1 had an exclusive preference for NAD ⁺ as the cofactor and was catalytically active toward short- and medium-chain aliphatic aldehydes, aromatic aldehydes, and the products of lipid peroxidation, 4-hydroxynonenal and malondialdehyde. Most importantly, human ALDH1B1 exhibited an apparent K_m of 55 μM for acetaldehyde, making it the second low K_m ALDH for metabolism of this substrate. The dehydrogenase activity of ALDH1B1 was sensitive to disulfiram inhibition, a feature also shared with ALDH2. The tissue distribution of ALDH1B1 in C57BL/6J mice and humans was examined by quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis. The highest expression occurred in the liver, followed by the intestinal tract, implying a potential physiological role for ALDH1B1 in these tissues. The current study is the first report on the expression, purification, and biochemical characterization of human ALDH1B1 protein. Copyright

Full text of DOI:10.1124/dmd.110.034678

0090-9556/10/3810-1679–1687$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:1679–1687, 2010
Vol. 38, No. 10
34678/3624360
Printed in U.S.A.
Aldehyde Dehydrogenase 1B1: Molecular Cloning and
Characterization of a Novel Mitochondrial Acetaldehyde-
Metabolizing Enzyme
Dimitrios Stagos,1 Ying Chen, Chad Brocker, Elizabeth Donald, Brian C. Jackson,
David J. Orlicky, David C. Thompson, and Vasilis Vasiliou
Departments of Pharmaceutical Sciences (D.S., Y.C., C.B., E.D., B.C.J., V.V.), Pathology (D.J.O.), and Clinical Pharmacy
(D.C.T.), University of Colorado Denver, Aurora, Colorado
Received May 24, 2010; accepted July 8, 2010
ABSTRACT:
Ethanol-induced damage is largely attributed to its toxic metabo- and was catalytically active toward short- and medium-chain ali-
lite, acetaldehyde. Clearance of acetaldehyde is achieved by its phatic aldehydes, aromatic aldehydes, and the products of lipid
oxidation, primarily catalyzed by the mitochondrial class II alde- peroxidation, 4-hydroxynonenal and malondialdehyde. Most im-
hyde dehydrogenase (ALDH2). ALDH1B1 is another mitochondrial portantly, human ALDH1B1 exhibited an apparent Km of 55 ␮M for
aldehyde dehydrogenase (ALDH) that shares 75% peptide se- acetaldehyde, making it the second low Km ALDH for metabolism
quence homology with ALDH2. Recent population studies in whites of this substrate. The dehydrogenase activity of ALDH1B1 was
suggest a role for ALDH1B1 in ethanol metabolism. However, to sensitive to disulfiram inhibition, a feature also shared with ALDH2.
date, no formal documentation of the biochemical properties of The tissue distribution of ALDH1B1 in C57BL/6J mice and humans
ALDH1B1 has been forthcoming. In this current study, we cloned was examined by quantitative polymerase chain reaction, Western
and expressed human recombinant ALDH1B1 in Sf9 insect cells.
blotting, and immunohistochemical analysis. The highest expres-
The resultant enzyme was purified by affinity chromatography to sion occurred in the liver, followed by the intestinal tract, implying
homogeneity. The kinetic properties of purified human ALDH1B1 a potential physiological role for ALDH1B1 in these tissues. The
were assessed using a wide range of aldehyde substrates. Human current study is the first report on the expression, purification, and
ALDH1B1 had an exclusive preference for NAD؉ as the cofactor biochemical characterization of human ALDH1B1 protein.
Introduction
tabolite of ethanol, is toxic and its accumulation contributes signifi-
cantly to ethanol-induced tissue damage (Eriksson, 2001; Meier and
Seitz, 2008). The toxicity of acetaldehyde is caused by it covalently
binding biological macromolecules, such as proteins and nucleic acids
and forming adducts that impair their functions (Niemela¨, 2007).
Acetaldehyde also induces lipid peroxidation, increased collagen syn-
thesis, and glutathione depletion (Esterbauer et al., 1991). Moreover,
protein adducts with acetaldehyde and products of lipid peroxidation
trigger autoimmune responses that are associated with alcoholic liver
disease (Viitala et al., 2000; Stewart et al., 2004). Therefore, metab-
olism and elimination of acetaldehyde is of great importance for
cellular defense against ethanol-induced toxicities.
Excessive alcohol consumption leads to a variety of psychological
and pathological consequences. Chronic alcohol intake is often
associated with damage to multiple organs, manifesting as hepa-
titis, liver cirrhosis, brain damage, and immune dysfunction. Many
of the toxic effects of ethanol are attributable to ethanol metabo-
lism. Ethanol is metabolized in the liver to acetaldehyde predomi-
nantly by class I alcohol dehydrogenase isoenzymes (ADH1A,
ADH1B, and ADH1C) located in the cytosol of hepatocytes. Alde-
hyde dehydrogenases (ALDHs) then oxidize acetaldehyde to acetate
and water (Crabb et al., 2004). Acetaldehyde, the intermediate me-
This work was supported by the National Institutes of Health National Institute
on Alcohol Abuse and Alcoholism [Grant AA017754]; and the National Institutes of
Health National Eye Institute [Grant EY17963].
Among the 19 known human ALDHs, mitochondrial ALDH2, and,
to a lesser extent, cytosolic ALDH1A1 has 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, namely 3.2 and 180 ␮M
for human ALDH2 and ALDH1A1, respectively (Klyosov et al.,
1996). The importance of ALDH2 in the clearance of acetaldehyde is
exemplified in humans carrying the ALDH2*2 allele, which encodes
D.S. and Y.C. contributed equally to this work.
1 Current affiliation: Department of Biochemistry and Biotechnology, University
of Thessaly, Larissa, Greece.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.034678.
ABBREVIATIONS: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenases; 4-HNE, 4-hydroxynonenal; MDA, malondialdehyde; ORF,
open reading frame; PCR, polymerase chain reaction; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; MS, mass spec-
trometry; PAGE, polyacrylamide gel electrophoresis; p-NPA, p-nitrophenyl acetate; Q-PCR, quantitative real-time PCR; IHC, immunohistochem-
ical; DAPI, 4,6-diamidino-2-phenylindole.
1679

1680
STAGOS ET AL.
and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless
otherwise specified,
a functional mutation (E487K) of ALDH2 and results in poor binding
affinity to cofactor NADϩ and Ͼ90% loss of enzyme activity (Yo-
shida et al., 1984; Larson et al., 2005). Compared with wild-type
individuals, blood acetaldehyde levels climb 20- and 6-fold higher in
homozygous and heterozygous individuals carrying this polymor-
phism, respectively, for equivalent levels of alcohol consumption
(Crabb et al., 1989). This causes the “alcohol flushing” syndrome
characterized by the reddening of the face, neck, and, in some cases,
the entire body due to dilation of capillaries (Eriksson, 2001). Similar
elevations in acetaldehyde have been observed in the Aldh2-null mice
(Yu et al., 2009). The second enzyme involved in acetaldehyde
metabolism is ALDH1A1. In addition to its crucial role in retinoic
acid biosynthesis (Duester, 2000), cytosolic ALDH1A1 has been
shown to be important in acetaldehyde metabolism and alcohol pref-
erence in rodent studies (Little and Petersen, 1983; Bond and Singh,
1990; Bond et al., 1991). Human studies also report the association of
ALDH1A1 variants with enzyme deficiency in white “flushers”
(Eriksson, 2001). Nevertheless, the contribution of ALDH1A1 to
acetaldehyde metabolism seems to be less important in humans than
in rodents, probably because rodent ALDH1A1 has a much lower Km
for acetaldehyde (15 ␮M) (Klyosov et al., 1996).
ALDH1B1, previously known as ALDHX and ALDH5, represents
another mitochondrial ALDH isoenzyme. The human ALDH1B1 gene
located on chromosome 9 spans a 5957-base pair region and is the
only known ALDH gene with an intronless coding region (Hsu and
Chang, 1991). The 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 an N-
terminal 19-residue mitochondrial lead signal. Based on peptide se-
Animals. Male C57BL/6J wild-type mice (ϳ12 weeks old) were purchased
from The Jackson Laboratory (Bar Harbor, Maine). Aldh2(Ϫ/Ϫ) knockout
mice were obtained from Dr. T. K. Kawamoto’s laboratory (Department of
Environmental Health, School of Medicine, University of Occupational and
Environmental Health, Yahatanishi-ku, Kitakyushu, Japan) (Yu et al., 2009)
and maintained in the C57BL/6J background. Mice were euthanized by CO2
inhalation followed by cervical dislocation. All procedures involving animals
were approved by the Institutional Animal Care and Use Committee at the
University of Colorado Denver.
Construction, Expression, and Purification of Recombinant Human
ALDH1B1. The pCMV-XL4-ALDH1B1 plasmid harboring the full length of
human ALDH1B1 cDNA (NM_000692.3) was purchased from Origene
(Rockville, MD). The open reading frame (ORF) of ALDH1B1 was amplified
by polymerase chain reaction (PCR) using the primer pair 5Ј-CAAGGTAC-
CTACAGGAAAGCCCACCATGCTGCGCTTCCTGGCA-3Ј (forward) and
5Ј-GTGAAGCTTTTACGAGTTCTTCTGAGGAACCTTGATGGTG-3Ј (re-
verse). The forward primer was designed to contain a KpnI site (GGTACC) for
subcloning and a sequence motif (CCACC) 5Ј-ward of the start codon ATG (in
bold) to ensure correct initiation of translation in eukaryotic cells (Ding and
Nam Ong, 2003). The reverse primer was designed to contain a HindIII site
(AAGCTT) 3Ј-ward of the stop codon TAA (in bold). The 1.6-kilobase PCR
product was then digested with KpnI and HindIII and subcloned into the
pBlueBac4.5 expression vector (Invitrogen, Carlsbad, CA). The correct se-
quence of the inserted ORF of ALDH1B1 was confirmed by DNA sequencing
analysis.
The pBlueBac4.5-ALDH1B1 plasmid was used to generate recombinant
baculoviruses, which were plaque-purified and amplified in Sf9 insect cells by
the Tissue Culture Core Facility at the University of Colorado Denver as
described previously (Manzer et al., 2003). Approximately 6 ϫ 108 Sf9 cells
were harvested 48 h after infection by centrifugation at 1000g for 5 min and
washed with phosphate-buffered saline, pH 7.4. Cell pellets were resuspended
quence alignment, human ALDH1B1 shares 65 and 75% homology in lysis buffer (100 mM phosphate buffer, 1 mM EDTA, 0.1 mM 2-mercap-
toethanol, 0.01% Triton X-100, 0.5 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, 0.5
␮g/ml aprotinin, and 100 ␮g/ml phenylmethylsulfonyl fluoride, pH 7.5), and
cell suspensions were homogenized by sonication on ice. The resulting crude
lysate was then centrifuged at 35,000g for 1 h at 4°C, and the supernatant was
subjected to affinity purification by fast protein liquid chromatography as
described previously (Pappa et al., 2003). In brief, 1 to 2 ml of Sf9 cell lysate
was applied to a 1.6 ϫ 6 cm 5Ј-AMP-Sepharose 4B affinity column (GE
Healthcare, Pittsburgh, PA) pre-equilibrated with the binding buffer (100 mM
potassium phosphate, 1 mM EDTA, 0.1 mM 2-mercaptoethanol, and 0.01%
Triton X-100, pH 7.4). The bound ALDH1B1 was eluted by applying a
gradient of 0 to 0.25 mM NADϩ dissolved in the binding buffer (0.005 mM
increment per minute). Elution fractions (5 ml) were collected and exam-
ined for the presence of ALDH1B1 protein by Coomassie Blue staining and
Western blot analysis. Fractions containing ALDH1B1 were then pooled
and concentrated in concentrating buffer (10 mM Tris-HCl, pH 7.4) at 4°C
using a Amicon concentrator (Millipore Corporation, Billerica, MA). The
identity and purity of concentrated ALDH1B1 protein were confirmed by
Coomassie Blue staining, Western blot analysis, and matrix-assisted laser
desorption ionization time of flight (MALDI-TOF) mass spectrometry
(MS) analysis (see below).
Western Immunoblot. Tissues from wild-type or Aldh2(Ϫ/Ϫ) knockout
mice were collected, flash-frozen in liquid nitrogen, and stored at Ϫ80°C until
use. Frozen tissues were homogenized in radioimmunoprecipitation assay
buffer (150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate,
0.1% SDS, 50 mM Tris, 1 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 1 ␮g/ml pepstatin, pH
7.4) on ice with a Tissue-Tearor (BioSpec Products, Bartlesville, OK).
Tissue homogenates were centrifuged at 10,000g at 4°C for 20 min, and the
supernatant was collected and used for tissue lysates. Proteins in tissue lysates
(16–40 ␮g) or purified recombinant human ALDH1B1 (25–200 ng) were
resolved by 10% SDS-PAGE and immunoblotted using primary antibodies as
with human ALDH1A1 and ALDH2, respectively. Northern blot
analysis in human tissues has shown high levels of ALDH1B1 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), in which cells are constantly exposed to UV-
induced lipid peroxidation. To date, little is known regarding the
biochemical properties and physiological roles of ALDH1B1. A study
using crude lysate from HuH7 hepatoma cells reported that mitochon-
drial ALDH1B1 contributed to the oxidation of short-chain aldehydes
including acetaldehyde and propionaldehyde, implying a role for
ALDH1B1 in ethanol 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
(A69V) and alcohol-induced hypersensitivity (A86V) in whites
(Husemoen et al., 2008; Linneberg et al., 2010). These findings
strongly suggest that ALDH1B1 enzyme may be involved in ethanol
detoxification and modifications in this enzyme may contribute to
alcohol-related diseases. To expand our current knowledge on the
catalytic properties of ALDH1B1, we cloned and purified human
recombinant ALDH1B1. Different aldehydes, including acetaldehyde,
were tested as substrates. In addition, we surveyed the expression
profile of ALDH1B1 in multiple mouse and human tissues. This is the
first report on the expression and biochemical characterization of
human ALDH1B1 enzyme.
Materials and Methods
Chemicals. 4-Hydroxynonenal (4-HNE) was obtained from Cayman Chem- specified under Results. Rabbit polyclonal ␣-ALDH1B1 was raised against the
ical (Ann Arbor, MI). Malondialdehyde (MDA) was synthesized according to human ALDH1B1 peptide sequence ELDTQQGPQVDKEQ FERVLGYIQL
a method described previously (Esterbauer et al., 1991). All other chemicals
GQKEGAKLLCGGERFGERGFFIKPTVFGGVQDDMR (amino acids 353–

INITIAL CHARACTERIZATION OF HUMAN ALDH1B1
1681
411) by GenWay Biotech Inc. (San Diego, CA). 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 monoclo-
cia, CA) according to the manufacturer’s protocol. cDNA was synthesized
with a SuperScript III RT Kit (Invitrogen, Carlsbad, CA) according to the
manufacturer’s instruction, using 5 ␮g of total RNA in a 20-␮l reaction
volume. Q-PCR reactions were carried out using 0.03 ␮g of cDNA by the
nal anti-glyceraldehyde-3-phosphate dehydrogenase (Ambion, Austin, TX), TaqMan gene expression assay (Applied Biosystems) for mouse Aldh1b1 gene
(primer identification number: Mm00728303_s1) and mouse Aldh2 gene
(primer identification number: Mm00477463_m1) according to the manufac-
turer’s protocol. Expression of ␤-actin was used for normalization of CT data
according to the ⌬⌬CT method (Livak and Schmittgen, 2001). The mRNA
level of ALDH1B1 in the liver was set as the control (ϭ 1), and relative mRNA
levels were expressed as fold of control. Data were reported as the mean Ϯ
S.E. of three to four animals.
and 1:10,000 for mouse monoclonal anti-␤-actin (Sigma-Aldrich, St. Louis,
MO). Blocking reagents and corresponding secondary antibodies were pur-
chased from LI-COR Biosciences (Lincoln, NE) or Calbiochem (San Diego,
CA) and used according to the manufacturer’s protocol. Protein bands were
visualized using an Odyssey Infrared Imaging System (LI-COR) or chemilu-
minescence (PerkinElmer Life and Analytical Sciences, Waltham, MA).
MALDI-TOF MS. Purified recombinant human ALDH1B1 protein (2 ␮g)
was resolved by 15% SDS-PAGE. The two protein bands, visualized by
Coomassie Blue staining, were excised and digested with trypsin (0.05 ␮g/␮l
in 25 mM ammonium bicarbonate) for 16 h at 37°C. Digested peptides were
then analyzed by MALDI-TOF MS on a 4800 plus MALDI TOF/TOF spec-
trometer (Applied Biosystems, Foster City, CA) equipped with a 200-Hz
solid-state laser using a matrix of ␣-cyano-4-hydroxycinnaminic acid at 10
mg/ml in 50% acetonitrile and 0.05% trifluoroacetic acid. The sample was
calibrated externally using a five-point peptide calibration mixture from
Sigma-Aldrich (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 1-kV acquisition method with collision-
induced dissociation turned on and air as the collision gas with a threshold of
1 ϫ 10Ϫ6 Torr. The laser intensity was set at 4300, and spectra were recorded
for 2000 shots or until the accumulated spectra reached an estimated signal/
noise ratio of 75 (five peaks and eight subspectra minimum). Database
searches were performed on the MASCOT search engine (www.matrixscience.
com; Matrix Science, London, UK) using Applied Biosystems GPS software.
Combined searches of both MS and tandem mass spectrometry data were
performed using the msdb database.
Enzyme Kinetic Studies. The enzymatic activity of purified recombinant
human ALDH1B1 was determined by monitoring the formation of NADH at 340
nm during the oxidation of aldehyde substrates using a Beckman DU-640 spec-
trophotometer. A molar extinction coefficient of 6.22 ϫ 10Ϫ3 was used to
calculate the rate of NADH formation (Pappa et al., 2003). Before the enzyme
assay, ALDH1B1 protein was first reactivated by incubation with 50 mM 2-mer-
captoethanol at 25°C in the dark for 1 h. Aldehyde substrates were prepared either
in double-distilled H2O or 20% ethanol (the final concentration of ethanol being
Ͻ1%). Eight to 10 concentrations of individual aldehyde substrate representing 3
to 5 concentrations below the apparent Km and 5 concentrations above the apparent
Km were used. The reaction was initiated by adding the substrate into the reaction
mixture containing 0.1 M sodium pyrophosphate, 1 mM NADϩ, 1 mM pyrazole,
1.0 mM 2-mercaptoethanol, and 5 ␮g of activated ALDH1B1 protein. Production
of NADH was monitored for 3 min at 25°C. For the Km study for NADϩ and
NADPϩ, propionaldehyde (10 mM) was used as the substrate to define the
activity. The disulfiram inhibition study was performed using propionaldehyde
(100, 500, and 1000 ␮M) as the substrate. In these studies, 100ϫ stock solution of
disulfiram dissolved in dimethyl sulfoxide was added to the reaction mixture and
preincubated with activated ALDH1B1 protein for 5 min before the addition of
substrate.
Immunohistochemistry and Immunofluorescence. Formalin-fixed and
paraffin-embedded normal human tissues were procured by IHCtech (Au-
rora, CO) in accordance with approvals and guidelines to use for immu-
nohistochemical (IHC) staining. Tissues from C57BL/6J wild-type male
mice were fixed in 4% paraformaldehyde, dehydrated in graded ethanol
solutions, and embedded in paraffin. Tissue sections (4-␮m) were then
deparaffinized, rehydrated, and processed for hematoxylin and eosin stain-
ing or for IHC staining of ALDH1B1 was performed using the TSA Biotin
System Kit (PerkinElmer, Waltham, MA) according to the manufacturer’s
protocol. Primary antibodies were used at a dilution of 1:200 for rabbit
polyclonal anti-ALDH1B1 (see above) or for rabbit preimmune serum
(serum control). Secondary antibody was used at a dilution of 1:250 for
goat polyclonal anti-rabbit IgG (Sigma-Aldrich). Images were obtained
using a Nikon upright microscope linked to a Nikon digital camera. For
immunofluorescence staining, tissue sections were blocked with 3% bovine
serum albumin in phosphate-buffered saline for 1 h at room temperature,
followed by incubation with rabbit polyclonal anti-ALDH1B1 (1:200)
overnight at 4°C. Samples were then washed and stained with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis,
MO). Tissue sections were costained with 4,6-diamidino-2-phenylindole
(DAPI) to identify the nucleus. Immunofluorescence staining was evalu-
ated using a fluorescence microscope (Nikon Eclipse E600).
Results
Baculovirus-Mediated Expression and Purification of Recom-
binant Human ALDH1B1. The ORF of human ALDH1B1 was
inserted into the pBlueBac4.5 vector for recombination with baculo-
virus 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). The recom-
binant virus with the highest expression of ALDH1B1 protein was
used for subsequent infection of Sf9 cells at a multiplicity of infection
of 1. Cell lysates from infected Sf9 cells were prepared at 48 h after
infection (as described under Materials and Methods) and used for the
purification of recombinant human ALDH1B1.
Purification of ALDH1B1 protein was performed by affinity
chromatography using a 5Ј-AMP-Sepharose 4B column as de-
scribed under Materials and Methods. This single-step process
yielded approximately 200 to 300 ␮g of purified protein from 500
ml of Sf9 cell cultures. When visualized by Coomassie Blue
staining on an SDS-PAGE gel (Fig. 1A), the purified proteins
revealed two peptide bands running slightly above 50 kDa, in
agreement with the predicted size of ALDH1B1 (Ϸ55 kDa). By
Western blot analysis (Fig. 1B), both peptides immunoreacted with
a rabbit polyclonal antibody raised against the human ALDH1B1
peptide sequence (amino acids 353–411). To further verify the
identity of the purified proteins, the two peptide bands were
excised from a Coomassie Blue-stained SDS-PAGE gel and sub-
jected to MALDI-TOF MS analysis after trypsin digestion (Fig. 2).
The MALDI-TOF MS analysis showed 13 peaks for both the upper
The esterase activity of human ALDH1B1 was determined by monitoring
the formation of p-nitrophenolate at 400 nm using a Beckman DU-640 spec-
trophotometer. A molar extinction coefficient of 18.3 ϫ 10Ϫ3 was used to
calculate the rate of p-nitrophenolate formation (Wang and Weiner, 1995). In
brief, activated ALDH1B1 protein (5 ␮g) was mixed with p-nitrophenyl
acetate (p-NPA) at various concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and
2.0 mM) in 0.1 M sodium phosphate buffer (pH 7.4); p-Nitrophenolate
formation was then monitored for 3 min at 25°C.
For all enzyme kinetic analyses, apparent kinetic constants (Vmax and Km)
were determined using the Sigma Plot Enzyme Kinetic Module (version 7.0,
2001; SPSS Inc., Chicago, IL) by means of the Michaelis-Menten model.
Enzyme activity is reported as nanomoles per minute per milligram of protein.
Data are reported as means Ϯ S.E. from three independent experiments carried
out in triplicate.
Reverse Transcription and Quantitative Real-Time PCR. Total RNA
was isolated from mouse tissues using an RNeasy Mini kit (QIAGEN, Valen- and lower band corresponding to a predicted amino acid sequence

1682
STAGOS ET AL.
zyme 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 char-
acteristic being shared by recombinant ALDH1B1. Indeed, after pu-
rification, recombinant human ALDH1B1 exhibited negligible alde-
hyde oxidation activity even at saturating concentrations of cofactor
and substrate (data not shown). Under these same reaction conditions,
recombinant human ALDH1B1, like recombinant ALDH2, was reac-
tivated by preincubation with the reducing agent 2-mercaptoethanol at
an optimal concentration at 50 mM (data not shown). Thus, for all the
enzyme kinetic studies, recombinant human ALDH1B1 was preacti-
vated by incubation with 50 mM 2-mercaptoethanol at 25°C
for 1 h.
The kinetic properties of human ALDH1B1 enzyme were stud-
ied using a wide range of aldehydes as substrates and using either
NADϩ or NADPϩ as the cofactor. The apparent Km and Vmax
values derived from these studies are summarized in Table 1.
ALDH1B1 showed an absolute preference for NADϩ (Km ϭ 3.6
␮⌴) as the cofactor; no catalytic activity occurred when NADPϩ
was used. The recombinant human ALDH1B1 exhibited a high
affinity for aliphatic aldehydes, including acetaldehyde, propional-
FIG. 1. Purified recombinant human ALDH1B1 revealed by SDS-PAGE. A, puri-
fied human ALDH1B1 protein was visualized by Coomassie Blue staining after
SDS-PAGE. Purified ALDH1B1 appeared as double protein bands running around
55 to 58 kDa. Lane 1, molecular weight marker; lane 2, recombinant human
ALDH1B1 (2 ␮g). B, increasing amounts of ALDH1B1 protein (25–200 ng) were
loaded and detected by Western blot (WB) using a rabbit polyclonal antibody
(anti-ALDH1B1) against human ALDH1B1 peptide (see Materials and Methods).
Both protein bands reacted with the antibody.
of ALDH1B1 protein (Fig. 2A), with a total sequence coverage of
30 and 37% for the higher and lower molecular weight band,
respectively (Fig. 2B).
Biochemical Characterization of Human ALDH1B1. Our previ-
ous study demonstrated that recombinant mitochondrial ALDH2 en- dehyde, hexanal, and nonanal, as reflected by apparent Km values
FIG. 2. Confirmation of the identity of purified ALDH1B1. A, the two protein bands visualized by Coomassie Blue staining were excised and analyzed by MALDI-TOF
MS. The two bands each showed 13 peaks corresponding to amino acid sequences of ALDH1B1 protein predicted from the ALDH1B1 cDNA. B, the predicted peptide
sequence of human ALDH1B1. The N-terminal 19 residues (in bold) are the predicted mitochondrial lead signal. Distinct peptide sequences of upper and lower protein
bands picked up by MALDI-TOF MS analysis are highlighted in light and dark gray, respectively. Peptide sequences positive for both protein bands are highlighted in
darkest gray. The peptide used to generate the polyclonal antibody against ALDH1B1 is underlined and highlighted in bold.

INITIAL CHARACTERIZATION OF HUMAN ALDH1B1
1683
TABLE 1
Mitochondrial ALDH2 has been reported to have esterase activity
(Sheikh and Weiner, 1997). Given the 75% peptide homology shared
by ALDH2 and ALDH1B1, we assessed whether ALDH1B1 also had
esterase activity by monitoring the formation of p-NPA. Human
ALDH1B1 exhibited maximal esterase activity of 1895 Ϯ 91 nmol/
(min ⅐ mg) and an apparent Km of 288 Ϯ 46 ␮⌴ 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 the Aldh1b1 gene in various tissues from male
C57BL/6J mice by Q-PCR and compared it with that of the Aldh2
gene (Fig. 4A). For easy comparison, the mRNA level of ALDH1B1
in the liver was set as the control (ϭ 1), and relative mRNA levels
were expressed as fold of control. The highest ALDH1B1 mRNA
expression was detected in liver and part of the small intestine (ileum
and jejunum); moderate levels (Ϸ0.5-fold) were observed in the testis,
distal colon, lung, heart, and duodenum and the lowest levels (Ϸ0.03-
fold) were found in the kidney and stomach. Compared with the
Aldh1b1 gene, the Aldh2 gene was comparably expressed in intestinal
tissues (Ͻ3-fold of ALDH1B1 mRNA level) but was more robustly
expressed in other tissues examined, including the kidney, liver,
testes, stomach, lung, and heart, namely 1135-, 145-, 64-, 63-, 16-, and
11-fold of ALDH1B1 mRNA, respectively.
The protein levels of ALDH1B1, ALDH2, and ALDH1A1 were
determined by Western blot analysis (Fig. 4B). To identify any
potential compensatory interactions between these closely related
ALDHs, we also investigated ALDH1B1 and ALDH1A1 expression
in tissues from Aldh2(Ϫ/Ϫ) knockout mice (Fig. 4B). Partially in
agreement with the mRNA expression profiles, ALDH1B1 protein
was expressed abundantly in liver, small intestine, and testes. In these
tissues, ALDH2 protein was also expressed at high levels. In large
intestine and lung, ALDH1B1 and ALDH2 were expressed moder-
ately and at comparable levels. Compared with ALDH1B1, ALDH2
seemed to be differentially expressed in certain tissues, including
stomach, kidney, and heart. In a manner similar to that of the mito-
chondrial ALDHs, cytosolic ALDH1A1 showed high expression in
liver, testes, and small intestine; interestingly, lung tissue had a
relatively high abundance of ALDH1A1 expression. As expected,
ALDH2 protein was undetectable in tissues from Aldh2(Ϫ/Ϫ) knock-
out mice. However, in these tissues ALDH1B1 and ALDH1A1 pro-
tein levels were no different from those obtained from wild-type mice,
except that ALDH1A1 appeared to be up-regulated in stomach from
Aldh2(Ϫ/Ϫ) knockout mice.
Kinetic properties of recombinant human ALDH1B1
Apparent Km and Vmax values were determined by fitting the data to the Michaelis-Menten
equation using SigmaPlot. Data represent mean Ϯ S.E. from three independent experiments
carried out in triplicate. The kinetic parameters for NADϩ and NADPϩ were determined
using propionaldehyde (10 mM) as the substrate. NADϩ (1 mM) was used for aldehyde
substrate experiments.
Substrate
Vmax
Km
V
max/Km
nmol/(min ⅐ mg)
␮M
NADϩ
1277 Ϯ 42
0
3.6 Ϯ 0.5
0
55 Ϯ 10
14 Ϯ 3
0.4 Ϯ 0.002 2398
0.8 Ϯ 0.1
50 Ϯ 8
359
0
12
56
NADPϩ
Acetaldehyde
665 Ϯ 55
789 Ϯ 155
1007 Ϯ 30
686 Ϯ 55
458 Ϯ 36
Propionaldehyde
Hexanal
Nonanal
Benzaldehyde
4-HNE
879
9.1
0.6
0.7
0
2043 Ϯ 163 3383 Ϯ 304
348 Ϯ 21
MDA
466 Ϯ 13
Unsaturated aldehydes (trans-2-hexenal,
trans-2-nonenal, trans-2-dodecenal)
p-NPA
0
0
1895 Ϯ 91
288 Ϯ 46
6.6
less than 100 ␮M; although there was a greater affinity toward medium-chain
saturated aldehydes (Km ϭ 0.4 and 0.8 ␮M for hexanal and nonanal,
respectively) relative to that for short-chain aldehydes (Km ϭ 55 and 14 ␮M
for acetaldehyde and propionaldehyde, respectively). Human ALDH1B1 had
an apparent Km of 50 ␮M for the aromatic aldehyde benzaldehyde. The order
of the catalytic efficiency of human ALDH1B1 for these aldehydes, as
reflected by Vmax/Km, was hexanal Ͼ nonanal Ͼ propionaldehyde Ͼ acet-
aldehyde Ͼ benzaldehyde. ALDH1B1 was also capable of metabolizing
products of lipid peroxidation, namely 4-HNE and MDA, albeit with low
affinity for substrate binding (Km Ͼ 400 ␮M) and poor catalytic efficiency
(Vmax/Km Ͻ 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. Because disulfiram is a potent inhibitor of
both ALDH1A1 and, to a lesser extent, ALDH2 in vitro, we also tested
whether ALDH1B1 was a target of disulfiram inhibition. As shown in Fig. 3,
ALDH1B1 was less sensitive to inhibition by disulfiram than was
ALDH1A1. At the lowest concentration of substrate tested (propionalde-
hyde ϭ 100 ␮M), ALDH1B1 was not affected by disulfiram. Only at higher
concentrations of propionaldehyde (500 and 1000 ␮M) was ALDH1B1
activity inhibited, with the extent of inhibition being Ͻ40%. On the other
hand, human ALDH1A1 activity was inhibited Ͼ80% by the same concen-
tration of disulfiram at all propionaldehyde concentrations.
The distribution of ALDH1B1 in several mouse tissues was further
investigated by immunohistochemical assay (Fig. 5A). In the liver,
ALDH1B1 displayed a pericentral distribution and was detected in the
cytoplasm of hepatocytes. In the small intestine, 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 cells reside. In the lung,
ALDH1B1 appeared to be expressed predominantly in the bronchiolar
epithelium and Clara cells of small airways, especially in the terminal
bronchioles. In the testes, intense ALDH1B1 immunoreactivity was
observed in the epithelial cells lining the seminiferous tubules. In the
thymus, ALDH1B1 seemed to be expressed specifically in the me-
dulla, which contains fewer lymphocytes but more epithelial cells. To
better visualize the cellular localization of ALDH1B1 protein, we
performed confocal immunofluorescent staining for ALDH1B1 in the
two tissues that showed the highest expression, i.e., liver and small
intestine (Fig. 5B). The staining of ALDH1B1 exhibited a cytoplas-
FIG. 3. Inhibition of the dehydrogenase activity of ALDH1B1 and ALDH1A1 by
disulfiram. Disulfiram (10 ␮M) was preincubated with recombinant human
ALDH1B1 or ALDH1A1 for 5 min before the initiation of the reaction. Various
concentrations of propionaldehyde were used as substrate. The ALDH activity of
enzymes in presence of disulfiram was expressed as a percentage of the activity
observed at the same concentration of substrate in the absence of disulfiram
(control). Data represent mean Ϯ S.E. from three independent experiments.

1684
STAGOS ET AL.
FIG. 4. Expression of ALDH1A1, ALDH1B1, and ALDH2 in mouse tissues. A, mRNA levels of ALDH1B1 and ALDH2 were measured by Q-PCR. The hepatic level
of ALDH1B1 mRNA was set as the control (ϭ 1) after normalization with ␤-actin. Relative levels of mRNA are reported as fold of control. The ratio of ALDH2/ALDH1B1
mRNA levels is indicated for each tissue examined. Data represent mean Ϯ S.E. from three to four mice. B, protein levels of ALDH1A1, ALDH1B1, and ALDH2 were
detected by Western blot in tissues from wild-type (ϩ/ϩ) and ALDH2-null (Ϫ/Ϫ) mice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
mic punctate pattern in both tissues, supporting an organelle- and alcohol hypersensitivity among whites to be polymorphisms in
associated localization of ALDH1B1.
the human ALDH1B1 gene, which encodes another mitochondrial
ALDH isoenzyme (Husemoen et al., 2008; Linneberg et al., 2010).
These studies and others (Stewart et al., 1995) are suggestive of a
potentially important role of ALDH1B1 in the metabolism of acetal-
dehyde, but more direct confirmation remains to be established.
Herein, we have cloned, expressed, and purified recombinant human
ALDH1B1 using a baculovirus expression system along with affinity
chromatography. Human ALDH1B1 is catalytically active toward a
wide range of aldehyde substrates and uses NADϩ, but not NADPϩ,
as the cofactor. The optimal substrates of human ALDH1B1 (Km Ͻ 1
␮M) are medium-chain aldehydes, including hexanal and nonanal.
Short-chain propionaldehyde and aromatic benzaldehyde are also
good substrates for ALDH1B1. Most importantly, human ALDH1B1
exhibits an intermediate Km (55 ␮M) for acetaldehyde compared with
human ALDH2 (Km ϭ 3.2 ␮M) and human ALDH1A1 (Km ϭ 180
␮M) (Klyosov et al., 1996), suggesting that ALDH1B1 indeed rep-
resents a low Km ALDH for acetaldehyde. Thus, our study provides
Expression of ALDH1B1 in Human Tissues. The expression of
ALDH1B1 in human tissues, including liver, pancreas, small intes-
tine, colon, and lung, was investigated by immunohistochemical stain-
ing (Fig. 6). The expression patterns of ALDH1B1, in terms of the
distribution and intensity of immunopositivity, in these human tissues
were quite consistent with what was seen in mouse tissues (Fig. 4A).
Furthermore, the punctate positive-staining of human ALDH1B1 was
clearly noted in the liver and pancreas.
Discussion
The major enzyme that metabolizes acetaldehyde, the most toxic
metabolite of ethanol, is mitochondrial ALDH2. The inactive variant
(E487K) of human ALDH2 is responsible for ethanol-induced hyper-
sensitivity, which serves as a deterrent against alcoholism in Asian
populations (Yoshida et al., 1984). Although approximately 50% of
Asians are carriers of this variant, it is nearly absent in whites. Recent direct evidence to support the hypothesis that ALDH1B1 represents
studies have identified the genetic determinants of drinking behavior the second mitochondrial ALDH, aside from ALDH2, that actively

INITIAL CHARACTERIZATION OF HUMAN ALDH1B1
1685
FIG. 5. Tissue distribution of ALDH1B1 protein
by immunohistostaining. Mouse tissues were pro-
cessed for immunohistochemical (A) or immuno-
fluorescent (B) analysis of ALDH1B1 as de-
scribed under Materials and Methods.
A, representative images of tissue sections stained
for general histology [hematoxylin and eosin
(H&E)], ALDH1B1 immunoreactivity (␣-
ALDH1B1), and serum control (Pre-Immune).
Liver displayed intense ALDH1B1 immuno-
positivity (brown) with a pericentral distribu-
tion. The absorptive epithelial cells of small
intestine also showed high staining. Immuno-
positivity in the large intestine appeared to co-
localize with stem cells. In the lung, ALDH1B1
was found to be expressed predominantly in the
bronchiolar epithelium and Clara cells of small
airways. In the testes, intense immunoreactivity
was observed in the epithelial cells lining the
seminiferous tubules. In the thymus, immuno-
positivity was detected specifically in the me-
dulla. B, confocal immunofluorescent staining
for ALDH1B1 (green) in the liver and small
intestine exhibited a cytoplasmic punctate pat-
tern, in agreement with an organelle-associated
localization of ALDH1B1. Sections were
costained with DAPI for nuclear staining. The
overlay represents the ALDH1B1 and DAPI
images combined. Original magnification,
400ϫ (A); 1000ϫ (B).
oxidizes acetaldehyde and consequently may be important for the et al., 1995). Such sensitivity of ALDH1B1 to disulfiram inhibition
metabolism of ethanol. may have therapeutic ramifications for the use of disulfiram
Disulfiram is a drug used to treat chronic alcoholism by produc- in vivo.
ing an undesirable acute sensitivity to alcohol. The effect of
An interesting observation is that recombinant human ALDH1B1
disulfiram is attributable to its action in elevating acetaldehyde expressed in Sf9 insect cells migrates as double bands by SDS-PAGE.
levels by inhibiting ALDH1A1 and ALDH2 (Petersen, 1992). Based on their locations relative to that of the molecular weight
Recombinant human ALDH1A1 (Ki ϭ 0.2 ␮M) is 60 times more marker, the two protein bands correspond to Ϸ55 and Ϸ58 kDa
sensitive to inhibition by disulfiram than recombinant human peptides. MALDI-TOF MS and Western blot analysis confirmed the
ALDH2 (Ki Ͼ 12 ␮M) (Lam et al., 1997). In the present study, identity of both peptides to be ALDH1B1. We propose that the
recombinant human ALDH1B1 was inhibited by disulfiram but 58-kDa protein band represents the 517-residue intact translated pep-
to a lesser extent than ALDH1A1 when propionaldehyde was used tide and the 55-kDa protein represents the mature form after the
as substrate, a finding consistent with a previous study (Stewart mitochondrial lead signal is excised. Alternatively, the 58-kDa protein
FIG. 6. Distribution of ALDH1B1 in human tissues by immunohistochemistry. Formalin-fixed and paraffin-embedded normal human tissues were processed in accordance
with approvals and guidelines for IHC staining using polyclonal anti-human ALDH1B1. The absorptive epithelium of small intestine revealed the highest intensity of
ALDH1B1 immunopositivity (brown). Hepatocytes and pancreatic acinar cells also showed strong positive staining for ALDH1B1, which displayed a cytoplasmic punctate
pattern. The immunopositivity of ALDH1B1 in colon appeared to colocalize with stem cells. In the lung, ALDH1B1 seemed to be expressed predominantly in the
bronchiolar epithelium and Clara cells of small airways. Original magnification, 400ϫ (top panels); 1000ϫ (bottom panels).

1686
STAGOS ET AL.
band could be a post-translationally modified form of ALDH1B1, medulloblastoma cell lines resistant to cyclophosphamide, suggesting
e.g., one that has been glycosylated or phosphorylated or both. Gly- a possible role of ALDH1B1 enzyme in cancer drug resistance (Ba-
cosylation has been documented previously for microsomal ALDH colod et al., 2008). Another study reported that the dynamic expres-
(Masaki et al., 1996). It has been determined that phosphorylation of sion of ALDH1B1 correlated with granulocytic development of he-
ALDH2 occurs. One study reported that ALDH2 is phosphorylated matopoietic stem cells; in this report, the author proposed a possible
and activated by the survival kinase protein kinase C␧ and that role of ALDH1B1 in retinoic acid metabolism mediating the effect
activation of ALDH2 is cardioprotective (Chen et al., 2008). On the (Luo et al., 2007). A transgenic mouse line having forced expression
other hand, it has also been reported that carbon tetrachloride expo- of the human ALDH2*2 variant exhibited distinct phenotypes, includ-
sure inhibits ALDH2 activity through c-Jun NH2-terminal kinase- ing small body size, reduced muscle mass, diminished fat content, and
mediated phosphorylation; inactivation of ALDH2 contributes to car- osteopenia (Endo et al., 2009), all of which are absent from ALDH2-
bon tetrachloride-induced liver damage (Moon et al., 2010). These null mice (Yu et al., 2009). One hypothesis to explain such a discrep-
studies suggest that the effect of phosphorylation of ALDH2 and ancy is that the ALDH2*2 mutant subunit not only inactivates the
probably of other ALDHs, is variable, leading to activation or inac- ALDH2 wild-type subunit but also inactivates other ALDHs by form-
tivation of ALDH depending on the specific stress. Further studies are ing heterotetramers, thereby blocking their functions. Given Ͼ75%
needed to determine whether phosphorylation is a mechanism regu- homology in peptide sequence and the same mitochondrial localiza-
lating ALDH1B1.
tion, the ALDH1B1 subunit could be a potential binding partner of the
ALDH1B1 messenger is actively transcribed in multiple human ALDH2 subunit. Taken together, these observations indicate the in-
tissues (Hsu and Chang, 1991; Stewart et al., 1996). In the present volvement of ALDH1B1 in multiple physiological or pathological
study, we observed a similar tissue expression profile in mouse processes, the biochemical mechanism(s) of which merits further
tissues. Liver had the highest level of ALDH1B1 and ALDH2 investigation.
mRNAs relative to those of other tissues examined, supporting an
In summary, the present study describes, for the first time, the
important role of the two mitochondrial ALDHs in this organ. In expression, purification, and biochemical characterization of the hu-
most tissues, except in the intestine, ALDH2 messenger is pre- man ALDH1B1 enzyme. Our results demonstrate that mitochondrial
dominant by up to 3 orders of magnitude. Much higher levels of ALDH1B1 represents a low Km for ALDH for acetaldehyde metab-
ALDH2 mRNA relative to ALDH1B1 mRNA have also been found olism and therefore is consistent with ALDH1B1 being actively
in human liver (Hsu and Chang, 1991). The predominance of hepatic involved in the metabolism of ethanol. The differential tissue distri-
ALDH2 over ALDH1B1 could possibly explain the inability of Asian bution of ALDH1B1 argues in favor of a role for ALDH1B1 in
carriers of the ALDH2*2 allele to efficiently metabolize acetaldehyde intestinal tissues. The observed biochemical properties of ALDH1B1
produced after alcohol consumption despite the presence of the are consistent with a physiological role(s) for this ALDH isoenzyme.
ALDH1B1 enzyme. Along the mouse gastrointestinal tract, there
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