Inactivation of human liver bile acid CoA:amino
acid N-acyltransferase by the electrophilic
E. M. Shonsey,1,†S. M. Eliuk,†M. S. Johnson,§S. Barnes,*,†,**,§§C. N. Falany,†
V. M. Darley-Usmar,§,††and M. B. Renfrow*,§§
Departments of Biochemistry and Molecular Genetics,* Pharmacology and Toxicology,†and Pathology,§
Comprehensive Cancer Center Mass Spectrometry Shared Facility,** Center for Free Radical Biology,††
and University of Alabama (UAB) Biomedical Fourier Transform-Ion Cyclotron Resonance Mass
Spectrometry Laboratory,§§University of Alabama at Birmingham, Birmingham, AL 35294
N-acyltransferase (BAT) catalyzes the formation of amino
acid-conjugated bile acids. In the present study, protein car-
bonylation of BAT, consistent with modification by reactive
oxygen species and their products, was increased in hepatic
homogenates of apolipoprotein E knock-out mice. 4-Hydroxy-
nonenal (4HNE), an electrophilic lipid generated by oxida-
tion of polyunsaturated long-chain fatty acids, typically reacts
with the amino acids Cys, His, Lys, and Arg to form adducts,
some of which (Michael adducts) preserve the aldehyde (i.e.,
carbonyl) moiety. Because two of these amino acids (Cys
and His) are members of the catalytic triad of human BAT,
it was proposed that 4HNE would cause inactivation of this
enzyme. As expected, human BAT (1.6 mM) was inactivated
by 4HNE in a dose-dependent manner. To establish the sites
of 4HNE’s reaction with BAT, peptides from proteolysis of
4HNE-treated, recombinant human BAT were analyzed by
peptide mass fingerprinting and by electrospray ionization-
tandem mass spectrometry using a hybrid linear ion trap Fou-
rier transform-ion cyclotron resonance mass spectrometer.
The data revealed that the active-site His (His362) dose-
dependently formed a 4HNE adduct, contributing to loss
of activity, although 4HNE adducts on other residues may
also contribute.—Shonsey, E. M., S. M. Eliuk, M. S. Johnson,
S. Barnes, C. N. Falany, V. M. Darley-Usmar, and M. B.
Renfrow. Inactivation of human liver bile acid CoA:amino
acid N-acyltransferase by the electrophilic lipid, 4-hydroxy-
nonenal. J. Lipid Res. 2008. 49: 282–294.
The hepatic enzyme bile acid CoA:amino acid
Supplementary key words
tions & mass spectrometry
coenzyme A & post-translational modifica-
Inflammation plays a major role in many types of liver
disease, including cholestasis (1) and both nonalcoholic
fatty liver disease (2) and alcoholic fatty liver disease (3).
In cholestasis, it has been hypothesized that the inflam-
matory cytokine pathway that is activated during inflam-
mation causes a functional defect in bile secretion and
flow at the hepatocellular level (4). Release of excessive
reactive oxygen species (ROS) and reactive nitrogen spe-
cies (RNS) induces neutrophil and Kupffer cell activation
and leads to liver injury (5). ROS and RNS species also
play important physiological roles, including release by
immune cells to aid in pathogen killing and cell signaling
associated with mitochondrial electron transport (6, 7).
Normally, the level of oxidative stress is tightly regulated
and a balance is maintained between oxidant status and
antioxidant capacity. The liver cells’ endogenous anti-
oxidant systems consist of enzymes such as superoxide
dismutase and catalase (8) that metabolize ROS, and the
glutathione-dependent systems that regulate protein thiol
modification (9, 10). Nonetheless, these antioxidant sys-
tems can be overwhelmed, and the reactive species within
the systems can rise to a level at which damage can occur.
An increase in the rate of production of ROS or RNS,
or a decrease in their rate of removal, results in direct
damage to DNA and proteins, as well as indirectly by re-
action with ROS/RNS-modified lipids (11). Modifications
of DNA or proteins may be repaired, or they are removed
through degradation (11). Patients suffering from various
forms of liver disease, including nonalcoholic fatty liver
disease (12, 13) and chronic hepatitis C with steatosis
(14), show a marked decrease in the level of polyunsatu-
rated fatty acids (PUFAs) and an increase in the products
of lipid peroxidation (12, 13).
Lipid peroxidation occurs when PUFAs undergo free
radical-initiated oxidation that starts a chain reaction re-
sulting in the formation of electrophilic lipids (15). The
most biologically relevant reactive electrophilic lipids are
Manuscript received 4 May 2007 and in revised form 25 September 2007
and in re-revised form 26 October 2007.
Published, JLR Papers in Press, October 27, 2007.
1To whom correspondence should be addressed.
Copyright D2008 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
282Journal of Lipid Research
Volume 49, 2008
by guest, on June 3, 2013
the 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes
(15). One of the most thoroughly studied of these is 4-
hydroxynonenal (4HNE). Kawamura et al. (16) showed
an increase in levels of 4HNE protein adducts through
immunohistochemical staining by use of a polyclonal 4HNE
antibody in patients with primary biliary cirrhosis. Seki
et al. (17) detected 4HNE protein adducts in the hepa-
tocytes of patients suffering from alcoholic liver disease by
use of a monoclonal 4HNE antibody.
Although several studies have reported an increase in
levels of protein-aldehyde adduct formation in hepato-
cytes, relatively few modifications of specific proteins have
been characterized (18–20). To identify cellular targets
affected by inflammatory responses, models of sepsis have
been employed to identify the genes that undergo differ-
ential regulation. In a study of late sepsis induced for
18 h by a cecal ligation and puncture (CLP) method
in rats, three upregulated genes and six downregulated
genes werefound (21). The six downregulated genes in rat
liver were hydroxysteroid dehydrogenase, EST/189895/
mouse RNase4, IF1, albumin, a2u-globulin, and rat bile acid
CoA:amino acid N-acyltransferase (rBAT) (21). Furutani
et al. (22) also showed downregulation of rBAT follow-
ing partial hepatectomy and during sepsis. BAT is re-
sponsible for the conjugation of bile acids with amino
acids. This is a two-step reaction in which bile acids are
first converted to CoA thioesters by bile acid CoA li-
gase and are then conjugated with an amino acid by
BAT (23, 24).
The loss of BAT message is thought to play a role in the
disturbance of bile acid metabolism that contributes to
the liver failure observed in the CLP model of sepsis (21).
This leads to the hypothesis that a decrease in BAT activity
would play a role in the overall failure of the liver as well.
Similarly, in a case in which excessive lipid peroxidation
occurs, modification of the enzyme by electrophilic lipids
may result in a decrease in activity of human BAT (hBAT)
that could also contribute to liver damage and failure.
This raises the following questions: does 4HNE decrease
BAT activity and would protein-aldehyde adduct for-
mation cause disruption of protein structure significant
enough to affect the enzyme’s activity?
hBAT belongs to a protein family of a/b fold hydro-
lases and has an enzymatic mechanism based on a cata-
lytic triad, Cys-His-Asp (24). Two of these amino acid
residues (His and Cys) are potential targets for modifica-
tion by electrophilic lipids. Mutation studies have dem-
onstrated that replacement of each member of the
catalytic triad eliminates the activity of the enzyme (24).
hBAT activity has been detected in both the peroxisomes
and the cytosol of hepatocytes, indicating that BAT is
localized in these two regions of the cell (25–29).
Traditionally, posttranslational modifications have been
identified on proteins through the use of specific anti-
bodies; although this technique is useful in gaining an
understanding of overall modification within a system,
it cannot provide unequivocal information on the actual
site of modification, particularly because of an antibody’s
epitope bias. More recently, mass spectrometry (MS) has
become a standard tool in the identification of adduct
formation. 4HNE adducts are identified by an increase
in the expected mass of proteolytic peptides by either
a Michael adduct (156.1150 Da), a Schiff base adduct
(138.1045 Da), or a 2-pentylpyrrole adduct (120.0939 Da)
(30–32). The presence of an aldehyde adduct is then con-
firmed by use of tandem mass spectrometry (MS/MS),
which allows direct assignment of the modification to a
specific amino acid (32). Fourier transform-ion cyclotron
resonance mass spectrometry (FT-ICR MS) provides high
resolution and high mass accuracy and has become widely
used in the characterization of posttranslational modifica-
tions, including phosphorylation (33, 34), glycosylation
(35, 36), and others (37, 38). The combination of FT-ICR
MS with the rapid MS/MS scan rate of a two-dimensional
linear ion trap provides a powerful tool for the investiga-
tion of site-specific posttranslational modifications.
In this study, hBAT was incubated with a range of 4HNE
concentrations, producing changes in the activity of the
enzyme that were correlated with the extent of posttrans-
lational modifications induced by 4HNE. FT-ICR MS/MS
was performed on proteolytic digests of the untreated and
4HNE-treated hBAT to identify sites of modification. The
potential for these modifications to occur in vivo was as-
sessed in the liver in the apolipoprotein E (apoE) knock-out
mouse using protein carbonyl formation as a surrogate
index of modification by reactive aldehydes. This animal
model of hypercholesterolemia is associated with increased
oxidative stress and protein modification (39). Increased
protein carbonyls were associated with BAT in the apoE
knock-out mouse liver homogenates compared with those
from wild-type mice, thus providing a potential mecha-
nism linking oxidative stress and regulation of BAT activity.
MATERIALS AND METHODS
Sodium chloride, Trizma base, Luria Bertani broth (LB),
ampicillin, isopropylthio-b-D-galactoside (IPTG), and 1-butanol
were purchased from Fisher Scientific (Norcross, GA). Biotin
was purchased from Research Organics (Cleveland, OH). 4HNE
was purchased from Calbiochem (San Diego, CA). Trypsin (se-
quencing grade) and Softlink avidin (avi) were purchased from
Promega (Madison, WI). Chymotrypsin and protease inhibitor
cocktail were purchased from Roche (Nutley, NJ). Biotin hydra-
zide was purchased from Pierce Chemical Co. (Rockford, IL).
Diethylaminoethyl (DEAE) -Sephacryl was obtained from Bio-
Rad (Hercules, CA). Chloramphenicol was purchased from
Acros Organics (Belgium). Bugbuster solution was obtained
from Novagen (San Diego, CA). Cholyl CoA was synthesized as
described by Shah and Staple (40). Its purity was assessed by its
absorbance at 230 nm and by negative-ion electrospray ioniza-
tion mass spectrometry (ESI MS) (m/z 1,159 [M-H]2; m/z 579
[M-2H]22; there was no m/z 407 ion that would indicate the pres-
ence of unreacted cholic acid).
Preparation of mouse liver homogenates. Fresh livers from apoE
knock-out and C57/BL6 mice were minced and homogenized
in a glass tissue grinder using ice-cold isolation medium contain-
Inactivation of hBAT by 4HNE 283
by guest, on June 3, 2013
ing 0.25 M sucrose, 1 mM EDTA, and 5 mM Tris-HCl, pH 7.4.
Nuclear debris and cellular debris were removed by centrifuga-
tion at 3,000 g for 10 min at 4jC.
Biotin hydrazide labeling of protein carbonyls and protein electro-
phoresis. Liver homogenate (50 mg as determined by Bradford
assay) was reacted with biotin hydrazide (1 mM in phosphate-
buffered saline) at 37jC for 1 h and then precipitated with 4 vols
methanol, followed by 3 vols water, followed by 1 vol chloroform.
Samples were centrifuged for 20 min at 16,000 g at 4jC.
Protein pellets were allowed to air dry at 37jC. Controls were
treated under identical conditions. The precipitated protein
was subjected to SDS-12.5% polyacrylamide gel electrophoresis
according to Laemmli (41). Dried protein samples were
resolubilized in 25 ml 23 SDS-PAGE sample buffer (20 ml b-
mercaptoethanol/ml buffer) and 25 ml water to give a 1 mg/ml
protein concentration. The hydrazide-linked proteins (15 mg)
were separated by SDS-PAGE (12.5% polyacrylamide at 100 V)
followed by Western blotting (overnight at 35 V). The mem-
brane was blocked with 5% blotting-grade blocker nonfat dry
milk (Bio-Rad) in TBS-T (0.25 M Tris, 35 mM NaCl, 27 mM KCl,
0.5% Tween 20) for 2 h at room temperature. The membranes
were then incubated with streptavidin HRP conjugated to goat
anti-rabbit IgG (Amersham) at 1/10,000 for 1 h at room tem-
perature. The signal was detected using chemiluminescence
(Pierce). Quantitation was done using AlphaEase FC software
(AlphaInnotech; San Leandro, CA), and the amount of car-
bonyl adduct formed was calculated using a biotinylated
cytochrome-C internal standard (42).
The precipitated biotin hydrazide-labeled protein was also
subjected to 2D-electrophoresis. The proteins were focused on
linear pH 5–8 isoelectrofocusing strips in the first dimension
and then resolved by electrophoresis on an SDS-12.5% poly-
acrylamide gel. The proteins were transferred to Immobilon-
FL polyvinylidene fluoride membrane (Millipore), which was
blocked with Odyssey blocking buffer. The blot was then probed
with a polyclonal rabbit anti-mouse BAT antibody (43), followed
with streptavidin Alexa fluor 680 conjugate and a fluorescent
secondary antibody, Alexa fluor 800 goat anti-rabbit IgG (In-
vitrogen). These two tags fluoresce at different wavelengths,
which permits simultaneous analysis of two targets on the same
blot. The membrane was scanned with a fluorescence imager
(Odyssey LiCor Infrared Imager) at wavelengths of both 700 nm
and 800 nm to detect both fluorescent components.
In vitro expression and biotinylation of hBAT in Escherichia
coli. Escherichia coli cells (BL-21) transformed with a modified
pET21a1 expression vector encoding hBAT with a C-terminal
avi-tag (24), as well as an expression vector containing the gene
for biotin ligase, were incubated in 5 ml cultures of LB at 37jC
and shaken at 180 rpm for 18 h. The 5 ml cultures contained
5 ml of 0.1 mg/ml ampicillin and 5 ml of 0.01 mg/ml chlor-
amphenicol. The cultures were placed in 400 ml LB contain-
ing 400 ml of 0.1 mg/ml ampicillin and 400 ml of 0.01 mg/ml
chloramphenicol. The cultures were incubated for 2.5 h at
37jC with shaking at 250 rpm. After 2.5 h, expression was in-
duced through the addition of IPTG to a final concentration of
400 mM. To achieve biotinylation, biotin was added to a final
concentration of 250 mM. The cultures were incubated at 30jC
with shaking at 200 rpm for a further 3 h. Following the incu-
bation time, the cultures were centrifuged at 3,000 g for 20 min
at 25jC to obtain a bacterial pellet.
Purification of hBAT. Cell pellets were resuspended in 5 ml
Bugbuster solution and 5 ml benzonase per milligram bacterial
protein, and incubated at room temperature for 30 min for
lysis. One half of a protease inhibitor cocktail tablet (Roche)
was also added. The lysates were centrifuged at 140,000 g for
40 min at 4jC. The supernate was applied to a DEAE-Sephacryl
column (20 ml bed volume) that had been preequilibrated with
5 bed volumes of 50 mM Tris-HCl, pH 8.5, containing 5 mM
NaCl. The DEAE column was then washed with 10 bed volumes
of 50 mM Tris-HCl, pH 8.5, to remove unbound proteins. Elu-
tion of bound proteins was performed by use of a 40 ml gra-
dient of 5–1,000 mM NaCl in 50 mM Tris-HCl, pH 8.5, collecting
2 ml fractions. The fractions were tested for BAT activity, and
the active fractions were applied to a Softlink avi column pre-
equilibrated with 5 bed volumes of 5 mM NaCl in 50 mM Tris-
HCl, pH 8.5. The avi column was washed with 20 bed volumes
of 50 mM Tris-HCl, pH 8.5, to remove nonbound proteins. Bio-
tinylated hBAT was eluted from the column by use of 3 bed
volumes of 50 mM Tris-HCl, pH 8.5, containing 10 mM biotin.
Preparation of 4HNE-modified hBAT. hBAT was modified by
4HNE using a method described by Isom et al. (32). Purified
hBAT (160 pmol in a total volume of 100 ml) was mixed in a glass
tube with 50 mM potassium phosphate buffer, pH 7.4, and a
range of 4HNE concentrations (4 mM to 128 mM) in ethanol
and incubated at 4jC for 1 h. The reaction was terminated by
quenching with 1 mM histidine (for the hBAT enzyme assay) or
5 ml 0.1% (v/v) formic acid (for protein mass spectrometry
experiments). A vehicle control was performed with the addi-
tion of ethanol in place of 4HNE and incubation under the
Radioassay of BAT activity. hBAT enzyme activity was assayed as
described by Johnson, Barnes, and Diasio (44). Briefly, 12.5 ml
of 0.8 mM cholyl-CoA, 12.5 ml of
12.5 ml of 100 mM potassium phosphate buffer, pH 8.4, and
2 pmol of enzyme were mixed in an Eppendorf tube and incu-
bated at 37jC for 15 min. The reaction was terminated by the
addition of 0.5 ml of butanol-saturated K2HPO4, pH 2.0, con-
taining 2% w/v SDS. The mixture was vortexed thoroughly, and
then 0.5 ml of water-saturated butanol was added. Following a
1 min vortex, the mixture was centrifuged at 2,000 g for 5 min
at room temperature. The butanol layer was transferred to a
clean Eppendorf tube and backwashed with 0.5 ml of the potas-
sium phosphate buffer. The mixture was vortexed for 1 min
and centrifuged at 2,000 g for 5 min at room temperature. An
aliquot (250 ml) of the butanol layer was mixed with 4.5 ml
of scintillation fluid, and the radioactivity was measured in a
liquid scintillation counter.
3H-taurine (2 mCi/mmol),
Proteolysis of hBAT and matrix-assisted laser desorption ionization
time-of-flight mass spectrometry peptide mass fingerprinting. Both the
native and modified forms of hBAT (30 ml of a 1.6 mM solution)
were digested with either trypsin (10 ml of a 12.5 ng/ml solution)
at 37jC or chymotrypsin (10 ml of a 12.5 ng/ml solution) at room
temperature overnight. The peptide digests were analyzed by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF MS). Aliquots (1 ml) were mixed with
3 ml of a saturated solution of a-cyano-4-hydroxycinnamic acid
in a 50/50 mixture of 0.1% trifluoroacetic acid and acetoni-
trile. An aliquot (1 ml) of the mixture was spotted on the MALDI
target plate and allowed to dry for 5 min. The molecular ions
were generated by a pulsed N2laser at 337 nm and analyzed by
delayed extraction in the positive reflector ion mode using an
Applied Biosystems (Foster City, CA) Voyager DE-PRO mass spec-
trometer with an acceleration voltage of 20 kV. The acquired
spectra, an accumulation of 300 laser shots, were analyzed using
284 Journal of Lipid Research
Volume 49, 2008
by guest, on June 3, 2013
DataExplorer: (Applied Biosystems). Internal calibration was
carried out using one of the digestive enzyme autolysis peaks,
m/z 2,162.05 for trypsin and m/z 1,528.58 for chymotrypsin. Spec-
tra were baseline corrected and noise filtered.
Liquid chromatography-ESI FT-ICR MS/MS analysis of 4HNE-
modified peptides. The proteolytic digests of hBAT (20 pmol)
were separated by nanoLC (Eksigent; Dublin, CA) on a 15 cm 3
75 mm inner diameter reverse-phase C18column with a linear
gradient of 5–95% acetonitrile in 0.1% formic acid at a flow
rate of 200 nl/min21. FT-ICR MS analysis was performed on a
linear quadrupole ion trap (LTQ) FT ICR hybrid mass spec-
trometer (LTQ FT; Thermo Electron, San Jose, CA). Eluted
tryptic and chymotryptic peptides were electrosprayed at 2 kV.
Peptide fragmentation was induced by collision-induced disso-
ciation (CID) in the ion trap, and fragment ions were also
analyzed in the ion trap. The LTQ FT mass spectrometer was
operated in a “top three” data-dependent acquisition mode.
The mass spectrometer was set to switch between an FT-ICR MS
full scan (200 m/z–2,000 m/z) followed by successive FT-ICR
MS single-ion monitoring scans and LTQ MS/MS scans of the
three most abundant precursor ions in the FT-ICR MS full
scan as determined by the Xcalibur software (Thermo Electron).
Dynamic exclusion was enabled after a repeat count of three
for a period of 90 s.
Data analysis. The MALDI-TOF MS peaks were compared
with in silico digests obtained from ProteinProspector (http://
prospector.ucsf.edu) for both hBAT and the digestive enzymes
in order to eliminate any autolysis peaks as potential modified
hBAT peptides. The liquid chromatography-electrospray ioniza-
tion (LC-ESI) LTQ MS/MS data were searched against a cus-
tom FASTA sequence database containing hBAT, as well as
unrelated human proteins as a negative control, by use of the
TurboSEQUEST algorithm within Bioworks 3.2 (Thermo Elec-
tron). Monoisotopic precursor and fragment ion masses were
searched with a mass tolerance of 5 ppm and 5 ppm, respectively.
For identification of 4HNE-modified peptides, the TurboSEQUEST
searches were amended to search for the mass additions of
156.1150 Da, 138.1045 Da, and 120.0939 Da for Michael, Schiff
base, and 2-pentylpyrrole adducts, respectively. Additionally, FT-
ICR MS spectra were extracted out of each sample data set for
manual identification of modifications based on high mass ac-
curacy. The modified peptides were manually validated by their
absence in the unmodified FT-ICR MS spectra; a mass accuracy
cut-off of 2 ppm was used.
Formation of BAT protein carbonyls is increased in
the liver of the apo E2/2mouse
Homogenates were prepared from the livers of apoE
knock-out and C57/BL6 mice. Protein carbonyls were then
measured by their derivatization using biotin hydrazide,
followed by 1D-electrophoretic separation of the proteins
and assessment of the biotin signal with fluorescent strep-
tavidin. Under these conditions, protein carbonyls could
be formed from ROS and/or by modification with reactive
lipid aldehydes such as 4HNE (45, 46). There was a sig-
nificant increase in protein carbonylation (P , 0.05) in
the liver homogenates of apoE knock-out mice compared
with C57/BL6 mice (Fig. 1A). Analysis by 2D-isoelectric
focusing/SDS-PAGE and Coomassie blue staining dem-
onstrated that equal protein loadings had occurred
(Fig. 1B). Western blot analysis with a specific polyclonal
anti-mouse BAT antibody revealed that mouse BAT was
present in several isoforms with decreasing isoelectric points
In apoE knock-out liver homogenates, more acidic iso-
forms of mouse BAT were present (Fig. 1D, right panel).
Visualization with fluorescent streptavidin demonstrated
that many liver proteins were carbonylated (Fig. 1C). Colo-
calization of the biotin signals and those immunoreactive
to BAT occurred for the apoE knock-out mice, but not
for the C57/BL6 mice (Fig. 1E).
Purification of hBAT
hBAT, subcloned into a modified pET21a1 vector that
contains a C-terminal avi-tag allowing for affinity purifica-
tion, was coexpressed with biotin ligase, which adds biotin
to the avi-tag during protein expression. Following overex-
pression in E. coli BL-21 cells, the recombinant protein was
purified on a DEAE-Sephacryl column by anion exchange
chromatography. hBAT eluted at 120 mM NaCl, with a
recovery of approximately 90% of its enzyme activity. The
active fractions were pooled, and proteins were subjected
to affinity chromatography on a Softlink avi column. The
Softlink avi column uses competitive elution with free biotin,
allowing the eluted enzyme to retain activity. Following the
affinity purification, 69% of enzyme activity was recovered.
The purification resulted in a 0.2 mg/ml solution of greater
than 95% pure hBAT-avi that gave a single band by SDS-
PAGE analysis (Fig. 2). The purified form of hBAT was used
in subsequent activity and mass spectrometry experiments.
Inactivation of hBAT-avi following modification
Purified hBAT (1.6 mM) was incubated with 4HNE over
a range of concentrations to determine its effect on en-
zyme activity. hBAT (1.6 mM) was incubated with increas-
ing concentrations of 4HNE (4 mM, 8 mM, 16 mM, 32 mM,
and 64 mM) for 1 h. The 4HNE concentrations were
chosen to mimic physiologically relevant levels of 4HNE
within the liver, which have been reported in ranges
from the nanomolar to millimolar level (47). Reduction of
hBAT activity was observed to be dose dependent (Fig. 3).
At the two highest levels of 4HNE treatment (128 mM and
64 mM), hBAT activity was not detected. At 32 mM 4HNE,
the treated protein retained 5.0 6 3.5% activity when com-
pared with untreated hBAT. At the lower concentrations
of 4HNE (16 mM, 8 mM, and 4 mM), reduction in enzyme
activitywaslesssevere(25.76 3.1%,61.96 7.8%,and84.26
8.6%, respectively). These results indicate that 4HNE-
dependent modifications occur to the protein that reduce
the enzyme activity in a dose-dependent manner. Next, we
determined the sites of modification by 4HNE in hBAT.
4HNE modification of hBAT peptide mass fingerprinting
To assess the presence of 4HNE modifications, both
tryptic and chymotryptic digests of native and modified
Inactivation of hBAT by 4HNE 285
by guest, on June 3, 2013
Fig. 1. Increased oxidation of mouse bile acid CoA:amino acid N-acyltransferase (BAT) in the liver of apo-
lipoprotein E (apoE) knock-out mice. A: Wild-type and apoE knock-out mouse liver homogenates (50 mg
protein) were treated with 1 mM biotin hydrazide. Proteins (15 mg) were separated by SDS-polyacrylamide
gel electrophoresis, followed by Western blot analysis. Biotin incorporation corresponding to the degree
of carbonyl formation was quantified using biotinylated cytochrome C as a standard. Results are expressed
as mean 6 SE, n 5 3. *P , 0.05 relative to wild-type. B–E: Liver homogenate (50 mg) was subjected to
2D isoelectric focusing SDS-PAGE. The proteins were resolved in the first dimension over a linear pH range
of 5–8 and separated in the second dimension using a 12.5% polyacrylamide gel and either stained with
comassie blue (B) or transferred to a polyvinylidene difluoride membrane and blocked with 5% milk. Biotin
hydrazide-labeled proteins (red) were detected with fluorescent-streptavidin (C), and mouse BAT (green) was
detected with a polyclonal rabbit anti-mouse BAT antibody followed by a fluorescent goat anti-rabbit anti-
body (D). Cofluorescence, indicating oxidation of BAT on the merged gel images, is shown in yellow (E).
286 Journal of Lipid Research
Volume 49, 2008
by guest, on June 3, 2013
hBAT were analyzed by use of MALDI-TOF MS. The
spectra obtained for the modified peptides were com-
pared against the spectra obtained for the peptides de-
rived from the unreacted hBAT to identify new peaks that
correlated to mass shifts associated with 4HNE modifica-
tions. Figure 4 shows the tryptic peptide mass fingerprint
MALDI-TOF MS spectra for untreated hBAT (Fig. 4A),
as well as hBAT following modification with 1 mM 4HNE
Comparison of the native hBAT MALDI-TOF mass spec-
trum with the 4HNE-modified BAT spectrum revealed the
presence of a new ion species (m/z 1,478) in the modified
BAT spectrum that corresponded to a 156 Da mass addi-
tion to the ion at m/z 1,322. This represents a putative
4HNE adduct to the peptide AHAEQAIGQLKR (residue
numbers 335–347). Because 4HNE modification to a lysine
or an arginine could prevent trypsin cleavage (32), a sec-
ond proteolytic enzyme (chymotrypsin) was used to de-
crease the chance of missing such modifications.
Figure 5 shows both the native (Fig. 5A) and modified
(Fig. 5B) chymotryptic MALDI-TOF MS for hBAT. As in
the tryptic mass spectrum, new ion species were detected
in the modified spectrum that corresponded to 156 Da
mass additions to unmodified ions. These peptide ions, at
m/z 1,195.7, 1,320.6, 1,719.9, and 1,914.5 in the modified
mass spectrum represent putative 4HNE adducts to hBAT.
For all four peptide ions (K345-W353, K93-F101, H271-
L284, and R51-L66), the mass difference corresponded
to a single Michael adduct to the peptides; however, each
of these peptides contains more than one possible site of
4HNE adduct formation. This initial analysis of 4HNE
adducts in the hBAT showed that modifications occurred
to hBAT and led to the putative identification of five sites
of modification; however, the precise residues could not
Analysis of 4HNE-modified hBAT peptides by use of
LC-ESI LTQ FT-ICR MS and MS/MS
After initial MALDI-TOF MS analysis indicated the pres-
ence of 4HNE adducts on hBAT peptides, sites of modi-
fication were localized for tryptic and chymotryptic digests
of 4HNE-modified hBAT by use of LC-ESI LTQ FT-ICR
MS/MS. This analysis was performed over a range of 4HNE
concentrations (8 mM–128 mM). The data were analyzed
by use of the TurboSEQUEST algorithm and by accurate
mass assignment of modified peptides from the FT-ICR
mass spectra. The high resolution and high mass accu-
racy (,2 ppm) of FT-ICR MS allowed manual inspection of
the mass spectra of tryptic and chymotryptic hBAT peptides
for the known mass increases of 156.1150 Da, 138.1045 Da,
and 120.0939 Da, corresponding to 4HNE Michael, Schiff
base, and 2-pentylpyrrole additions, respectively. At 128 mM
4HNE, 14 modifications were identified on 8 different
peptides. At the lowest concentration of 4HNE (8 mM),
7 modifications were found on 5 peptides. The results for
all concentrations of 4HNE are listed in Table 1.
Fig. 2. RecombinanthumanBAT(hBAT)-avidin(avi)isolatedfrom
Escherichia coli. Recombinant hBAT-avi was isolated from bacterial
cytosol (lane 1)in a two-steppurification. Cytosol was initiallyloaded
onto a DEAE column and eluted with NaCl gradient. Fractions re-
taining hBAT activity (pooled fractions, lane 2) were then loaded
onto an avi column (lane 3, flow through) and washed with 50 mM
Tris, pH 8.0 (lane 4). hBAT-avi was competitively eluted with biotin,
resulting in .95% purification (lane 5).
Fig. 3. Inhibition of hBAT activity by 4-hydroxynone-
nal (4HNE). hBAT (1.6 mM) was pretreated by in-
cubation with 4HNE (4 mM–64 mM) for 1 h at 4jC,
and then the mixture was quenched with 1.0 mM
histidine. The remaining enzyme activity of hBAT
was measured by use of a radioassay (see Materials
and Methods), with cholyl CoA (0.8 mM) and 1,2-3H-
taurine (0.1 mCi; 1 mM) as substrates. Each bar repre-
sents the average of three independent measurements
of hBAT-avi activity, with the results reported as a
percentage of the activity measured for unmodified
hBAT-avi. Error bars indicate 6 SD.
Inactivation of hBAT by 4HNE287
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MS/MS can unambiguously map specific sites of post-
translational modification (35), including amino acids
trometer fragments the tryptic and chymotryptic peptides
by CID in the ion trap. CID of peptide ions results in
the fragmentation of the peptides at the amide bonds to
produce b- and y-type fragment ions. When 1.6 mM hBAT
was reacted with 32 mM 4HNE, ten different sites of modi-
fication were localized with a mass accuracy of 2 ppm
by both TurboSEQUEST and manual inspection of the
FT-ICR mass spectra (Table 1). Figure 6 shows the LTQ
MS/MS spectrum of the triply charged m/z 625.6877 ion
from the chymotrypsin digest of 4HNE-treated hBAT.
The parent mass from the FT-ICR spectrum (not shown)
indicated that the ion species corresponded to the hBAT
chymotryptic peptide [271HGQIHQPLPHSAQL2831 2
Michael adducts 1 2H]21(1,875.0487 theoretical mass,
1,875.04856 observed mass, 0.07 ppm error). Inspection of
the LTQ MS/MS spectrum confirmed the peptide se-
quence with the addition of two Michael adducts as fol-
lows. From the N terminus, b1–b4ions are present with a
mass increase of 156.1, corresponding to a 4HNE Michael
adduct on the H271 residue. The remaining b ions (b5, b6,
b8, and b9) show no additional mass increase of 156.1.
This eliminates H274 as a site of 4HNE modification. From
the C terminus, the initial y2ion corresponds to the first
two amino acids, leucine and glutamine. The subsequent
observed y ions (y6–y9) have a mass increase of 156.1, cor-
responding to a 4HNE Michael adduct on H279. The re-
maining y ions (y10–y12), containing both H279 and H274,
show no additional mass increase of 156.1. This is in agree-
ment with analysis of the N-terminal b ions. The combina-
tion of fragment ions unambiguously assigns the two sites
of 4HNE Michael adducts as H271 and H279.
Figure 7 shows the LTQ MS/MS spectrum for a second
doubly charged 927.488521ion from the chymotrypsin
digest of 4HNE-treated hBAT. The parent mass from the
FT-ICR spectrum (not shown) indicated that the ion spe-
cies corresponded to the hBAT chymotryptic peptide
[356SYPGAGHLIEPPYSPL3711 Michael adduct 1 2H]21
(1,853.9689 theoretical mass, 1,853.9684 observed mass,
0.27 ppm error). This peptide includes H362, a member
of the catalytic triad. The LTQ MS/MS spectrum con-
firmed the peptide sequence with the addition of a sin-
gle Michael adduct. CID product ions from both N- and
C-terminal directions, b8–b14and y10–y14, confirm the ex-
Fig. 4. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) pep-
tide mass fingerprints of native and 4HNE-modified hBAT following trypsin digestion. After treatment with
1 mM 4HNE, tryptic digests of the untreated protein (A) and the 4HNE-modified hBAT (B) were analyzed
by MALDI-TOF MS. Several hBAT tryptic peptides were identified in the unmodified spectra (A). An addi-
tional tryptic peptide corresponding to [335A-R3461156]1was detected in the 4HNE-treated hBAT spectrum.
288 Journal of Lipid Research
Volume 49, 2008
by guest, on June 3, 2013
pected sequence with the addition of 156.1 to H362. This
mass addition is not seen on b3and b5ions N-terminal to
the addition or y3and y5–y9ions C-terminal to the addi-
tion. This unambiguously localizes the 4HNE Michael
adduct to the H362 residue. Table 1 shows the residues
modified at each concentration of 4HNE treatment.
Sequence coverage by two proteolytic digests
Analysis of 4HNE-modified peptides was performed by
tryptic and chymotryptic digests of hBAT and indicated
that 49 out of the 55 sites that could be modified by 4HNE
were observed. MS analysis of the hBAT trypsin digest
yielded peptides corresponding to 70% of the hBAT
Fig. 5. MALDI-TOF MS peptide mass fingerprints of native and 4HNE-modified hBAT following chymo-
trypsin digestion. hBAT was treated with 4HNE as described in the legend to Fig. 3. Following that reac-
tion, the untreated protein (A) and the 4HNE-modified hBAT (B) were digested with chymotrypsin and
analyzed by use of MALDI-TOF MS. Modified peptides are labeled in B.
TABLE 1.4HNE-modified peptides identified by LTQ FT-ICR MS/MS in hBAT at 4HNE concentrations from 128 mM to 8 mM
Modified Amino Acid
128 lM 64 lM 32 lM 16 lM8 lM
H271, H274, H279
NNWTLLSYPGAGHLIEPPYSPLCCASTTHDLRH362, C372, C373,
hBAT, human bile acid CoA:amino acid N-acyltransferase; 4HNE, 4-hydroxynonenal; LTQ FT-ICR MS/MS, linear quadrupole ion trap Fourier
transform-ion cyclotron resonance tandem mass spectrometry.
Inactivation of hBAT by 4HNE289
by guest, on June 3, 2013
amino acid sequence and localized 10 sites of modifica-
tion. Although the inclusion of an hBAT chymotrypsin
digest resulted in only a modest increase in sequence
coverage, the difference in the number of observed sites
of adduct formation was significant. Analysis of the chy-
motrypsin digest of hBAT increased the sequence cover-
age modestly, to 78.7%, but identified four additional sites
Semiquantitation of 4HNE-modified His362
The ion abundance of the chymotryptic peptide
356SYPGAGHLIEPPYSPL371containing the 4HNE-modi-
fied H362 versus the same peptide in an unmodified form
was compared at all levels of 4HNE treatment. The abun-
dances of each pair of ions were normalized against two
peptides (m/z 639.8212 and m/z 653.8654) that were not
modified at any level of 4HNE treatment. The semiquan-
titative analysis demonstrated a dose-dependent effect on
the level of modified and unmodified peptide detected. At
128 mM 4HNE, there was no detectable level of unmod-
ified peptide (Fig. 8). At 64 mM 4HNE, there was a low
level of unmodified peptide in comparison to the modi-
fied form of the peptide. The level of unmodified peptide
increased as the concentration of 4HNE decreased. At
8 mM 4HNE, the lowest concentration used, the modified
form of the peptide decreased, whereas the level of un-
modified peptide increased.
The present study demonstrates that carbonylation of
the important liver enzyme BAT occurs in a model of
oxidative stress, the apoE knock-out mouse. Furthermore,
the electrophilic lipid 4HNE, a product of oxidative stress
in the liver, causes a dose-dependent reduction of the
activity of recombinantly expressed human liver BAT.
More importantly, even at the lowest concentrations of
4HNE, amino acids that are part of or adjacent to the
active site are modified, as determined by high-resolution
FT-ICR-MS. These experiments, therefore, also provide
structural insight into the hBAT active site.
Bile acid amidation with the amino acids glycine and
taurine is a vital process in normal hepatobiliary and
gastrointestinal functions. Amidated bile acids form mixed
micelles in both the biliary tract and the lumen of the
small intestine. Mutations in hBAT have been identified in
patients who have decreased levels of amidated bile acids
(47). This results in a variety of symptoms associated with
a decrease in the ability to absorb fat and fat-soluble
Fig. 6. Linearquadrupoleiontraptandemmassspectrometry(LTQMS/MS)spectrumofthetriplycharged
4HNE-modified hBAT peptide NH2-SYPGAGHLIEPPYSPL-COOH at m/z 625.688. A single LTQ MS/MS
spectrum from the liquid chromatography-electrospray ionization LTQ Fourier transform-ion cyclotron
resonance mass spectrometry (LC-ESI LTQ FT-ICR MS) analysis of 4HNE-modified hBAT-avi digested with
chymotrypsin successfully localized a site of modification. Peptide backbone bonds (9 of 13) are cleaved.
Sites of 4HNE modification were identified from a series of differentially Michael adduct (156.1) -modified
fragment ions. The fragment ions uniquely localize two sites of 4HNE Michael adduction, to His271 and
His279, and eliminate His274 as a modified site. N-terminal fragment ions (b) and C-terminal fragment
ions (y) are indicated above and below the hBAT-avi chymotryptic peptide sequence, respectively. Stars,
290Journal of Lipid Research
Volume 49, 2008
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vitamins. In some forms of liver disease, including cho-
lestasis (1), nonalcoholic fatty liver disease (2), alcoholic
fatty liver disease (3), and atherosclerosis, oxidative stress
plays a key role in the progression of the disease. Sub-
sequently, lipid peroxidation increases the level of 4HNE
within the liver, and hBAT and other liver enzymes are
at a higher risk of modification by 4HNE. In the apoE
knock-out mouse, 4HNE production has been observed
through increased immunohistochemical staining using a
4HNE-specific antibody and is decreased by treatment
with antioxidants, which also have a beneficial effect on
the development of the disease (48). In the present study,
there was increased protein carbonyl formation in the
mouse liver in apoE knock-out mice compared with wild-
type mice. It was accompanied by marked carbonyl for-
mation associated with different isoforms of BAT in apoE
knock-out mouse liver, but not in the C57/BL6 mice.
To pursue the effect of an electrophilic lipid, in vitro
experiments were carried out with 4HNE and recom-
binantly expressed hBAT. At high concentrations of
4HNE (aldehyde-to-protein ratio of 80:1), BAT activity
was completely abolished. Over the range from 4 mM–
32 mM 4HNE, a dose-dependent decrease in hBAT ac-
tivity was observed, with 80% of the activity remaining at
an aldehyde-to-protein ratio of 2.5:1 (Fig. 2). This dose-
dependent effect was characterized at the molecular level
by use of LC-ESI LTQ FT-ICR MS and MS/MS to identify
and localize sites of modification.
4HNE modifies amino acids that contain nucleo-
philic groups, including histidines, lysines, and cysteines,
through the formation of Michael adducts (49). Lysine
residues also form both Schiff bases and 2-pentylpyrrole
adducts with 4HNE (30, 31). Recently, 4HNE has also been
shown to modify arginine residues, forming 2-pentylpyrrole
adducts (32). We hypothesize that as the inflammatory
process progresses, hBAT activity is diminished, leading to
an increase of unconjugated bile acids in the liver, a de-
crease in bile flow, and a decrease in conjugated bile acids
within the intestinal tract. All of these pathologies could
contribute to increased liver damage in pathologies asso-
ciated with increased hepatic ROS/RNS.
At the highest level of 4HNE treatment of hBAT, a total
of 14 distinct modifications were found. As expected, the
number of sites of modification decreased as the con-
centration of 4HNE treatment decreased (Table 1). The
observed modifications can be evaluated in the context
of what is known about the structure of hBAT, specifically
the active site. hBAT belongs to a protein family of a/b
fold hydrolases that are characterized by an enzyme mech-
anism based on a Cys-His-Asp catalytic triad (50). Previous
mutational studies have identified Cys235, Asp328, and
His362 as the residues critical to hBAT activity (24). These
three residues are located on three loops between a b-
strand and an a-helix. These loops are hypothesized to
fold, creating a substrate binding pocket. This a/b fold
structure was first characterized in the crystal structure of
Fig. 7. LTQ MS/MS spectrum of the 4HNE-modified doubly charged hBAT peptide NH2-SYPGAGH-
LIEPPYSPL-COOH at m/z 927.088. Peptide backbone bonds (13 of 15) are broken (notation as in Fig. 6).
The fragment ions unambiguously localize the 4HNE Michael adduct to the His362 active site residue.
This and six other sites were modified across all concentrations of 4HNE treatment (Table 1).
Inactivation of hBAT by 4HNE 291
by guest, on June 3, 2013
dienelactone hydrolase (51). As many as 11 enzymes have
been identified as members of this family, based on three-
dimensional crystal structures and the presence of a cata-
lytic triad consisting of a nucleophile-His acid-amino acid
triad positioned on the loops of the protein (50, 51). Se-
quence similarity searches using NCBI BLAST have found
significant similarity between hBAT, dienelactone hydro-
lase, and other a/b hydrolases (24).
Within hBAT, the catalytic triad is in the C-terminal
half of the protein. Two of the residues are susceptible
to 4HNE modification (Cys235 and His362). At all levels
of 4HNE incubation where hBAT activity was reduced
(Fig. 3), His362 was modified with a 4HNE Michael
adduct. Mutation studies that replaced the active site His
with an Ala residue abolished enzyme activity (24). This
suggests that modification of the His by 4HNE would have
a similar result on the activity of the enzyme. It was ob-
served, however, that partial hBAT activity remained
intact, even with the presence of the modified His362.
Semiquantitative analysis of the His362-modified peptide
across all concentrations of 4HNE revealed that there is
a dose-dependent change in the ratio of modified to un-
modified His362-containing peptide (Fig. 8). The rela-
tionship between the forms of the His362-containing
peptide is correlated with hBAT activity levels (Figs. 3, 8).
This demonstrates the cumulative effect that prolonged
exposure to 4HNE can have on the enzyme. Of the 55 pos-
sible residues that can be modified by 4HNE within hBAT,
the modification of the active site His362 at even low ratios
of 4HNE demonstrates the active site’s accessibility to
oxidative modification/damage. The remaining modifi-
cations can be evaluated in the context of dividing hBAT
into two halves, the C-terminal half that contains the cat-
alytic triad and the N-terminal half that has not been
reported as being vital to hBAT enzymatic activity.
Fewer modifications were found on the N-terminal half
of the protein, with modifications at the highest level of
4HNE treatment on His18, His62, and His74. These are
hypothesized to lie within the unstructured region at the
N-terminus of the protein, and as such would probably be
more susceptible to posttranslational modification, owing
to increased accessibility of the ROS to that region of
the protein. These are the only modifications seen within
the N-terminal half of the protein, with only two addi-
tional potential sites of modification unaccounted for
in the sequence coverage. The localization of fewer modi-
fications to the N-terminal half of the protein may be due
to poor accessibility of potential sites of modification,
whereas in the C-terminal half of the protein, the acces-
sibility to the residues increases with the location of the
binding pocket within this region of the protein.
At the highest level of 4HNE modification, nine resi-
dues in close proximity to the members of the catalytic
triad are also modified. These include 4HNE Michael ad-
ducts on His271, His274, His279, Lys329, Lys334, His336,
Cys372, Cys373, and His378. Among these additional
sites of modification, Cys372 has been shown to be es-
sential to hBAT enzyme activity (24). Mutation of Cys372
to Ala372 decreased the N-acyltransferase activity of the
enzyme, but with a small amount of activity remaining
(24). Cys372, Cys373, and His378 are all in the catalytic
histidine-containing loop. Modification of these sites is
only observed at the highest concentration of 4HNE treat-
ment. Although their modification may result in further
reduction in hBAT activity, they are probably not the sites
that 4HNE adduction would damage first. In contrast,
Lys329, Lys334, His336, His271, and His279 are modified
at all concentrations of 4HNE treatment. Lys329, Lys334,
and His336 are in the hypothesized acid loop, with Lys329
directly adjacent to the catalytic triad member Asp328.
Fig. 8. Semiquantitation of the modified and unmodified peptide containing the active site residue
His362. The relative abundances of the chymotryptic modified and unmodified His362-containing peptides
were compared across all concentrations of 4HNE treatment. To account for variability in the amount of
chymotryptically digested hBAT-avi loaded onto the column, individual ion abundances in each sample
were normalized against two control peptides within the same LC-ESI LTQ FT-ICR MS analysis (m/z 639.8212
and 514.7689) that were lacking in amino acids that could be modified by 4HNE. As the concentration of
4HNE was increased, the ion abundance of the unmodified peptide decreased and the modified peptide
increased in a dose-dependent manner.
292Journal of Lipid Research
Volume 49, 2008
by guest, on June 3, 2013
His271 and His 279 are hypothesized to be in the loop
between the sixth b-sheet and fourth a-helix. Modification
of these residues with the same level of susceptibility as the
catalytic triad member His362 would suggest that they
have a role in substrate access to the active site. Therefore,
damage to these sites could have as much significance in
terms of reduction of hBAT activity as does modification to
the active site His362 itself. These modifications may block
substrate accessibility to the essential members of the
catalytic triad directly, by infiltrating the binding pocket of
the protein, or indirectly, by shifting the relative position
of the members of the catalytic triad through structural
disturbance of the loops containing them. Of these five
amino acids, we hypothesize that modification of Lys329
in such close proximity to the catalytic triad may have the
most significant role in the inhibition of enzyme activity.
The addition of a bulky Michael adduct could disrupt the
charge relay mechanism that hBAT uses for its conjuga-
tion activity. This modification, in combination with the
His362 modification at all concentrations, may combine to
explain how 4HNE blocks hBAT activity, even at relatively
low levels of 4HNE.
Based on the location of the majority of the modifica-
tions in hBAT and the subsequent activity analysis, the
C-terminal half of the protein is important for its enzy-
matic function. Posttranslational modifications occurring
block substrate access to the active site residues specifically,
or to completely block access to the binding pocket.
Whether the modifications observed in hBAT are block-
ing substrate access, or the charge relay mechanism that
hBAT uses (24), needs further examination. Studies also
need to be performed to determine the residues within
order to better understand the role that the modifications
observed are playing in the inhibition of hBAT activity.
This study was supported by Grants-in-aid from the National
Institute of Diabetes, Digestive and Kidney Diseases, R01 DK-
46390-08 (S.B.), and from the National Heart, Lung and Blood
Institute, PO1 HL-70610 (V.M.D-U.). The mass spectrometers
were purchased following Shared Instrumentation Awards from
the National Center for Research Resources, S10 RR-13795 and
at Birmingham School of Medicine. Funds for the operation
of the University of Alabama at Birmingham Comprehensive
Cancer Center Mass Spectrometry Shared Facility were provided
by a National Cancer Institute Core Support Grant, P30 CA-
of Alabama at Birmingham Biomedical FT-ICR MS Laboratory
were provided in part by the Supporters of the University of
Alabama at Birmingham Comprehensive Cancer Center and the
Department of Biochemistry and Molecular Genetics.
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294 Journal of Lipid Research
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