Download Amino Acid Differences in the Deduced 5

Amino Acid Differences in the Deduced 5-Lipoxygenase
Sequence of CAST Atherosclerosis-Resistance Mice Confer
Impaired Activity When Introduced Into the Human Ortholog
Hartmut Kuhn, Monika Anton, Christa Gerth, Andreas Habenicht
Downloaded from http://atvb.ahajournals.org/ by guest on April 29, 2017
Objectives—The mouse strain CON6, which was generated by breeding athero-resistant CAST mice into an atherosusceptible B6 background, exhibits almost complete resistance to atherosclerosis. An athero-resistance gene cluster has
been localized at the central region of chromosome 6, and among the candidate genes of this locus, the 5-lipoxygenase
has attracted particular attention because of its involvement in the biosynthesis of proinflammatory leukotrienes.
Comparison of 5-lipoxygenase genomic sequences of B6 and CON6 mice indicated 2 conserved amino acid exchanges
in the CON6 animals, but the functional impact of these mutations has not been defined.
Methods and Results—We analyzed the functionality of these amino acid exchanges relative to essential catalytic
properties (specific activity, substrate affinity, and reaction specificity) and found that these mutations confer an
impaired lipoxygenase and leukotriene A4-synthase activity when introduced into the human enzyme. In contrast,
substrate affinity, enantiomer selectivity, and positional specificity remained unchanged.
Conclusions—These data are consistent with the possibility that naturally occurring conservative mutations in the coding
region of the murine 5-lipoxygenase gene can significantly affect enzyme activity and that this loss of function may be
involved in CAST/CON6 athero-resistance. (Arterioscler Thromb Vasc Biol. 2003;23:1072-1076.)
Key Words: eicosanoids 䡲 leukotrienes 䡲 inflammation 䡲 mutation 䡲 atherogenesis
T
he 5-lipoxygenase (5-LOX) is the key enzyme in the
biosynthesis of leukotrienes (LTs), which constitute
powerful proinflammatory mediators.1,2 Expression of the
enzyme has been reported in different types of leukocytes,2
including in foamy macrophages of atherosclerotic lesions3
and in the aorta of apolipoprotein E– deficient and LDL
receptor– deficient mice.4 Moreover, the number of transcripts of both 5-LOX and LT-receptors seems to expand
during lesion progression,3 suggesting a causal relation between 5-LOX expression and atherogenesis. The genes for
human and murine 5-LOX were mapped to syntenic regions
of chromosomes 10 and 6, respectively.5 To identify genes
with potential impact on atherogenesis, athero-resistant
CAST mice were crossed with athero-susceptible B6 animals.
This crossbreeding yielded the congenic mouse strain CON6,
which exhibits a similarly pronounced athero-resistance as
the parent CAST strain.6 Importantly, CON6 mice retained
the athero-resistance gene locus of the CAST animals,6 and
genotyping of the 2 strains identified the 5-LOX gene
immediately underneath the linkage peak.4 Moreover, bone
marrow transplantation suggested the importance of leukocytes in the athero-resistance of CON6 mice.4 Thus, the
leukocyte 5-LOX emerged as an attractive candidate gene
that could contribute to athero-resistance of CAST and CON6
mice. To address this hypothesis, 5-LOX expression was
compared between athero-resistant (CON6) and atherosusceptible (B6) mouse strains, and it was found that CON6
mice exhibited 5-fold lower expression levels of both 5-LOX
mRNA and protein than the corresponding B6 control animals.4 Moreover, comparison of the genomic 5-LOX sequences indicated 2 nucleotide exchanges in the 5-LOX
coding region, which resulted at the protein level in 2
adjacent point mutations: isoleucine 645 of B6 mice was
exchanged to valine (I645V) and valine 6461 was mutated to
isoleucine (V646I).4 These mutations are rather conservative
in nature, and, thus, no major functional consequences were
initially expected. However, amino acid alignments of all
mammalian 5-LOX species indicated an absolute conservation of these residues, suggesting their functional importance.
To explore whether these amino acid exchanges may be
associated with alterations of 5-LOX functionality, we overexpressed the human enzyme as recombinant nonfusion
protein in Escherichia coli, mutated the corresponding amino
acids, and characterized the resulting LOX species with
respect to major enzymatic properties, such as specific
activity (LOX and LTA4-synthase activity), positional speci-
Received March 5, 2003; revision accepted April 9, 2003.
From the Institute of Biochemistry (H.K., M.A., C.G.), Humboldt University Medical School Charité, Berlin and Institute for Vascular Medicine
(A.H.), Friedrich-Schiller-University of Jena, Germany.
Correspondence to Dr Hartmut Kuhn, Institute of Biochemistry, University Clinics Charité, Humboldt University, Monbijoustr. 2, 10117 Berlin, F.R.
Germany. E-mail [email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1072
DOI: 10.1161/01.ATV.0000074167.01184.48
Kuhn et al
5-LOX and Atherogenesis
1073
ficity, and substrate affinity. We found that each point
mutation (I645V and V646I) as well as the double mutant
(I645V⫹V646I) exhibited reduced catalytic activities but
retained their positional specificity and substrate affinity.
These data suggest that athero-resistant CON6 mice express a
catalytically less active 5-LOX, and they also raise the
important possibility that similar loss of function mutations
may occur in other species. Thus, our findings may encourage
experimental approaches to search for functional 5-LOX
open reading frame mutations in human populations with a
modified risk of cardiovascular disorders.
Methods
Chemicals
Downloaded from http://atvb.ahajournals.org/ by guest on April 29, 2017
The chemicals used were from the following sources:
(5Z,8Z,11Z,14Z)-eicosa-5,8,11,14-tetraenoic acid (arachidonic
acid), (5S,6E,8Z,11Z,14Z)-5-hydro(pero)xyeicosa-6,8,11,14tetraenoic acid [5S-H(p)ETE], CaCl2, EDTA, ATP, and sodium
borohydride from Serva; ampicillin from Gibco; dipalmitoyl phosphatidylcholine, isopropyl-␤-D-thiogalactopyranoside (IPTG), and
ATP-sepharose from Sigma-Aldrich; and HPLC standards of hydroxy fatty acids and LT A4 methyl ester from Cayman Chemical
(distributed by Alexis GmbH). HPLC solvents were obtained from
Merck. Restriction enzymes were purchased from New England
Biolabs. Phage T4 ligase, PWO-polymerase, and sequencing kits
were bought at Boehringer Mannheim, and the E. coli strain HB 101
was purchased from Invitrogen. Oligonucleotide synthesis was
carried out by TiB-Molbiol.
Bacterial Expression, Site-Directed Mutagenesis,
and Enzyme Purification
The human 5-LOX was overexpressed as nonfusion protein in E.
coli,7 and for this purpose its cDNA was subcloned into the bacterial
expression plasmid PKK 233-2. First, we introduced an NcoI
restriction site at the starting ATG and a HindIII site just behind the
stop codon. The NcoI/HindIII restriction fragment was then ligated
into the expression vector, and bacteria (HB 101) were transformed
with the recombinant plasmid. Then bacteria were cultured at 37°C
in 5 mL of LB medium containing 0.1 mg/mL ampicillin to an
optical density at 600 nm of approximately 0.5. LOX expression was
induced by addition of IPTG (1 mmol/L final concentration). After
12 hours at 30°C, bacteria were spun down, washed with PBS,
resuspended in 0.5 mL of 0.1 mol/L phosphate buffer, pH 7.4,
containing 1 mmol/L EDTA, and kept on ice for 10 minutes. Then
the cells were lysed by sonication with a Labsonic U tip-sonifier, cell
debris was removed by centrifugation, and the lysis supernatant was
used for activity assay, Western blotting, or additional purification.
Site-directed mutagenesis was carried out using the QuickChange
site-directed mutagenesis kit (Stratagene). Bacteria were transformed
with recombinant wild-type and mutant plasmids and plated for
selection. For each mutant, 3 different clones were picked and
sequenced. One clone carrying the mutation was selected and
replated. To assay the 5-LOX activity, 3 well-separated colonies
were picked for each mutant and 5 mL bacterial liquid cultures was
grown. When the cultures had reached an optical density at 600 nm
of 1.0, expression of the recombinant enzymes was induced by
addition of 1 mmol/L isopropyl-␤-D-thiogalactopyranoside. After an
additional 12 hours at 30°C, bacteria were spun down, washed with
PBS, resuspended in 0.5 mL of 0.1 mol/L Tris-HCl buffer, pH 7.4,
containing 1 mmol/L EDTA, and kept on ice for 10 minutes. The
cells were lysed by sonication with a Labsonic U tip-sonifier
(Braun), cell debris was spun down, and the lysis supernatant was
used for activity assay (see below). For more detailed investigations
of the enzymatic properties, the recombinant enzyme species were
purified from large-scale fermentations (500 mL liquid cultures) as
described previously.8
Figure 1. Quantification of expression of 5-LOX mutants in E.
coli. Five-milliliter liquid cultures of E. coli expressing the different 5-LOX mutants were grown, and lysis supernatants were
prepared as described in the Methods section. Aliquots of the
supernatants representing equal amounts of total lysis proteins
were applied to SDS-PAGE, and the Western blot was developed using a polyclonal anti-human 5-LOX antibody. As reference, the purified human 5-LOX was used.
Activity Assays
The enzymatic activity was assayed by RP-HPLC quantification of
the arachidonic acid oxygenation products (sum of 5-HpETE and
LTA4-hydrolysis products).7 For this purpose, the bacterial lysate
supernatants or the purified enzyme preparations were incubated for
10 minutes in 0.5 mL Tris-HCl buffer, pH 7.4, containing
0.1 mmol/L arachidonic acid, 0.4 mmol/L CaCl2, 40 ␮g/mL dipalmitoyl phosphatidylcholine, and 0.1 mmol/L ATP. The hydroperoxyl
compounds formed were reduced with sodium borohydride, the
mixture was acidified to pH 3, and 0.5 mL of ice-cold methanol was
added. The protein precipitate was spun down, and aliquots of the
clear supernatant were injected to HPLC for quantification of the
LOX products. HPLC was carried out on a Shimadzu system
connected to a Hewlett Packard diode array detector 1040. Reversephase HPLC was performed on a Nucleosil C-18 column (MachereyNagel, KS-system, 250⫻4 mm, 5-␮m particle size) coupled with an
appropriate guard column (30⫻4 mm, 5-␮m particle size). A solvent
system of methanol/water/acetic acid (80/20/0.1, by volume) was
used at a flow rate of 1 mL/min. The chromatographic scale was
calibrated (5-point calibration) by injecting known amounts of
15S-HETE (conjugated dienes) and LTB4 (conjugated trienes).
5(R/S)-HETE enantiomers were separated on a Chiralcel OB column
(250⫻4 mm, 5-␮m particle size), and the hydroxy fatty acid methyl
esters were eluted with the solvents system n-hexane/2-propanol/
acetic acid (100/4/0.1, by volume) at a flow rate of 1 mL/min.
Immunoblotting
For immunoblotting, the bacteria were lysed as described for the
activity assay. Aliquots of the lysis supernatants containing similar
amounts of total protein were applied to SDS gel electrophoresis.
Proteins were transferred to a nitrocellulose membrane by a semidry
procedure, and the blots were probed with a polyclonal rabbit
antibody raised against the recombinant human 5-LOX. For generation of the antiserum, E. coli– expressed 5-LOX protein was
purified by SDS polyacrylamide gel electrophoresis. The electroeluted antigen was then directly injected into lymph nodes of a rabbit
with one boost. The immunoreactive bands (see Figure 1) were
quantified densitometrically, and a linear calibration curve (0.08 to
0.3 ␮g) was established using an electrophoretically homogenous
human 5-LOX preparation.
Miscellaneous Methods
Protein concentration was determined with the Roti-Quant detection
system (Roth) that is based on the Bradford method. For chiral phase
HPLC, the carboxylate group of the free hydroxy fatty acids was
methylated with diazomethane in diethylether, and the resulting
1074
Arterioscler Thromb Vasc Biol.
June 2003
Downloaded from http://atvb.ahajournals.org/ by guest on April 29, 2017
Figure 2. RP-HPLC analysis of arachidonic acid oxygenation
products formed by recombinant 5-LOX species. Five-milliliter
liquid cultures of E. coli expressing wt 5-LOX and its
V645I⫹I646V double mutant were grown, and lysis supernatants
were prepared as described in the Methods section. Aliquots of
the supernatants (normalized to an equal amount of 5-LOX protein) were incubated with 0.1 mmol/L arachidonic acid. Product
preparation and RP-HPLC were performed as described in the
Methods section.
methyl esters were repurified by RP-HPLC. KM-values were determined by varying substrate concentrations in the range of 10 to 80
␮mol/L, evaluating the data with Lineweaver-Burk plots. A reference mixture of the LTA4-hydrolysis products was prepared by
incubating 5 ␮g of LTA4 methyl ester for 2 hours at pH 2 (epoxide
ring opening). Then KOH was added to a final concentration of 0.5
mol/L, and the methyl esters of the resulting diol isomers were
hydrolyzed to the free acids. After acidification to pH 3, the
hydrolysis mixture was directly injected for RP-HPLC and the
hydrolysis products were prepared.
Results
When we expressed the wild-type (wt) human 5-LOX and the
3 mutants I645V, V646I, and I645V⫹V646I in E. coli, we
observed by immunoblotting variable expression levels of the
recombinant enzyme species (9 independent small-scale
preparations). When all expression experiments are taken
together, the wt enzyme showed always the highest expression level (approximately 100 ␮g 5-LOX protein/L liquid
culture; was set 100%), whereas expression of the mutants
varied between 55% and 90% in different fermentation
samples. An immunoblot of a representative experiment is
shown in Figure 1. Because of the expression variability, the
results of the activity assays had to be normalized to 5-LOX
protein, as determined by densitometry of 5-LOX immunoblotting. To obtain reliable activity data, 3 clones were
randomly selected for wt 5-LOX and for each mutant, and
activity assays were carried out. In Figure 2, representative
chromatograms obtained for wt human 5-LOX and for the
I645V⫹V646I double mutant are compared. It can be seen
that wt LOX produced significantly higher amounts of
arachidonic acid oxygenation products, ie, 5-HETE and the
LTA4 hydrolysis products, than the double mutant. In the
Table, the normalized 5-LOX activities of lysis supernatants
are summarized. These data indicated that the specific activities of the 5-LOX mutants are strongly reduced compared
with wt 5-LOX.
These results were clearly surprising, because the mutations carried out were rather conservative in nature. To
exclude methodological artifacts, an additional set of experiments determining the normalized arachidonic acid oxygenase was performed. For this purpose, bacteria were retransformed with sequenced recombinant plasmids, and wellseparated clones of wt enzyme and of each mutant were
randomly selected. In this series of experiments (4 independent measurements using single clones of the wt 5-LOX and
of each mutant; mean of the wt activity was set 100%, ⫾
represents SD), the following results were obtained: Wt,
100⫾12.9%; I645V, 12.9⫾9.2%; V646I, 5.6⫾3.5%; and
I645V⫹V646I, 14.7⫾9.8%. The differences between wt
5-LOX and the mutant enzyme species were significant at
P⬍5⫻10⫺5. By contrast, normalized activities were not
significantly different when the various mutants were compared with each other. These data revealed that each single
amino acid exchange as well as the double mutation exhibited
an impaired 5-LOX activity compared with the wt enzyme.
To explore the possibility of whether the impaired activities of the mutant enzymes are the result of a reduced
substrate affinity, basic kinetic parameters were determined.
For the wt enzyme, a KM of 55.6 ␮mol/L and a Vmax of 1.90
␮g oxygenation products/␮g 5-LOX protein was calculated
(Table). This KM is somewhat higher than the corresponding
Enzymatic Properties of 5-LOX Mutants
Normalized 5-LOX Activity of Crude Enzyme Preparations
Absolute 5-LOX Activity,
␮g product/␮g
Relative 5-LOX
Enzyme Species
5-LOX per min
Activity, %
Wild-type
1.47⫾0.05
I645V
0.25⫾0.01
V646I
0.15⫾0.01
I645V⫹V646I
0.18⫾0.01
100
KM,
␮mol/L
Purified Enzyme
Vmax,
␮g products/␮g Enantioselectivity,
5-LOX per min
S/R ratio
Relative
5-LOX
Activity, %
55.6⫾18.1
1.90⫾0.33
88:12
100
17.0
47.9⫾21.4
0.36⫾0.08
82:18
11.0⫾1.1*
10.4
24.8⫾5.3
0.14⫾0.01
75:25
18.9⫾3.0*
11.2
49.9⫾7.3
0.22⫾0.02
88:12
11.8*†
Five-milliliter cultures were grown for each LOX species, and activity assays with the bacterial lysis supernatants were carried out
as described in Methods (columns 2 and 3: n⫽5, ⫾ indicates SD). KM and Vmax values (columns 4 and 5) were determined by
measuring the reaction rates in the substrate concentration range between 10 and 80 ␮mol/L, and the data were evaluated with
Lineweaver-Burk plots (0.96⬍R2⬍0.98; ⫾ indicates SD). 5-HETE enantiomers were resolved as methyl esters by chiral phase HPLC
(column 6). For enzyme purification, two independent experiments were performed (column 7); *⫾indicates the error range; †one
purification experiment only.
Kuhn et al
Downloaded from http://atvb.ahajournals.org/ by guest on April 29, 2017
value determined for the purified native enzyme,9 and this
may be attributable to the presence of fatty acid– binding
proteins in the E. coli lysate supernatant. KM values for the
mutant enzyme species were found to be in the same range.
Moreover, the Vmax, values for the 5-LOX mutants were
strongly reduced (Table), indicating their impaired catalytic
activities even under Vmax conditions.
In many cases, LOX mutants with low specific activity
show a reduced reaction specificity, eg, arachidonic acid is
oxygenated to a complex mixture of stereo-random oxygenation products. To examine whether the 5-LOX mutants
prepared in this study retained stereochemical control, the
enantiomer composition of the major oxygenation product
(5-HETE) was analyzed. We found that the S/R ratio determined by chiral phase HPLC was similar for the wt enzyme
and the mutants (Table). These data indicated that the high
degree of stereo-specificity of the wt enzyme was retained in
the mutant 5-LOX species, supporting the conclusion that
these mutations did not alter the substrate alignment at the
active site.
To exclude that non-LOX proteins in the bacterial lysate
may have impacted the activity assay, bacterially expressed
enzymes were purified by affinity chromatography on ATPsepharose.9 Comparable amounts of the purified enzyme
preparations were used for activity assays, and the activity
data were normalized for an equal LOX content. Here again,
we observed that the 5-LOX mutants exhibited a strongly
impaired enzymatic activity (Table).
The 5-LOX is a multifunctional enzyme that exhibits an
arachidonic acid 5-oxygenase activity but also is capable of
converting 5(S)-HpETE, its primary oxygenation product, to
LT A4.10 To find out whether mutations of the I645 and V646
may also impact the LT synthase activity, the enzyme was
incubated in our standard assay system, leaving out arachidonic acid but using 50 ␮mol/L 5(S)-HpETE as substrate
instead. It can be seen from Figure 3 that large amounts of
LTA4 hydrolysis products were detected when the wild-type
enzyme was used. In contrast, with the mutant enzyme
species, much smaller amounts were analyzed. These data
suggest that I645V and V646I exchange does not only impair
the oxygenase but also LTA4 synthase activity of the enzyme.
Discussion
Because LOXs are lipid-peroxidizing enzymes, they have
been implicated in the pathogenesis of atherosclerosis in the
frame of the LDL oxidation hypothesis.11,12 During the last
decade, research was focused predominantly on the 12/15LOX family, because these LOXs are capable of oxidizing
low-density lipoprotein to an atherogenic form. However, the
precise role of these enzymes has not yet been completely
understood. In some animal models, 12/15-LOXs seem to
exhibit a proatherogenic activity,13–15 and in others they act
antiatherogenic.16,17 Apart from these unresolved questions
concerning 12/15-LOXs, 5-LOX metabolites such as LT have
been discussed for more than 10 years as possible mediators
in atherogenesis.18 Today atherosclerosis is well recognized
as an inflammatory disorder,19,20 and, thus, proinflammatory
LTs have been suggested to promote the disease.3 Several
months ago we reported that 5-LOX and LT receptors3 are
5-LOX and Atherogenesis
1075
Figure 3. RP-HPLC analysis of LTA4 synthase activity of 5-LOX
mutants. Five-milliliter liquid cultures of E. coli overexpressing
wt and mutant 5-LOX species were grown, and lysis supernatants were prepared as described in the Methods section. Aliquots of the supernatants were incubated in the standard oxygenase assay. However, arachidonic acid was left out, and 50
␮mol/L 5S-HpETE was used as substrate instead. RP-HPLC of
the LTA4 hydrolysis products was performed under the same
conditions as 5-HETE, and the compounds of interest were
identified by comigration with an authentic standard (see Methods section) and by their characteristic uv-spectra. It should be
stressed that the peak eluting at 4.5 minutes constitutes a twin
peak (5,12-DiHETE diastereomers), which is not resolved under
these conditions. In contrast, the 5,6-DiHETE diastereomers
(peak doublet eluting at approximately 6 minutes) are partly
resolved.
abundantly expressed in human atherosclerotic plaques, and
similar data were recently obtained in our laboratory for
murine lesions. When LDL receptor– deficient mice that
lacked just one 5-LOX allele (LDL-R⫺/⫺/5-LOX⫹/⫺) were fed
a cholesterol-rich diet for 8 weeks, they developed significantly fewer lesions than their 5-LOX–sufficient counterparts.4 These data indicated that a modest decrease in 5-LOX
expression might have major antiatherogenic effects in vivo.
CON6 mice that had been generated by crossing the
atheroresistant CAST mice onto a B6 background carry an
antiatherogenic gene cluster in the central region of chromosome 6 that confers almost complete athero-resistance.4
Among the genes located in this region, the 5-LOX was
particularly attractive because it is involved in the biosynthesis of proinflammatory mediators.1,2 Reverse-transcriptase–
polymerase reaction, Northern blots, and immunoblotting of
CON6 bone marrow cells indicated a 3- to 5-fold lower
expression level of 5-LOX mRNA and protein, respectively.
Interestingly, bone marrow cells prepared from CON6 mice
only produced less than 5% LTB4 compared with the corresponding B6 cells, suggesting the importance of posttranslational mechanisms. Comparison of the coding sequences of
the 5-LOX of CON6 and B6 mice revealed 2 amino acid
exchanges (I645V and V646I). These mutations are rather
conservative, and, thus, no functional consequences were
immediately apparent. Moreover, modeling of the 5-LOX
structure based on the X-ray coordinates of rabbit 15-LOX21
suggested that I645 and V646 are located in a surface helix
with no obvious connection to the active site, casting addi-
1076
Arterioscler Thromb Vasc Biol.
June 2003
Downloaded from http://atvb.ahajournals.org/ by guest on April 29, 2017
tional doubt on the functionality of these mutations. On the
other hand, I645 and V646 and the entire primary structure
region surrounding these residues are absolutely conserved
among all mammalian 5-LOXs. Such a high degree of amino
acid conservation suggests a functional importance of this
structural element. Here we report that even conservative
amino acid exchanges, such as I645V and V646I, may lead to
mutant enzyme species that exhibited strongly impaired
catalytic activities, whereas other important enzyme properties (substrate affinity and positional specificity) remained
unaltered. Considering the low expression levels of the
5-LOX in CON6 mice4 and the impaired specific activities of
the mutant enzyme expressed in these animals (data reported
here), a strongly reduced 5-LOX pathway must be postulated
for the athero-resistant CON6 mice. This conclusion is
consistent with low-level formation of LTB4 by CON6 bone
marrow cells.4
It has been reported before that a subpopulation of human
asthma patients carrying mutations in the central promoter
region of the 5-LOX gene are less susceptible for treatment
with drugs impacting LT metabolism.22 Thus, this regulatory
polymorphism seems to impact the pathogenesis of an allergic disease. It would be of particular interest to find out
whether a structural polymorphism (open reading frame
mutations) also exists for the 5-LOX gene and whether such
polymorphisms may correlate with the risk for cardiovascular
disorders. Identification of such functional mutants and the
corresponding correlation studies may help to examine
whether 5-LOX is related to the risk of developing inflammatory cardiovascular disorders in humans.
Acknowledgments
This study was supported by grants to A.H. from the Deutsche
Forschungsgemeinschaft (Ha 1083/13-1/13-2), the EU (QLG1-CT2001-01521), and the Interdisziplinäres Zentrum für Klinische Forschung Jena (TP4.4; TP4.9).
References
1. Samuelsson B, Dahlen SE, Lindgren JA, Rouzer CA, Serhan CN. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects.
Science. 1987;237:1171–1176.
2. Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid
biology. Science. 2001;294:1871–1875.
3. Spanbroek R, Gräbner R, Lötzer K, Hildner M, Urbach A, Rühling K,
Moos MPW, Kaiser B, Cohnert TU, Wahlers T, Zieske A, Plenz G,
Robenek H, Salbach P, Kühn H, Radmark O, Samuelsson B, Habenicht
AJR. Expanding expression of the 5-lipoxygenase pathway within the
arterial wall during human atherogenesis. Proc Natl Acad Sci U S A.
2003;100:1238 –1243.
4. Mehrabian M, Allayee H, Wong J, Shih W, Wang XP, Shaposhnik Z,
Funk CD, Lusis AJ. Identification of 5-lipoxygenase as a major gene
contributing to atherosclerosis susceptibility in mice. Circ Res. 2002;91:
120 –126.
5. Funk CD, Chen XS, Johnson EN, Zhao L. Lipoxygenase genes and their
targeted disruption. Prostaglandins Other Lip Med. 2002;68 – 69:
303–312.
6. Mehrabian M, Wong J, Wang X, Jiang Z, Shih W, Fogelman A, Lusis
AJ. Genetic locus in mice that blocks development of atherosclerosis
despite extreme hyperlipidemia. Circ Res. 2001;89:125–130.
7. Schwarz K, Gerth C, Anton M, Kuhn H. Alterations in leukotriene
synthase activity of the human 5-lipoxygenase by site-directed
mutagenesis affecting its positional specificity. Biochemistry. 2000;39:
14515–14521.
8. Denis D, Falgueyret JP, Riendeau D, Abramovitz M. Characterization of
the activity of purified recombinant human 5-lipoxygenase in the absence
and presence of leukocyte factors. J Biol Chem. 1991;266:5072–5079.
9. Aharony D, Stein RL. Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase. J Biol Chem. 1986;261:11512–11519.
10. Shimizu T, Izumi T, Seyama Y, Tadokoro K, Radmark O, Samuelsson B.
Characterization of leukotriene A4 synthase from murine mast cells:
evidence for its identity to arachidonate 5-lipoxygenase. Proc Natl Acad
Sci U S A. 1986;83:4175– 4179.
11. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond
cholesterol: modification of low density lipoprotein that increases its
atherogenicity. N Engl J Med. 1989;320:915–924.
12. Chisolm GM, Steinberg D. The oxidative modification hypothesis of
atherogenesis: an overview. Free Radic Biol Med. 2000;28:1815–1826.
13. Cyrus T, Witztum JL, Rader DJ, Tangirala R, Fazio S, Linton MF, Funk
CD. Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice. J Clin Invest. 1999;103:1597–1604.
14. George J, Afek A, Shaish A, Levkovitz H, Bloom N, Cyrus T, Zhao L,
Funk CD, Sigal E, Harats D. 12/15-lipoxygenase gene disruption
attenuates atherogenesis in LDL receptor-deficient mice. Circulation.
2001;104:1646 –1650.
15. Harats D, Shaish A, George J, Mulkins M, Kurihara H, Levkovitz H,
Sigal E. Overexpression of 15-lipoxygenase in vascular endothelium
accelerates early atherosclerosis in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol. 2000;20:2100 –2105.
16. Shen J, Henderick E, Cornhill JF, Zsigmond E, Kim HS, Kuhn H,
Guevara NV, Chan L. Macrophage-mediated 15-lipoxygenase expression
protects against atherosclerosis development. J Clin Invest. 1996;98:
2201–2208.
17. Trebus F, Heydeck D, Schimke I, Gerth C Kühn H. Transient experimental anemia in cholesterol-fed rabbits induces systemic overexpression
of the reticulocyte-type 15-lipoxygenase and protects from aortic lipid
deposition. Prostaglandins Leukot Essent Fatty Acids. 2002;67:419 – 428.
18. Wissler RW. Update on the pathogenesis of atherosclerosis. Am J Med.
1991;91:3S-9S.
19. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;
340:115–126.
20. Lusis AJ. Atherosclerosis. Nature. 2000;407:233–241.
21. Gillmor SA, Villasenor A, Fletterick R, Sigal E, Browner MF. The
structure of mammalian 15-lipoxygenase reveals similarity to the lipases
and the determinants of substrate specificity. Nature Struct Biol. 1997;4:
1003–1009.
22. Drazen JM, Yandava CN, Dube L, Szczerback N, Hippensteel R, Pillari
A, Israel E, Schork N, Silverman ES, Katz DA, Drajesk J. Pharmacogenetic association between ALOX5 promoter genotype and the response
to anti-asthma treatment. Nat Genet. 1999;22:168 –170.
Downloaded from http://atvb.ahajournals.org/ by guest on April 29, 2017
Amino Acid Differences in the Deduced 5-Lipoxygenase Sequence of CAST
Atherosclerosis-Resistance Mice Confer Impaired Activity When Introduced Into the
Human Ortholog
Hartmut Kuhn, Monika Anton, Christa Gerth and Andreas Habenicht
Arterioscler Thromb Vasc Biol. 2003;23:1072-1076; originally published online May 1, 2003;
doi: 10.1161/01.ATV.0000074167.01184.48
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2003 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/23/6/1072
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the
Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for
which permission is being requested is located, click Request Permissions in the middle column of the Web
page under Services. Further information about this process is available in the Permissions and Rights
Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online
at:
http://atvb.ahajournals.org//subscriptions/