(Received for publication, May 10, 1995)
From the
5-Lipoxygenase of mouse macrophages and bone marrow-derived mast
cells (BMMC) was investigated. Indirect immunocytofluorescence combined
with confocal microscopy provided evidence for distinct intracellular
expression patterns and trafficking of 5-lipoxygenase upon cellular
activation. In resting BMMC, 5-lipoxygenase was found within the
nucleus co-localizing with the nuclear stain Yo-Pro-1. When BMMC were
IgE/antigen-activated the 5-lipoxygenase immunofluorescence pattern was
changed from nuclear to perinuclear. The absence of divalent cations in
the incubation medium, or calcium ionophore A23187 challenge, altered
the predominantly nuclear expression pattern to new sites both
cytosolic and intranuclear. The cDNA for murine macrophage
5-lipoxygenase was cloned by the polymerase chain reaction and would
predict a 674 amino acid protein. Using control cells obtained from
5-lipoxygenase-deficient mice it was determined that a single isoform
accounts for both soluble and membrane-bound and nuclear and
cytosolic-localized enzyme in macrophages and BMMC. A mutation at amino
acid 672 (Val The enzyme 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase,
EC 1.13.11.34) catalyzes the formation of
5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) In
alveolar macrophages there is evidence for the existence of two
5-lipoxygenase ``pools,'' cytosolic and membrane-bound
forms(7) . Recent data by the same investigators has
established nuclear soluble and nuclear bound 5-lipoxygenase expression
patterns in rat basophilic leukemia cells(8) . Lepley and
Fitzpatrick (9) have obtained in vitro data that
5-lipoxygenase can bind to cytoskeletal proteins and the signaling
protein Grb2 via an SH3-binding domain interaction. Thus, in addition
to the carrier-mediated export of
leukotrienes(10, 11) , the concept is emerging that
5-lipoxygenase may have novel intracellular functions, possibly
independent of leukotriene biosynthesis, and its intracellular location
may be dictated by specific protein-protein interactions. cDNAs
encoding the human (12, 13) and rat (14) 5-lipoxygenases have been isolated, and the human
5-lipoxygenase genomic structure (15) has been elucidated.
Recently, in our efforts to better understand the biology of
5-lipoxygenase and leukotrienes we created 5-lipoxygenase-deficient
mice by gene targeting(16, 17) . We realized the
importance of ascertaining more clearly the existence, or not, of
5-lipoxygenase isoforms and their relationship to distinct
intracellular pools of 5-lipoxygenase in resting and activated cells.
Our studies have focused primarily on bone marrow-derived mast cells
(BMMC) and macrophages. Here we show evidence for different
5-lipoxygenase intracellular locations depending on the state of cell
activation in BMMC. Moreover, studies with 5-lipoxygenase-deficient
mice show that the different 5-lipoxygenase pools in alveolar
macrophages derive from the same gene product. Additionally, we
demonstrate the importance of the amino acid residue two positions
upstream of the carboxyl terminus for 5-lipoxygenase activity, the
chromosomal location of the murine 5-lipoxygenase gene, and studies
with macrophages and BMMC of 5-lipoxygenase-deficient mice designed to
examine compensatory expression of other proteins key to the formation
of leukotrienes (FLAP and LTA
Slides were examined under oil immersion with a Zeiss LSM410 laser
scanning confocal microscope using
Figure 5:
Immunoblot detection of 5-lipoxygenase,
FLAP and LTA
A second construct was also prepared for
expression studies. The codon for Met
Figure 1:
Cloning of mouse 5-lipoxygenase
cDNA. The stippled box represents the coding region. Primers
(represented by arrows) used for polymerase chain reaction
analysis are displayed above or below the amplified cDNAs (dark
bars). Some restriction sites for cloning and construction of
expression vectors are shown (sites in italics are polylinker
derived). A line plot depicting the four putative non-heme iron ligands
is displayed above the cDNA map. The importance of Val
A description of the probes
and restriction fragment length polymorphisms for the loci linked to
the Alox5 locus including microphthalmia (mi),
ras-related fibrosarcoma oncogene (Raf1), and ret
proto-oncogene (Ret) has been reported
previously(28, 29) . Recombination distances were
calculated as described (30) using the computer program SPRETUS
MADNESS. Gene order was determined by minimizing the number of
recombination events required to explain the allele distribution
patterns.
Figure 2:
Expression of recombinant mouse
5-lipoxygenase in HEK 293 cells. A, HEK 293 cells were
transfected with m5LO/Met
Figure 3:
Immunofluorescence localization of
5-lipoxygenase in resting and stimulated BMMC. Non-challenged (panels A-C), EDTA (2 µM)-treated (panel
D), IgE plus antigen-treated (panel E), and A23187 (0.5
µM)-stimulated BMMC were incubated in modified
Tyrode's buffer for 30 min, washed two times with complete
medium, and cytospun onto glass microscope slides. After fixation and
permeabilization, cells were incubated with an anti-human
5-lipoxygenase antiserum (1:2500; panels A and C-F)
or non-immune rabbit serum (1:2500, panel B), followed by a
donkey anti-rabbit Cy3-conjugated antibody (1:4000). All samples were
incubated with the nuclear stain Yo-Pro-1 (green fluorescent
signal). Slides were examined by confocal microscopy and overlay
digital contrast images obtained. Cells observed in panel A are from 5-lipoxygenase-deficient mice. Cells in all other panels
are from wild-type mice. Yellow-orange signals indicate
co-localization with nuclear staining, whereas red signal is
specific 5-lipoxygenase immunofluoresence independent of nuclear
staining. A23187-treated cells (0.5 µM) changed shape
significantly, and cell viability was approximately 70% as previously
observed under these conditions(45) . Cells in panel D are 1.2 times larger than in other panels to accentuate the
cytoplasmic 5-lipoxygenase
immunofluorescence.
Figure 4:
RP-HPLC detection of lipoxygenase products
synthesized by IgE plus antigen-stimulated and calcium ionophore A23187
challenged BMMC cells. +/+, cells obtained from
wild-type mouse; -/-, cells obtained from
5-lipoxygenase-deficient mouse. No detectable leukotrienes were
synthesized by -/- cells (bottom panel), resting
or EDTA-treated cells (not shown). The products eluting at 11.5 (peak I) and 24 (peak II) min co-elute with authentic
LTB
We also examined the expression of genes involved in
the production and regulation of leukotriene formation (FLAP and LTA
hydrolase) at the protein level to see if there was a compensatory
increase, or decrease, in expression in the absence of 5-lipoxygenase.
Analysis of BMMC and macrophages from three separate mice using various
amounts of protein indicated that leukotriene A
Figure 6:
Chromosomal localization of mouse
5-lipoxygenase gene. Alox5 gene maps to the central region of
mouse chromosome 6 by interspecific backcross analysis. The segregation
patterns of Alox5 and flanking genes from 134 backcross
animals that were typed for all loci are shown at the top of the
figure. Each column represents the chromosome identified in the
backcross progeny that was inherited from the (C57BL/6
5-Lipoxygenase exists as a single isoform in mice and
traffics to various intracellular sites in activated BMMC. Using
indirect immunocytofluorescence labeling combined with confocal
fluorescence microscopy, 5-lipoxygenase was found almost exclusively
within the nucleus of resting BMMC. A similar expression pattern was
seen with the transformed RBL-1 rat basophilic leukemia-derived cell
line. These basophilic cells bear some resemblance to mucosal mast
cells since they secrete mast cell protease
II(8, 32) . Interestingly, when resting BMMC were
incubated in the absence of divalent cations (2 mM EDTA) for
30 min there was diffusion or leakage of 5-lipoxygenase from the
nucleus throughout the cytoplasm. Enzyme also remained within the
nucleus. The EDTA treatment probably caused a depletion of
intracellular divalent cations, in addition to extracellular depletion,
by disruption of ion pumps and transporter proteins. Although not
proven, these results are suggestive of a primary or secondary divalent
cation requirement to maintain nuclear 5-lipoxygenase localization. An
intracellular Ca Activation of BMMC by IgE/antigen, a
challenge known to elicit transient elevation of intracellular
Ca The murine 5-lipoxygenase cDNA was cloned by PCR based on homology
with the human (12, 13) and rat (14) sequences. All are the same size, taking into account a
putative error noted at the deduced COOH terminus of the rat
sequence(14, 37) . The mouse sequence is 96% identical
to the rat sequence and 93% identical to the human 5-lipoxygenase. It
shares the conserved histidine and COOH-terminal isoleucine residues
found in all lipoxygenases. Based on the crystal structure of the
soybean 15-lipoxygenase, these residues act as ligands for the non-heme
iron atom(37, 38) . Serendipitously, a PCR-generated
cloning error revealed the stringent requirements of the amino acid 2
residues upstream of the COOH-terminal isoleucine during expression
experiments in HEK 293 cells. A conservative valine to methionine
substitution abolished 5-lipoxygenase activity at position 672. This
alteration would result in a small increased side chain volume in the
vicinity of isoleucine 674, perhaps perturbing the ability of this
residue to coordinate the iron atom. Previously, we carried out
deletion of the COOH-terminal isoleucine and mutagenesis to 8 different
residues using mouse 12-lipoxygenases and verified the essential
importance of this residue for enzyme activity(39) . Only
valine could be substituted for isoleucine with minimal loss of
activity. Moreover, Zhang et al.(40) had found that
deletion of 6 amino acids from the COOH terminus of human
5-lipoxygenase abolished enzyme activity. Taken together these results
indicate the importance of the integrity of the 3 COOH-terminal amino
acids of lipoxygenases which probably relates to the ability of the
polypeptide chain to fold back and interact with the essential iron
atom. 5-Lipoxygenase exists as a single form in the mouse unlike
12-lipoxygenase which has two distinct isoforms encoded by separate,
linked genes(39) . First, we were unable to clone any cDNA
variants indicative of splice variants. Second, disruption of the
5-lipoxygenase gene removed all 5-lipoxygenase protein and
enzyme activity in alveolar macrophages (known to contain both soluble
and membrane-bound species) and in IgE/antigen-activated BMMC (where
nuclear and perinuclear expression patterns were seen). The polyclonal
antibody used in these studies cross-reacts with human,
rat(7, 8) , and mouse 5-lipoxygenases and would
predictably detect alternative isoforms, if generated by different
genes since 5-lipoxygenases display very high homology across species.
Finally, our past data using Southern blot analysis with genomic DNA
has indicated a single copy gene with no related cross-hybridizing
bands at moderate stringency conditions in mice and
humans(15, 16) . A single report describing human
5-lipoxygenase alternative transcripts (41) could mean there is
heterogeneity in the 3`- or 5`-untranslated regions, or possibly, that
these transcripts were not entirely processed after transcription. Disruption of the 5-lipoxygenase gene in mice did not alter
expression of leukotriene A The
5-lipoxygenase gene maps to mouse chromosome 6 by interspecific
backcross analysis. We have compared the interspecific map of
chromosome 6 with a composite mouse linkage map that reports the map
location of many uncloned mouse mutations (compiled by M. T. Davisson,
T. H. Roderick, A. L. Hillyeard, and D. P. Doolittle and provided from
GBASE, a computerized data base maintained at The Jackson Laboratory,
Bar Harbor, ME). Alox5 mapped in a region of the composite map
that lacks mouse mutations with a phenotype that might be expected for
an alteration in this locus (data not shown). Consistent with this
finding was the lack of an observable mutated phenotype in
5-lipoxygenase-deficient mice under normal, non-stressed physiological
conditions(17, 44) . However, these mice exhibited
blunted inflammatory responses in certain models of inflammation. The central region of mouse chromosome 6 shares regions of homology
with human chromosomes 3 and 10 (summarized in Fig. 6). The
human homolog of Alox5 has previously been assigned to human
chromosome 10(43) . The placement of the mouse gene in this
region of mouse chromosome 6 confirms and extends this region of
homology between mouse and human chromosomes. In conclusion, the
murine 5-lipoxygenase has been characterized at several levels. A
single 5-lipoxygenase form is distributed within the nucleus in mast
cells and apparently traffics to different sites upon cellular
activation. The availability of 5-lipoxygenase-deficient mice should
prove useful in the elucidation of putative nuclear functions. The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
L42198[GenBank Link].
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Met) introduced serendipitously during the cloning
process was found to completely abolish 5-lipoxygenase enzyme activity
when the enzyme was expressed in human embryonic kidney 293 cells. This
subtle change is proposed to affect the ability of the COOH-terminal
isoleucine to coordinate the essential non-heme iron atom. In
macrophages and BMMC obtained from 5-lipoxygenase-deficient mice,
compensatory changes in expression of genes involved in the
biosynthesis of leukotriene B
were investigated.
5-Lipoxygenase-activating protein expression was reduced by 50%, while
leukotriene A
hydrolase expression was unaltered. The
5-lipoxygenase gene was mapped to the central region of mouse
chromosome 6 in a region that shares homology with human chromosome 10
by interspecific backcross analysis. These studies provide a global
picture of the murine 5-lipoxygenase system and raise questions about
the role of 5-lipoxygenase and leukotrienes within the nucleus.
(
)and its subsequent conversion to leukotriene
(LT)A
(5,6-oxido-7,9,11,14-eicosatetraenoic acid).
LTA
is a pivotal intermediate in the biosynthesis of
inflammatory and anaphylactic mediators which include leukotriene
B
(5S,12R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic
acid and the peptidyl leukotrienes (LTC
, LTD
,
and LTE
; see Refs. 1, 2 for reviews). In human neutrophils,
5-lipoxygenase undergoes a Ca
-dependent translocation
from the cytosol to a membrane site which appears to be the nuclear
envelope(3, 4) . 5-Lipoxygenase activating protein
(FLAP), an 18-kDa membrane protein found in the nuclear envelope, acts
apparently as an arachidonate-binding protein to facilitate the
concerted formation of LTA
(5, 6) .
hydrolase).
Mice
C57BL/6 129 mixed genetic
background mice were maintained in the animal barrier facility of
Vanderbilt University on a 12 h light/12 h dark cycle with water and
food provided ad libitum. The generation of
5-lipoxygenase-deficient mice has been described(17) .
Isolation of Cells
BMMC were prepared from cells
flushed from femurs and tibiae of wild-type and
5-lipoxygenase-deficient mice and were cultured in the presence of 50%
WEHI-3b conditioned medium, 50% RPMI 1640 (complete medium) for
3-6 weeks(18) . Cell purity estimated by cell morphology
and staining with toluidine blue was 90-95%. Peritoneal
macrophages (19) were obtained by lavage from the peritoneal
cavity with 3 ml of Dulbecco's modified Eagle's medium
containing 10% fetal calf serum and 5 units/ml heparin. Cells were
plated in a humidified 95% air, 5% CO atmosphere at 37
°C in tissue culture dishes. After adherence for 1 h, the cells
were washed three times and used for experiments. Cell purity was
estimated to be >97% based on cell morphology and staining with
nonspecific esterase. Pulmonary alveolar macrophages were isolated by a
published procedure(20) .
Intracellular Localization of 5-Lipoxygenase in
BMMC
Cultured BMMC obtained from wild-type and
5-lipoxygenase-deficient mice were washed three times with modified
Tyrode's buffer (contains 0.32 mM
Ca; (18) ) at 4 °C. One
group of cells was sensitized with monoclonal IgE directed against
DNP-human serum albumin (Sigma; 100 µg/ml) for 1 h at 37 °C,
followed by three washes, and subsequent incubation with DNP-BSA (50
ng/ml) for 30 min. Other groups of cells were incubated with calcium
ionophore A23187 (0.5 µM), EDTA (2 mM) or no
additions for 30 min in modified Tyrode's. The cells were quickly
washed two times with complete medium, with or without EDTA, at 4
°C. Cells were placed on glass microscope slides using a Shandon
cytocentrifuge (550 revolutions/min for 5 min). Cells were fixed with
4% paraformaldehyde in phosphate-buffered saline for 15 min and
permeabilized with 0.2% Triton X-100 for 10 min. Cells were incubated
with 3% bovine serum albumin for 30 min followed by 5% donkey serum for
30 min to block nonspecific binding. The cells were incubated with a
rabbit polyclonal anti-5-lipoxygenase antiserum (1:2500 dilution; see
below) for 5 h at room temperature or overnight at 4 °C. The slides
were washed three times with phosphate-buffered saline and incubated
with Cy3-labeled donkey anti-rabbit antibody (Jackson ImmunoResearch
Laboratories; 1:4000 dilution). The slides were washed three times
with phosphate-buffered saline and incubated with the nuclear stain
Yo-Pro-1 (Molecular Probes; 1:5000 dilution) for 15 min. The slides
were air-dried and mounted with Aqua-Poly/Mount (Polysciences Inc.).
40 or
63 objectives.
Excitation/emission settings were 543/650 nm (HeNe laser) for Cy3 and
488/520 nm for Yo-Pro-1 (ArKr laser). Raw data images were processed
further using Adobe Photoshop (MacIntosh) and Showcase (Silicon
Graphics Indigo
) programs.
Protein Immunoblot Analysis
Cells were sonicated
for two bursts of 15 s on ice and centrifuged at 10,000 g at 4 °C. Soluble and pellet fractions were obtained. Protein
was quantitated by Bradford assay (Bio-Rad reagent) and prepared for
SDS-polyacrylamide gel electrophoresis. Immunoblot analysis was carried
out as described in the Fig. 5legend using previously
characterized polyclonal anti-human 5-lipoxygenase, FLAP and
leukotriene A
hydrolase antisera, and purified recombinant
human 5-lipoxygenase as standard (generous gifts of Dr. J. Evans, Merck
Frosst). Detection was by enhanced chemiluminescence combined with
autoradiography using reagents from Amersham Corp. Relative band
densities were estimated by densitometric analysis of x-ray films using
Image 1.38 software (Wayne Rasburn, NIH, Bethesda, MD).
hydrolase in mouse macrophages and BMMC
obtained from wild-type (+/+) and 5-lipoxygenase-deficient
(-/-) mice. The specified amounts of protein were
electrophoresed in SDS-polyacrylamide gels (10% for 5-lipoxygenase and
LTA
hydrolase, 15% for FLAP), transferred to nitrocellulose
membrane, and probed with anti-human 5-lipoxygenase (1:1000),
anti-human FLAP (1:1000), and anti-human LTA
hydrolase
(1:2000) antisera. Detection was by enhanced chemiluminescence and
autoradiography. A, 5-lipoxygenase in alveolar macrophages. Lanes 1 and 2, +/+, 10 µg and 2 µg; lanes 3 and 4, -/-, 10 µg and 2
µg; lane 5, peritoneal macrophage protein from control
mouse (2 µg); lane 6, purified recombinant 5-lipoxygenase
standard (5 ng). B, FLAP and LTA
hydrolase in
peritoneal macrophages and BMMC. 5 µg of protein was loaded in each
lane. For estimation of relative band densities at least four different
amounts of protein (between 1-15 µg) were loaded. Results are
from cells obtained from one mouse. Similar results were obtained from
three additional mice. Positions of molecular weight markers are
displayed at the right of each blot.
Enzyme Assays
BMMC (10 cells) in 1 ml
of modified Tyrode's buffer were incubated in the presence and
absence of 0.5 µM A23187, or IgE/antigen for 20 or 30 min
at 37 °C as described above. No exogenous arachidonic acid was
added. Incubations were terminated with 4 volumes of ethanol, extracted
with ODS-silica columns, and products were separated by RP-HPLC as
described(17) .
PCR Cloning of Mouse 5-Lipoxygenase cDNA
To
isolate the complete coding region for the murine 5-lipoxygenase cDNA
degenerate oligonucleotide primers based on the known human and rat
sequences (12, 13, 14) were designed (Primer
1, 5`-GCCATGCCNTCCTACACNGTCAC-3` and Primer 2,
5`-TTAGATGGCYACACTGTTYGGAAT-3`; underlined bases indicate start and
stop codons, respectively). Two additional primers were prepared based
on on the sequence we had obtained from a genomic clone containing
exons 4-6 of the murine gene (Primer 3,
5`-ATGGATGGAGTGGAACCCCGG-3` and Primer 4, 5`-CTGTACTTCCTGTTCTAAACT-3`).
RNA was prepared by the method of Chomczynski and Sacchi(21) .
Total RNA (1 µg) obtained from resident peritoneal macrophages of
C57BL/6 129 F
mice was used as the starting
material for reverse transcriptase-PCR (RT-PCR) carried out by standard
procedures(22) . Amplification conditions for primer 2/primer 3
set using one-fifth of the cDNA mixture were: 94 °C, 45 s; 46
°C, 45 s; 72 °C, 1 min 30 s for 35 cycles. A 1.6 kb band was
purified by agarose gel electrophoresis and glass powder extraction
(Qiagen), and an aliquot was reamplified for an additional 25 cycles. A
0.8-kb product was amplified using primer 1/primer 4 set using the same
conditions mentioned above except without subsequent reamplification.
Both PCR products were cloned into the pCRII vector (Invitrogen), and
the inserts were entirely sequenced by the dideoxy chain termination
method.
5-Lipoxygenase Expression in HEK 293 Cells and
Mutagenesis
An expression construct (m5LO/Met672) was prepared
in the pcDNA1 vector (Invitrogen) by ligation of the two PCR-generated
5-lipoxygenase cDNA fragments. First, the 5` end of the 5-lipoxygenase
cDNA was inserted into the vector as an EcoRI (polylinker
derived)/EcoRV 0.5-kb fragment. After verification of the
preceding construct, the 3` end was inserted as an EcoRV/NsiI
(polylinker site) 1.5-kb fragment. DNA purified by ion-exchange resin
(Qiagen) was transfected into human embryonic kidney 293 cells as
described previously by the calcium phosphate
method(23, 24) . 48 h later enzyme activity was
assayed(25) . in the original 3`
end PCR product was changed to Val
in a PCR reaction
using primers 5 and 6 (Fig. 1;
5`-AAGTCTAGATTTAGATGGCCACACTGTTTGG-3` and
5`-TTCAAGCTGCTGGTA-3`; altered base is underlined and restriction site
for cloning is italics). The change was verified by DNA sequencing, and
a 0.4-kb PstI/XbaI fragment was replaced into
m5LO/Met
to produce m5LO/Met
Val.
Authenticity of the construct was checked by restriction site mapping
and DNA sequencing. Subsequently, a 3` RACE protocol using an oligo(dT)
adapter-primer and primer 7 (5`-ATCAGCGTGATCGCCGAG-3`) with newly
synthesized cDNA was employed to verify the codon at position 672.
is
discussed in the text.
Chromosomal Mapping
Interspecific backcross
mapping was performed as described (26) by using progeny
generated from mating (C57BL/6J Mus spretus)F
females and C57BL/6J males. A total of 205 N
mice
were used to map the Alox5 locus (see ``Results''
for details). DNA isolation, restriction enzyme digestion, agarose gel
electrophoresis, Southern blot transfer, and hybridization were
performed as essentially described(27) . The probe was a
403-base pair cDNA (bases 432-834) derived by PCR with primer
3/primer 4 set, labeled with [
-
P]dCTP using
a nick translation labeling kit (Boehringer Mannheim). Washing was done
to a final stringency of 1.0
SSCP, 0.1% SDS at 65 °C. A
fragment of 3.8 kb was detected in HindIII-digested C57BL/6J
(B) DNA, and a fragment of 10.0 kb was detected in HindIII-digested M. spretus (S) DNA. The presence or
absence of the 10.0-kb HindIII M. spretus-specific
fragment was followed in backcross mice.
Cloning of Mouse Macrophage 5-Lipoxygenase
cDNA
Previously, we had isolated a mouse genomic 5-lipoxygenase
clone that coded for a small region of 5-lipoxygenase(16) . To
examine potential 5-lipoxygenase isoforms, we sought to clone the
complete murine 5-lipoxygenase cDNA by RT-PCR from macrophage RNA. Two
overlapping fragments were obtained (Fig. 1). The 2.0-kb cDNA
encodes a protein of 674 amino acids (including the initiator Met
residue) with a molecular weight of 78,000. Various PCR primer
combinations revealed no evidence for splice variants by agarose gel
electrophoresis size selection of amplified products and subsequent
sequence analysis. Moreover, hybridization of mouse genomic DNA with
various 5-lipoxygenase cDNA restriction fragments under reduced
stringency conditions did not reveal cross-hybridizing bands or complex
band patterns (data not shown). These results are consistent with our
previous data that there is only a single 5-lipoxygenase
gene(16) .Expression and Mutagenesis of Mouse 5-Lipoxygenase
cDNA
An expression construct (m5LO/Met) was
prepared by splicing together the two PCR fragments at an unique EcoRV site (Fig. 1). Introduction of this expression
vector into human embryonic kidney 293 cells led to expression of
immunoreactive 5-lipoxygenase protein of the correct size, but devoid
of enzyme activity (Fig. 2). Alignment of the mouse sequence
with human and rat sequences and examination of the primer 2 sequence
indicated a PCR-based error in the codon for the amino acid two
residues from the COOH terminus (amino acid 672), a residue that is
conserved in most mammalian lipoxygenases. Methionine would be present
at this position instead of valine. Mutagenesis to introduce valine and
reconstruction of the expression construct (m5LO/Met
Val)
was carried out. In contrast to m5LO/Met
expression in
HEK 293 cells, m5LO/Met
Val exhibited high 5-lipoxygenase
enzyme activity (measured as 5-H(P)ETE) with comparable immunoreactive
protein levels (Fig. 2, A and B). No
5-lipoxygenase protein and enzyme activity were detected in
mock-transfected cells. A 3` RACE procedure starting with newly
synthesized mouse macrophage cDNA and an alternate upstream primer
(primer 7) verified the presence of a valine codon at position 672.
(top) or
m5LO/Met
Val (bottom) expression vectors and
sonicated extracts were assayed for the formation of 5-H(P)ETE 48-h
post-transfection with 100 µM arachidonic acid substrate
and the lipoxygenase activator 13-HPODE. The peaks at 18 and 20 min are
13-HODE and 13-HPODE, respectively. B, immunoblot detection of
5-lipoxygenase from: 1, purified recombinant 5-lipoxygenase
standard (5 ng); 2, mock-transfected; 3,
m-5LO/Met
-transfected; and 4,
m5LO/Met
Val-transfected cells (5 µg each for lanes 2-4).
Intracellular Localization of 5-Lipoxygenase in
BMMC
The intracellular expression pattern of 5-lipoxygenase in
paraformaldehyde-fixed, cytospun BMMC preparations was studied by
indirect immunocytofluorescence labeling and confocal fluorescence
imaging microscopy. In resting, unstimulated BMMC from wild-type mice
5-lipoxygenase was localized primarily within the nucleus (Fig. 3C). This was evident by the extensive
co-localization with the nuclear stain Yo-Pro-1 (Fig. 3C) and Z-plane sectioning at 1-µm intervals
followed by image reconstruction (not shown). No specific
immunofluorescence signal was detected in BMMC from
5-lipoxygenase-deficient mice or when the primary antiserum was
substituted with non-immune serum (Fig. 3, A and B). If the divalent cation chelator EDTA was added to the
modified Tyrode's buffer for the 30-min incubation period, there
was a marked difference in the pattern of 5-lipoxygenase expression.
Besides significant enzyme within the nucleus there was now clear
evidence for 5-lipoxygenase throughout the cytoplasm (Fig. 3D). Washing the cells with, or without, divalent
cations prior to the cell fixation step which took approximately
5-10 min did not appreciably alter this pattern (not shown). When
BMMC were activated with IgE/antigen the 5-lipoxygenase
``translocated'' to a perinuclear location along the nuclear
envelope but what appeared to be now mainly on the cytoplasmic side (Fig. 3E). In contrast, if the cells were stimulated
with Ca ionophore A23187 the immunofluorescence
pattern was different. Signal was detected as a punctate/reticular
pattern, possibly associated with structural proteins, both nuclear and
perinuclear localized (Fig. 3F). Leukotriene synthesis
was associated with IgE/antigen- and A23187-challenged, but not resting
and EDTA-treated, BMMC obtained from wild-type mice (Fig. 4).
All stimuli using BMMC obtained from 5-lipoxygenase-deficient mice
failed to produce detectable leukotriene products (Fig. 4).
and LTC
standards,
respectively.
5-Lipoxygenase, FLAP, and LTA
Recent data on the
localization of 5-lipoxygenase in alveolar macrophages indicated the
presence of enzyme in both membrane and soluble fractions, including
nuclear localization in rat basophilic leukemia
cells(7, 8, 31) . This raised the possibility
of the existence of distinct 5-lipoxygenase isoforms. Protein blot
analysis indicated a single immunoreactive 5-lipoxygenase band in
alveolar macrophages from normal wild-type mice but no band in
5-lipoxygenase-deficient mice generated by gene targeting (Fig. 5A). Moreover, a product co-eluting with
leukotriene C Hydrolase
Expression in Macrophages and BMMC
was synthesized by A23187-stimulated alveolar
macrophages from wild-type mice but not 5-lipoxygenase-deficient mice
(not shown).
hydrolase
protein levels were not changed; however, FLAP levels dropped
approximately 50% in 5-lipoxygenase-deficient mice (Fig. 5B) as revealed by densitometric analysis.
Chromosomal Localization of the Mouse 5-Lipoxygenase
Gene
The 5-lipoxygenase chromosomal location (designated Alox5) was determined by interspecific backcross analysis
using progeny derived from matings of ((C57BL/6J M. spretus)F
C57BL/6J) mice. This
interspecific backcross mapping panel has been typed for over 1700 loci
that are well distributed among all the autosomes as well as the X
chromosome(26) . C57BL/6J and M. spretus DNAs were
digested with several enzymes and analyzed by Southern blot
hybridization for informative restriction fragment length polymorphisms
using a mouse cDNA Alox5 probe. The 10.0-kb HindIII M. spretus fragment length polymorphisms (see
``Experimental Procedures'') was used to follow the
segregation of the Alox5 locus in backcross mice. The mapping
results indicated that Alox5 is located in the central region
of mouse chromosome 6 linked to mi, Raf1, and Ret. Although 134 mice were analyzed for every
marker and are shown in the segregation analysis (Fig. 6), up to
175 mice were typed for some pairs of markers. Each locus was analyzed
in pairwise combinations for recombination frequencies using the
additional data. The ratios of the total number of mice exhibiting
recombinant chromosomes to the total number of mice analyzed for each
pair of loci, and the most likely gene orders are: centromere - mi - 15/175 - Raf1 - 0/138 - Alox5 - 1/151 - Ret. The recombination
frequencies (expressed as genetic distances in centiMorgans (cM)
± the standard error) are - mi - 8.6
± 2.1 - (Raf1, Alox5) - 0.7 ± 0.7
- Ret. No recombinants were detected between Raf1 and Alox5 in 138 animals typed in common suggesting that
the two loci are within 2.1 cM of each other (upper 95% confidence
limit).
M.
spretus)F
parent. The black boxes represent
the presence of a C57BL/6J allele, and white boxes represent
the presence of a M. spretus allele. The number of offspring
inheriting each type of chromosome is listed at the bottom of each
column. A partial chromosome 6 linkage map showing the location of the Alox5 gene in relation to linked genes is shown at the bottom
of the figure. Recombination distances between loci in centimorgans are
shown to the left of the chromosome, and the positions of loci in human
chromosomes are shown to the right.
change with ionophore stimulation
resulted in a significant rearrangement of nuclear 5-lipoxygenase to a
punctate/reticular pattern around the nuclear envelope. Given the
recent data that 5-lipoxygenase can bind cytoskeletal proteins by SH3
domain interactions (9) and previous data that 5-lipoxygenase
undergoes a Ca
-dependent translocation to membrane
sites that requires extracellular Ca
(3, 33) it is possible that 5-lipoxygenase is
associating with nuclear filament proteins (lamins) or other
cytoskeletal proteins that attach to the nuclear envelope through
protein-protein interactions. More precise localization data should be
achieved with high resolution electron microscopy. In fact, recent
findings in human alveolar macrophages using this technique indicated
5-lipoxygenase association with the euchromatin in resting cells.
A23187 stimulation resulted in translocation to the nuclear
envelope(34) .
in these and rat basophilic leukemia
cells(35) , also caused translocation of 5-lipoxygenase. The
enzyme shifted predominantly to a juxtanuclear position with some
localized distribution in the cytoplasm. Malaviya et al.(36) noticed a reversible translocation of 5-lipoxygenase
in mast cells upon IgE/antigen stimulation from a supernatant to pellet
fraction by Western blot analysis. The detection of membrane bound
5-lipoxygenase was dependent upon quick-freezing of the
cells(36) . How their results correlate with ours is uncertain
due to the different means of analysis. Although many questions remain
to be answered including: (i) what sequence-specific signals (e.g. nuclear localization signal) control trafficking of
5-lipoxygenase; (ii) what protein-protein interactions govern
localization and movement; (iii) how Ca
ion or other
divalent cation fluxes regulate 5-lipoxygenase compartmentalization;
and (iv) how in vitro data obtained on fixed, immobilized
cells correlate with in vivo cellular activation, it is
becoming clear that the simple 5-lipoxygenase-initiated generation of
leukotrienes and their subsequent extracellular transport will have to
be modified with novel roles of 5-lipoxygenase within the nucleus.
hydrolase, an enzyme downstream
in the pathway of leukotriene B
synthesis, in macrophages
and BMMC. However, FLAP expression was reduced about 50%. FLAP may act
as an arachidonic acid transfer protein(6) . The reason, or
mechanism, for the reduced FLAP expression is unknown. The human FLAP
and 5-lipoxygenase genes reside on different chromosomes and have
different promoter elements(15, 42, 43) .
Perhaps, intracellular leukotrienes can act by a feedback mechanism to
positively regulate FLAP gene expression, or inhibit degradation, and
this pathway is abrogated in 5-lipoxygenase-deficient mice.
We thank Ginger Griffis and Mary Barnstead for
excellent technical assistance and Drs. Bill Serafin, Lee Limbird, and
Alan Brash for helpful discussions. We are grateful to Dr. Tom Jetton
and the Vanderbilt Imaging Resource Center for assistance with the
immunocytofluorescence experiments and laser scanning confocal
microscopy. Dr. Jilly Evans (Merck Frosst) is kindly acknowledged for
supplying antisera.
dmark, O., Hg, J.-O., Jrnvall, H., and Samuelsson, B. (1988) Proc. Natl. Acad. Sci. U. S. A.85, 26-30 and correction p.3406
[Abstract]
dmark, O., and Samuelsson, B.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2587-2591
[Abstract]
dmark, O., and Samuelsson, B.(1992)Proc. Natl. Acad. Sci. U. S. A. 89, 485-489
[Abstract]
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.