©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cDNA Cloning, Expression, Mutagenesis, Intracellular Localization, and Gene Chromosomal Assignment of Mouse 5-Lipoxygenase (*)

(Received for publication, May 10, 1995)

Xin-Sheng Chen (1) Todd A. Naumann (1) Usha Kurre (1) Nancy A. Jenkins (2) Neal G. Copeland (2) Colin D. Funk (1)(§)

From the  (1)Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232 and the (2)Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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)()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) .

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 hydrolase).


EXPERIMENTAL PROCEDURES

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.).

Slides were examined under oil immersion with a Zeiss LSM410 laser scanning confocal microscope using 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).


Figure 5: Immunoblot detection of 5-lipoxygenase, FLAP and LTA 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) .

A second construct was also prepared for expression studies. The codon for Met 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/MetVal. 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.


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 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.

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.


RESULTS

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/MetVal) was carried out. In contrast to m5LO/Met expression in HEK 293 cells, m5LO/MetVal 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.


Figure 2: Expression of recombinant mouse 5-lipoxygenase in HEK 293 cells. A, HEK 293 cells were transfected with m5LO/Met (top) or m5LO/MetVal (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/MetVal-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).


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 and LTC standards, respectively.



5-Lipoxygenase, FLAP, and LTA Hydrolase Expression in Macrophages and BMMC

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 was synthesized by A23187-stimulated alveolar macrophages from wild-type mice but not 5-lipoxygenase-deficient mice (not shown).

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 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).


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 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.




DISCUSSION

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 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) .

Activation of BMMC by IgE/antigen, a challenge known to elicit transient elevation of intracellular Ca 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.

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 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.

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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM15431 and the National Cancer Institute, Department of Health and Human Services, under contract NO1-CO-46000 with ABL. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) L42198[GenBank Link].

§
Recipient of a Research Career Development Award HL02710[GenBank Link]. To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University, Nashville, TN 37232. Tel.: 615-343-4496; Fax: 615-322-4707.

The abbreviations used are: 5-H(P)ETE, 5-hydro(pero)xy-eicosatetraenoic acid; LT, leukotriene; FLAP, 5-lipoxygenase-activating protein; BMMC, bone marrow-derived mast cell; PCR, polymerase chain reaction; HEK, human embryonic kidney; RACE, rapid amplification of cDNA ends; DNP-BSA, dinitrophenyl bovine serum albumin; 13-H(P)ODE, 13-hydro(pero)xy-octadecadienoic acid; RP-HPLC, reversed phase-high performance liquid chromatography; kb, kilobase(s).


ACKNOWLEDGEMENTS

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.


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