From the Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, September 7, 2000, and in revised form, October 9, 2000
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ABSTRACT |
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5-Lipoxygenase is the key enzyme in the formation
of leukotrienes, which are potent lipid mediators of asthma
pathophysiology. This enzyme translocates to the nuclear envelope in a
calcium-dependent manner for leukotriene biosynthesis.
Eight green fluorescent protein (GFP)-lipoxygenase constructs,
representing the major human and mouse enzymes within this family, were
constructed and their cDNAs transfected into human embryonic kidney
293 cells. Of these eight lipoxygenases, only the 5-lipoxygenase was
clearly nuclear localized and translocated to the nuclear envelope upon
stimulation with the calcium ionophore A23187. The N-terminal " Leukotrienes are potent lipid mediators of inflammation and
anaphylaxis (1). They are generated by an initial reaction with the
enzyme 5-lipoxygenase from arachidonic acid that has been liberated
from membrane lipids (1-3). 5-Lipoxygenase is a 78-kDa protein found
predominantly within inflammatory cell types (e.g.
macrophages, mast cells, neutrophils, and eosinophils). The location of
the enzyme is cell-type specific. In unstimulated neutrophils, it is
found in the cytoplasm, whereas in alveolar macrophages and bone
marrow-derived mast cells it is situated mainly in the nucleus
(4-7). Transfection of 5-lipoxygenase cDNA leads to nuclear
localized protein in HEK1
293, COS, NIH-3T3, and Chinese hamster ovary cells, as well as bone
marrow-derived mast cells and RAW macrophages (8, 9).
Regardless of the cellular localization, 5-lipoxygenase undergoes a
calcium-dependent translocation event to the nuclear
membrane in activated inflammatory cells (5). Early studies documented a reversible, soluble to membrane compartment transition in neutrophils challenged with the calcium ionophore A23187 (10, 11). Experiments with
RBL-2H3 cells demonstrated that the translocation to membranes required
influx of extracellular calcium, which could be induced either with
ionomycin or cross-linking of IgE receptors (12-14). The neutrophil
5-lipoxygenase cytosol to membrane translocation was associated with a
loss of activity (11). However, in alveolar macrophages and RBL cells
the translocation to membrane fractions activated 5-lipoxygenase
activity (12, 15). It is generally regarded that the translocation
event to the nuclear envelope is a necessary event in leukotriene
formation. At this site there is an apparent transfer of substrate to
5-lipoxygenase by unknown mechanisms, via the integral membrane protein
referred to as 5-lipoxygenase activating protein (16, 17).
5-Lipoxygenase was recently shown to directly bind two calcium ions
(18). It has long been known that this enzyme is unique among
lipoxygenases in its ability to have its activity stimulated by calcium
(19, 20). Although this calcium stimulatory effect is not an absolute
requirement for the purified enzyme, it is essential when the enzyme is
present in intact cells, or in isolated preparations incubated with
membranes or phospholipids (21, 22). 5-Lipoxygenase also possesses a
nucleotide-binding site of unknown function and ATP is known to
stimulate activity (23, 24).
There has been little, if any, insight to document the domain(s) within
mammalian lipoxygenases that govern membrane translocation. The
lipoxygenases are known to possess two domains based on the crystal
structures of two soybean enzymes and the rabbit reticulocyte 15-lipoxygenase (25-28). The N terminus contains a Here, we show strong proof that the 5-lipoxygenase
putative2 Plasmid Constructs
5-Lipoxygenase Constructs--
The coding region for green
fluorescent protein (GFP) from the vector pEGFP-C2
(CLONTECH) was ligated with the coding region for
5-lipoxygenase to make pEGFP-5LO as described previously (8). pEGFP-5LO
was used for preparation of chimeric molecules and truncated 5-lipoxygenase cDNA constructs. GFP-tagged truncated 5LO constructs (see Fig. 2C), including pEGFP-5LO-(1-114),
pEGFP-5LO-(115-673), pEGFP-5LO (N6-deletion), and pEGFP-5LO
(N17-deletion) were prepared using polymerase chain reaction (PCR) and
standard subcloning techniques (30). Chimeric lipoxygenases (see Fig.
4B) were engineered from pEGFP-5LO (8), pEGFP-12LO, and
pEGFP-15LO (see below for preparation) and pEGFP-truncated 5LO
templates. To prepare a GFP-tagged chimera, generally a restriction
enzyme site was incorporated in a region of interest in the
lipoxygenase DNA sequence by using the QuikChange Site-directed
Mutagenesis kit (Stratagene) or PCR. Subsequently, ligation of
restriction fragments was carried out using a Rapid DNA Ligation kit
(Roche Molecular Chemicals). For example, to obtain
pEGFP-N-15LO-(1-111)-C-5LO-(115-673), an EcoRI site was
incorporated between the region encoding amino acid residue 111 and 112 of 15-LO through site-directed mutagenesis. EcoRI-cut fragment DNA encoding N-terminal 111 amino acids of 15-LO was cloned in
EcoRI-digested pEGFP-5LO-(115-673). Ligation boundaries and
PCR-generated DNA sequences in the above constructs were verified by
automated DNA sequencing using the facilities of the Department of
Genetics, University of Pennsylvania. More detailed information of these DNA constructs and mutagenic primers may be obtained upon
request. The pEGFP-5LO-(1-80), pEGFP-5LO-(1-127), pEGFP-5LO-(1-166), pEGFP-5LO-(81-673), pEGFP-5LO-(C6-deletion), pEGFP-5LO-(H367Q), and
pEGFP-5LO-(H550Q) were prepared previously (8).
pEGFP-12LO--
The XbaI insert from
pcDNA1-6His12LX (31) was blunt end ligated into the blunted
HindIII site of pEGFP-C2. The six-histidine tag was shown
previously not to affect enzyme activity (31). The EGFP-tagged protein
displayed high level 12-lipoxygenase activity in HEK 293-transfected
cells (exclusive formation of 12-HPETE from arachidonic acid).
pEGFP-15LO-1--
An EcoRI/BglII fragment
of the coding region for human 15-lipoxygenase (32) was cloned into the
EcoRI and BamHI sites of pEGFP-C2. This construct
yielded both 15-HETE and 12-HETE (9:1 ratio) from arachidonic acid in
transfected cells similar to studies without the GFP tag (32).
pEGFP-15LO-2--
The coding region for human 15-lipoxygenase-2
was amplified by RT-PCR using primers and conditions based on the
published sequence (33) starting with human hair RNA template (34). The
PCR products were first cloned in pCR2.1 (Invitrogen) and sequences
verified. Two fragments, a 0.9-kilobase
EcoRI/AseI piece encoding the N-terminal region,
and a 1.1-kilobase AseI/BamHI insert encoding the
C terminus, were ligated in a three-fragment reaction with
EcoRI/BamHI-digested pEGFP-C2. The construct when transfected into HEK 293 cells and subsequently incubated with arachidonic acid synthesized exclusively 15-HPETE.
pEGFP-12(R)LO--
The construction and characterization of this
plasmid was described previously (34).
pEGFP-e12LO--
The original expression construct described
previously (35) was cut with EcoRI, filled in with a Klenow
reaction, and blunt end ligated into the SmaI site of
pEGFP-C2.
pEGFP-8LO--
The coding region for murine 8-lipoxygenase was
amplified by RT-PCR using primers and conditions based on the published
sequence (36) starting with phorbol ester-treated epidermal RNA
template from a 6-day-old mouse (35). The PCR products were first
cloned in pCR2.1 (Invitrogen) and sequences verified. The
EcoRI fragment with the correct sequence was first cloned
into the expression vector pcDNA3 (Invitrogen) and then in
pEGFP-C2. When transfected into HEK 293 cells 8-lipoxygenase activity
was detected.
pEGP-eLO-3--
The sequence for this novel lipoxygenase was
cloned exactly as done for 8-lipoxygenase described above based on the
published sequence (37). No enzyme activity was detected with
arachidonic acid substrate as described, presumably since this enzyme
utilizes a different, as of yet undetermined, substrate.
Cell Culture and Transfection
HEK 293 cells were cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Cells growing in culture dishes, cover glass-bottomed culture dishes (for photography of living cells) or glass chamber slides were
transfected with FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. The transfected cells (15-20 h post-transfection) were used for
translocation studies, fluorescence microscopy, and protein preparation
for Western blot and lipoxygenase activity assays.
Translocation Studies and Fluorescence Microscopy
The HEK 293 cells on chamber slides, 15-20 h post-transfection,
were washed twice with pre-warmed Dulbecco's modified Eagle's medium.
For data documentation, the washed cells, 20-30 min post-incubation with 10 µM A23187 (Sigma) or 0.1% Me2SO
(vehicle) in serum-free Dulbecco's modified Eagle's medium, were
fixed with 2% paraformaldehyde in phosphate-buffered saline for 20 min. Slides were mounted with Gel/Mount and kept at 4 °C. Cell
images were examined as described previously (8) for conventional
fluorescence microscopy or using a Nikon E600 upright microscope
equipped with Bio-Rad 1024 confocal imaging system. For time course
analysis of translocation, the GFP fluorescence of living cells
incubated in a 37 °C stage chamber, 15-20 h post-transfection, was
recorded before and after addition of 10 µM A23187 using
a Nikon TE300 inverted microscope equipped with Bio-Rad 1024 confocal
system. Raw data photoimages were acquired by LazerSharp software
(Bio-Rad) and processed further by Confocal Assistant and Adobe
Photoshop programs.
Western Blot and Activity Assays
Cytosol proteins from transfected cells for Western blot and
lipoxygenase activity assay were prepared as described previously (6).
A mouse monoclonal antibody against GFP (Berkeley Antibody Co., 1:1000
dilution) was used for enhanced chemiluminescence detection. Activity
assay of 12- and 15-lipoxygenases was carried out with 10,000 × g supernatant proteins of transfected cells in
phosphate-buffered saline. The supernatants were incubated with 100 µM arachidonic acid for 15 min at 37 °C and the
reactions were terminated with 2 volumes of stop solution (8). Activity assay for 5-lipoxygenase with addition of Ca2+ and ATP and
reverse phase-high pressure liquid chromatography analysis of
arachidonate metabolites (HETEs and HPETEs) by lipoxygenases were done
as described previously (8) using an
acetonitrile:H2O:acetic acid (65:35:0.1) mobile phase with
additional assessment of leukotriene B4 production in
transfected cells using UV detection at 270 or 280 nm. Authentic
5-H(P)ETE, LTB4, and
6-trans-12-epi-LTB4 standards (Cayman Chemical
Co.) were chromatographed in parallel.
5-Lipoxygenase Is Unique among Human and Murine Lipoxygenases in
Its Ability to Translocate to the Nuclear Envelope--
Eight
different GFP-lipoxygenase fusion constructs representing the major
lipoxygenase forms unique to man and mouse were prepared and introduced
into HEK 293 cells. The cells were visualized 15-20 h
post-transfection either with or without calcium ionophore A23187
stimulation and were fixed for data documentation. In unstimulated
cells, GFP-5LO was localized primarily within the nucleus as we had
demonstrated previously (8). All other GFP-lipoxygenases were localized
primarily to the cytosol of HEK 293 cells (left panels, Fig.
1, A and C). The
lone exception, perhaps, was GFP-8LO, which distributed throughout the
cell, in a pattern more reminiscent of GFP alone. All GFP-lipoxygenases
were expressed as
When the cells were stimulated with A23187, there was a clear
translocation to the nuclear envelope in nearly all GFP-5LO expressing
cells as evidenced by a "ring-like" pattern (right panel, Fig. 1, A and C). There was little
evidence, if any, for translocation to the nuclear membrane for the
other seven GFP-lipoxygenase fusions (right panels, Fig.
1A).
The
The time course of translocation for both the GFP full-length and
Translocation Patterns and Enzymatic Activity of Chimeric
Translocation Patterns of GFP-5LO Constructs with Iron-binding
Ligand Mutations--
Mammalian lipoxygenases contain a non-heme iron
atom bound by three histidine residues and an oxygen molecule on the
C-terminal isoleucine (27). Point mutation of these critical
histidines, or truncation of the C terminus, can severely cripple iron
binding and abolishes enzyme activity (39). Most of these mutations also severely disrupt nuclear targeting indicative of specific folding
requirements for nuclear localization (8). We tested the same mutants
we made previously and found that the ability to translocate was
unaffected by these inactivating mutations, in stark contrast to our
results with nuclear targeting (Fig. 6).
We have demonstrated using GFP-lipoxygenase fusion constructs
transfected into HEK 293 cells and stimulated with calcium ionophore A23187 the following conclusions: 1) 5-lipoxygenase is unique in its
nuclear localization and ability to translocate to the nuclear envelope
when compared with other human and murine lipoxygenase family members;
2) the Lipoxygenases possess a two-domain structure (25-28). The N-terminal
Although other lipoxygenases did not translocate to the nuclear
envelope with A23187 stimulation in the transfected HEK 293 cells, this
finding does not exclude translocation to other membrane sites for
access of substrate. It is likely these other lipoxygenases interact in
defined ways with membranes that is not evident in this system. Indeed,
it has been shown that rat and human 12-lipoxygenases in platelets and
tumor cells, as well as 15-lipoxygenase in reticulocytes and
interleukin 4-treated monocytes translocate from cytosol fractions to
undefined membrane sites (48-50).
In this study and a previous one (8), we were able to visualize
5-lipoxygenase cellular localization in living cells in real time or
fixed with paraformaldehyde. The enzyme demonstrated an intrinsic
capability to enter the nucleus of transfected cells indicative of
specific NLS sequences. We showed that there were weak nontraditional
NLS sequences somewhere within the first 80 residues of the protein (8)
and Healy et al. (9) showed divergent results with weak
determinants residing in a C-terminal basic region that were important
for nuclear localization. The NLS determinants in the One other study has attempted to address the translocation of 5- and
15-lipoxygenases using truncation mutants and a chimeric approach with
transfection into RAW264.7 macrophages (51). This study, unlike ours,
was unable to assign a specific region within 5-lipoxygenase as
important for membrane targeting. We are not exactly sure why divergent
results were obtained but some points to consider are differences in
cell type, transfection methods, cellular localization, and choice of
construct design. We3 and Healy et al. (9) found
that GFP-5LO localizes to the nucleus of this macrophage cell line
instead of the cytosol as found in the study of Christmas et
al. (51). However, here and in other studies in alveolar
macrophages it has been shown that both cytoplasmic and nuclear pools
of 5-lipoxygenase are capable of translocation to the nuclear envelope
(5). We demonstrated with our chimeric approach that it was critical to
carefully assign the junction between domains for obtaining active
proteins. This finding indicates that important contacts between the
two domains must be maintained for proper functional responses.
Significant variability in enzyme activity and translocation capacity
was evident with junction shifts either direction (±30-40 residues)
from the initial chosen Although we did not investigate in detail parameters affecting the time
course of translocation, this process appeared to be slower than
expected if functionally correlated to 5-HPETE or leukotriene
biosynthesis in stimulated inflammatory cells. The GFP-5LO In summary, we have shown that the
-barrel" domain of 5-lipoxygenase, but not the catalytic domain, was
necessary and sufficient for nuclear envelope translocation. The
GFP-N-terminal 5-lipoxygenase domain translocated faster than
GFP-5-lipoxygenase.
-Barrel/catalytic domain chimeras with 12- and
15-lipoxygenase indicated that only the N-terminal domain of
5-lipoxygenase could carry out this translocation function. Mutations
of iron atom binding ligands (His550 or deletion of C-terminal
isoleucine) that disrupt nuclear localization do not alter
translocation capacity indicating distinct determinants of nuclear
localization and translocation. Moreover, data show that
GFP-5-lipoxygenase
-barrel containing constructs can translocate to
the nuclear membrane whether cytoplasmic or nuclear localized. Thus,
the predicted
-barrel domain of 5-lipoxygenase may function like the
C2 domain within protein kinase C and cytosolic phospholipase
A2 with unique determinants that direct its
localization to the nuclear envelope.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel region of ~110-115 (mammals) and 150 (plants) amino acids, in addition to
the large non-heme iron containing catalytic domain at the C terminus.
Sequence alignments between mammalian 15- and 5-lipoxygenases indicate
~33% identity in the predicted N-terminal domain and only around
10% homology between the corresponding region of plants and mammals.
The function of the
-barrel of lipoxygenases is unknown but it has
been suggested that this domain, which bears resemblance to the
C-terminal domain of certain lipases, is important for lipid binding
(27). In fact, a recent study showed that this region within a cucumber
lipoxygenase is important for binding to liposomes and lipid bodies
(29).
-barrel domain
is unique among the mammalian lipoxygenase members in its ability to
direct nuclear membrane translocation. Using green fluorescent
protein-lipoxygenase fusions we present evidence for its necessity and
sufficiency in nuclear translocation in real time using transfected cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
110-kDa proteins by Western blot analysis using an
anti-GFP antibody (Fig. 1B). Each lipoxygenase fusion
yielded enzyme activity consistent with expression in a native
nonfusion vector (e.g. pcDNA3). The three
"classical" lipoxygenases (5-LO, 12-LO, and 15-LO-1) gave the
highest enzyme activities, followed by 15-LO-2 and 8-LO and finally by
the three epidermal lipoxygenases (12(R)-LO, eLO-1, and eLO-3), which
gave very low to undetectable activity (Fig. 5 and data not shown).
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Fig. 1.
Cellular localization and calcium ionophore
A23187-induced translocation of 5-lipoxygenase is unique among
mammalian lipoxygenases. HEK 293 cells were transfected with
GFP-tagged mammalian lipoxygenase cDNA constructs. A,
cellular location and translocation patterns of fluorescence carried by
GFP-lipoxygenase fusion proteins were identified before (left
panels) and after (right panels) application of calcium
ionophore A23187 in living cells. Parafomaldehyde-fixed cells were used
for image photographs (see "Experimental Procedures" for details).
B, Western blot analysis. GFP-tagged lipoxygenase proteins
were detected with a monoclonal anti-GFP antibody (1:1000 dilution).
The sample number is equivalent to that in panel A. C, more
detailed images of 5-lipoxygenase location before (left
panels) and after (right panels) A23187-induced
translocation with differential interference contrast (DIC)
phase microscopy and fluorescence overlays. Note that separate fields
of cells are depicted in the left and right
panels for all images. Experiments were repeated at least three
times with similar results.
-Barrel Domain, but Not the Catalytic Domain, of
5-Lipoxygenase Is Essential for Nuclear Membrane
Translocation--
Since lipoxygenases are known to possess two
distinct domains (25-28), we prepared GFP-5LO fusions of the
N-terminal
-barrel and the C-terminal catalytic domains. The precise
demarcation between the two domains is somewhat arbitrary because there
is a "linking" region of about 15 amino acids between the last
-strand of the
-barrel and the first helical structure of the
catalytic domain (residues 110-125) based on the rabbit reticulocyte
15-lipoxygenase structure (27). We chose a division between residues
114 and 115 of 5-lipoxygenase. In unstimulated cells, the
GFP-5LO-(1-114)
-barrel construct was localized primarily to the
nucleus (Fig. 2A, top left
panel) consistent with our previous finding of weak nuclear
localizing signal (NLS) sequences within the first 80 residues of the
protein (8). The GFP-5LO-(115-673) catalytic domain fusion protein was
distributed throughout the cell (Fig. 2A, bottom left
panel). Only GFP-5LO-(1-114) translocated to the nuclear membrane
(Fig. 2A, top right panel) upon ionophore challenge indicating that the
-barrel was essential for translocation. Extending the
-barrel region (residues 1-127 or 1-166) yielded a
similar translocation pattern but shortening the region from the
C-terminal side (residues 1-80) abolished the ability to translocate (Fig. 2B). Truncation at the N terminus (N-6 deletion; 6 residues) did not affect translocation capacity but truncation of 17 amino acids did (Fig. 2B). Both truncations abolished enzyme
activity consistent with data showing that lipoxygenases can tolerate
N-terminal additions but not truncations (31, 38). The data for eight different constructs are summarized in Fig. 2C. The results
suggest that determinants both near the N terminus (residues 6-17) and the C terminus (residues 80-114) of the
-barrel are either directly involved or related to folding determinants that regulate interaction with the nuclear membrane.
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Fig. 2.
The -barrel, but not
the catalytic, domain of 5-lipoxygenase is essential for nuclear
membrane targeting upon A23187 cell stimulation. A, HEK
293 cells were transfected with GFP-5LO-(1-114)
-barrel or
GFP-5LO-(115-673) catalytic domain constructs and fluorescence was visualized in unstimulated (left
panels) and A23187 challenged (right panels) cells as
described in the legend to Fig. 1 and under "Experimental
Procedures." B, the GFP-5LO
-barrel domain was
shortened from the N terminus by six residues (N6-deletion), 17 residues (N-17 deletion), or C terminus (1-80) or extended to include
additional residues linking the two domains () or including part
of the catalytic domain (). Fluorescence was visualized as
mentioned above. Note that separate fields of cells are depicted in the
left and right panels for all images. Experiments
were repeated three times with similar results. C, summary
of results in tabular form depicting various constructs tested.
-barrel 5-LO fusions was monitored in living cells (Fig. 3). Both nuclear localized fusions
translocated, with earliest detectable events around 5 min after A23187
addition for the GFP-5LO
-barrel construct. Complete "ring-like"
patterns were discernable by 20-30 min for both. The translocation was
faster with the GFP-
-barrel construct than the full-length
fusion.
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Fig. 3.
Time course of A23187-induced translocation
of GFP-5LO full-length (top panels) and
()- -barrel (bottom
panels) fusion proteins in HEK 293 transfected cells.
The cells were maintained at 37 °C and data collected on living
cells using a Bio-Rad 1024 confocal imaging system.
-Barrel/Catalytic Domain GFP-lipoxygenases--
To assess further
the importance of the 5-lipoxygenase
-barrel with respect to
translocation ability, eight distinct chimeric lipoxygenase constructs
were made with 5-LO and either 15-LO-1 or 12-LO (the three so-called
classical lipoxygenases)(Fig. 4). First, all chimeric constructs were cytoplasmic localized. The NLS
sequences in 5-LO are complex and context dependent (i.e. there may be signals at both ends of the 5-LO molecule and not all
fusions to different reporter proteins (e.g. GFP
versus pyruvate kinase) are transported to the nucleus) (8,
9). Second, only chimeras with the complete
-barrel of 5-LO
translocated to the nuclear envelope. If the
-barrel was truncated
(1-80 residues), the chimeric protein no longer translocated. These
particular chimeras, however, displayed enzymatic activity of the
corresponding 12- or 15-lipoxygenase catalytic domains (Figs.
4B and 5). Third, chimeras with either the
-barrel of
12-LO or 15-LO did not translocate to the nuclear envelope. Enzyme
activity of these chimeras was dependent on the two-domain fusion
boundary that, in general, had to be shifted toward the N terminus
(Fig. 5). GFP-5LO and chimeras with the
extended 5-LO catalytic domain were able to make 5-HPETE, 5-HETE, and
products from LTA4 (LTB4 and
6-trans-LTB4 isomers) from arachidonic acid.
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Fig. 4.
A23187-induced translocation of various
GFP-lipoxygenase chimeras to the nuclear envelope requires the
-barrel region of 5-lipoxygenase.
A, the results from four different chimeras,
N5LO-(1-127)-CP12LO-(126-663) (a 12-lipoxygenase enzymatically active
chimera containing the N-terminal
-barrel and
interdomain connecting region of 5-lipoxygenase plus the
C-terminal catalytic domain of platelet-type
12-lipoxygenase; top panels),
NP12LO-(1-75)-C5LO-(81-673) (a 5-lipoxygenase enzymatically active
chimera; second level panels),
N5LO-(1-114)-C15LO-(112-663) (containing the
-barrel of
5-lipoxygenase and catalytic domain of 15-lipoxygenase type 1;
third level panels), and N15LO-(1-111)-C5LO-(115-673)
(containing the
-barrel of 15-lipoxygenase type 1 and catalytic
domain of 5-lipoxygenase; bottom panels), respectively.
B, summary of results in tabular form depicting these
constructs and additional chimeras tested. The data are representative
from experiments performed at least three times. 5-Lipoxygenase
contains additional residues within the N-terminal region with respect
to 12- and 15-lipoxygenases; thus, numbering may not always correspond
in linear sequence.
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Fig. 5.
Reverse phase-high pressure liquid
chromatography chromatograms depicting formation of arachidonic acid
metabolites in GFP-lipoxygenase-transfected HEK 293 cell
supernatants. Panel I, relative amounts of HETE
metabolites (UV absorbance at 235 nm) formed by: A,
N5LO-(1-80)-C15LO-(76-663); B,
N5LO-(1-80)-CP12LO-(76-663); D,
NP12LO-(1-75)-C5LO-(81-673); and E,
N5LO-(1-127)-CP12LO-(123-663). C, HETE standards. Results
are presented in a qualitative fashion only at the same attenuation. No
attempts were made to quantitate products. Panel II,
comparison of products made by GFP-5LO (B) and the
N15LO-(1-75) C5LO-(81-673) chimera (A) immediately after
incubation. C, LTB4 and 5-H(P)ETE standards. The
hydroperoxy compound, 5-HPETE, and its reduction product, 5-HETE
(analysis at 235 nm) were detected in addition to LTB4
(formed by endogenous leukotriene A4 hydrolase present in
incubations) and the two 6-trans-LTB4 isomers
(nonenzymatic breakdown products of LTA4). The three latter
products did not separate using the aqueous acetonitrile reverse
phase-high pressure liquid chromatography system. IIB inset,
using another system (methanol:H2O, 70:30 in 10 mM ammonium acetate, pH 7.8) better separation was
achieved. Results are representative from at least three
experiments.
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Fig. 6.
Mutations to iron-binding ligands of
5-lipoxygenase do not affect ability to translocate to the nuclear
envelope. A, mutations to disrupt iron ligands in
5-lipoxygenase H367Q (top panels), H550Q (middle
panels), and deletion of the six C-terminal residues including the
isoleucine oxygen ligand were prepared as described previously (8).
A23187-induced translocation was assessed as in the legend to Fig. 1.
Results are representative from at least three determinations.
B, summary of results in tabular form depicting these
constructs.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel region of 5-lipoxygenase is necessary and sufficient
for nuclear membrane translocation; 3) determinants at both ends of the
-barrel region are important for translocation; 4) catalytically
active functional chimeras of 5-lipoxygenase, 12-lipoxygenase, and
15-lipoxygenase type 1 can be created by correct association of the two
domains at or close to the junction of the two domains; 5)
5-lipoxygenase nuclear targeting and translocation determinants are
distinct; and 6) nuclear membrane translocation can occur from either
the nuclear or cytoplasmic side.
-barrel domain of rabbit reticulocyte 15-lipoxygenase revealed
striking homology with the C-terminal domain of various lipases. These
domains on the distinct proteins share approximately the same size
(115-125 amino acids) and both need to access lipid substrates at a
membrane surface. A detailed characterization of the role of the
-barrel in mammalian lipoxygenase function for substrate or membrane
interaction has not been addressed previously. However, a recent report
with a cucumber lipoxygenase indicated that the
-barrel was
necessary for transport from cytosol to lipid bodies (29). Among the
mammalian lipoxygenase members identified so far, only the
5-lipoxygenase has a definite binding requirement for calcium ions
(18). Calcium is essential for membrane association and has recently
been shown to bind to the
-barrel of 5-lipoxygenase
(40).3 Thus, in many respects
this domain appears to mimic the C2 domains of protein kinase C and
cytosolic phospholipase A2, both which bind calcium and
translocate to membranes (41, 42). What directs the 5-lipoxygenase, and
not other lipoxygenases, specifically to the nuclear membrane is not
clear. Besides the clear calcium binding and dependence on this cation
in vivo, the 5-lipoxygenase appears unique among other
lipoxygenases in having an accessory protein (5-lipoxygenase activating
protein) that is important for substrate presentation. Moreover,
certain interactions with other cytoskeletal and signaling molecules
may represent another mode of directing 5-lipoxygenase translocation
specifically to the nuclear envelope (43-45). The 5-lipoxygenase
contains an "insertion" sequence of 5 residues in the
-barrel
that is not present in most mammalian 12- and 15-lipoxygenases (46). An
aspartic acid residue within this stretch, as well as other
determinants at both ends of the domain, could be important for calcium
binding, and proper protein folding for optimum interactions with
membrane sites analogous to cytosolic phospholipase
A2 (47).
-barrel are
definitely distinct from the translocation determinants.
Mutations introduced to strong iron binding ligands (His-550 or
terminal Ile-673 truncation) (39) changed the cellular localization
from nuclear to cytoplasmic, whereas they had no effect on
translocation. We interpret these results to mean that proper folding
of the catalytic domain for appropriate contact with the
-barrel and
other potential chaperone proteins is essential for nuclear entry but
is not important for translocation and association with the attendant
membrane lipid and protein partners.
-barrel/catalytic domain junction (residue
115). Using this approach, translocation determinants could be
definitely localized to the
-barrel region in our studies. Previous
studies in our laboratory showed that it was difficult to construct
functional chimeras with 5-lipoxygenase and either 12- or
15-lipoxygenase in several locations within the center of the molecule
within the catalytic domain (38). However, we and several other groups
have been able to successfully alter oxygen insertional activity and
product profiles with limited point mutations or small substitutions
(38, 52-54).
-barrel
showed evidence of translocation by at least 5 min at 37 °C after
ionophore stimulation and continued to proceed over the next 10-20
min. It is possible that the earliest events after ionophore
stimulation may not be detectable by fluorescence assay of GFP due to
sensitivity problems. Alternatively, the GFP-tag may actually slow the
translocation process relative to nonfusion protein. Another aspect to
consider is that HEK 293 cells do not normally express 5-lipoxygenase
or 5-lipoxygenase activating protein and cellular machinery may not
function analogously as in activated inflammatory cells. For instance,
5-lipoxygenase phosphorylation, which can occur in vitro
(55), may not occur to the same extent or rapidity in these cells as in neutrophils.
-barrel domain of 5-lipoxygenase
is necessary for nuclear envelope translocation. Addressing the
concerted actions of calcium and ATP binding, in addition to
phosphorylation status, with respect to translocation and functional correlation with leukotriene biosynthesis and enzyme inactivation at
membrane sites will be important topics for future studies.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the kind assistance of Dr. James Sanzo and Irina Chernysh at the Biomedical Imaging Core facility for assistance with confocal microscopy and John Lawson and Ting-Ting Kong for analysis of arachidonate metabolites.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL58464.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology,
Center for Experimental Therapeutics, University of Pennsylvania, 421 Curie Blvd., Rm. 814, Philadelphia, PA 19104-6160. Tel.: 215-898-0254; Fax: 215-573-9004; E-mail: colin@spirit.gcrc.upenn.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M008203200
2
Since the crystal structure of 5-lipoxygenase
has not been determined, it can only be assumed that this enzyme has a
two-domain structure similar to other lipoxygenases with elucidated
structures. Instead of using "putative" throughout the manuscript
it is assumed that there is a N-terminal -barrel-like domain and a
C-terminal catalytic domain and the word putative will not be used for
the remainder of the text for simplicity.
3 X-S. Chen and C. D. Funk, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: HEK, human embryonic kidney; GFP, green fluorescent protein; LO, lipoxygenase; RBL, rat basophilic leukemia; PCR, polymerase chain reaction; RT, reverse transcription; HETE, hydroxyeicosatetraenoic acid; NLS, nuclear localizing signal.
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