From the Department of Immunology and Infectious
Diseases, Harvard School of Public Health and the § Division
of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital
and the Department of Medicine, Harvard Medical School, Boston,
Massachusetts 02115
Received for publication, September 27, 2000, and in revised form, March 9, 2001
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ABSTRACT |
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We report a novel phospholipase
A2 (PLA2), group XII (GXII)
PLA2, distinct from other cysteine-rich groups with a
catalytic histidine motif, by its 20-kDa size and distribution of the
14 cysteine residues within the protein. Alternative spliced forms with
distinct subcellular localization, designated GXII-1 and GXII-2, were
identified by reverse transcription-polymerase chain reaction.
Importantly, GXII PLA2s, in particular GXII-2
PLA2, and group V PLA2, but not group X
PLA2, were selectively expressed in murine type 2 helper T
(Th2) clones and in vitro differentiated mouse CD4 Th2
cells as compared with type 1 helper T clones and cells. Stimulation
with anti-CD3 appreciably up-regulated expression of GXII
PLA2s and group V PLA2 by steady state analysis
of the Th2 cells as compared with type 1 helper T cells. These results suggest that group XII and group V PLA2s might participate
in helper T cell immune response through release of immediate
second signals and generation of downstream eicosanoids.
The family of phospholipase A2
(PLA2)1 enzymes
is defined by the enzymatic activities of its members, which hydrolyze
the sn-2 ester bond of phospholipids to release free fatty
acids (1). Based on their protein structure and biochemical properties,
mammalian PLA2 enzymes can be divided into several classes:
low molecular weight secretory; 85-kDa cytosolic; selective acetyl
hydrolases of platelet-activating factor; and calcium-independent
PLA2. Thus far, eight low molecular weight mammalian
PLA2 enzymes have been described including group (G) IB,
five GII enzymes, GV, and GX (2-5).2 Each low
molecular weight PLA2 is 13-15 kDa in size and contains a
signal peptide and 12-14 cysteine residues, the positions and spacing
of which are conserved among the low molecular weight PLA2
members (6). In addition, an ~55-kDa mammalian protein containing a
region homologous to the 10-kDa bee venom PLA2 has been
described (7). Low molecular weight PLA2 enzymes require millimolar Ca2+ for activity and display no specificity for
the fatty acid in the sn-2 position of phospholipids (8).
Cytosolic PLA2, or GIV PLA2, is a large
cytosolic enzyme (~85 kDa), requires only micromolar Ca2+
for activity, and is the only cloned PLA2 that displays
high selectivity toward arachidonate-containing phospholipids (9, 10).
The hematopoietic calcium-independent PLA2, or GVI
PLA2, is an 85-kDa cytosolic enzyme (11). In addition to
the absence of a calcium requirement, the hematopoietic
calcium-independent PLA2 contains eight ankyrin motifs that
are not found in any other PLA2 (12). Groups IV and VI and
the platelet-activating factor acetyl hydrolases (GVII and GVIII)
exhibit a catalytic serine, whereas the cysteine-rich, low molecular
weight groups utilize a catalytic histidine (13).
GIV PLA2 has been established, by comparison of various
hematopoietic cell types derived from mice with a disruption of this gene with those of their normal littermates, as essential for providing
arachidonic acid to the downstream enzymes, such as cyclooxygenase (COX) and 5-lipoxygenase, for generation of
prostanoids and leukotrienes (14-16). GV PLA2,
although non-selective as to the sn-2 fatty acid, has been
implicated as a supporting enzyme for prostanoid biosynthesis in cell
systems by the kinetics of transcript induction and effects of generic
inhibitors for several related groups (17, 18), but definitive evidence
awaits cells with gene disruption. GIIA PLA2, a secretory
granule-stored species, has been shown to augment arachidonic acid
release by an extracellular membrane action that can be active
site-dependent or mediated by cell activation through
lectin-like receptors (19, 20). In addition to these downstream
eicosanoid mediators, there are metabolites with gene-inducing
functions via interaction with the intracellular receptors of the
peroxisome proliferator-activated receptor family (21, 22). Finally,
both released arachidonic acid and the concomitantly generated
lysophospholipid have second messenger functions (23-25). None of the
available information reveals a particular T cell distribution for any
PLA2 group; nor do the limited examples of gene disruption,
GIV and GIIA, provide evidence of an anomaly in the adaptive immune
response (14, 15, 26, 27).
Several lines of evidence suggest that the
PLA2/COX/prostaglandin cascade might play a critical role
in regulating the differentiation and function of helper T cells.
CD4+ helper T cells can be divided into two functional subsets
based on their secreted cytokines (28, 29). Type 1 helper T (Th1) cells
secrete interferon- Here, we report the molecular cloning of a novel PLA2,
designated group XII PLA2 (GXII PLA2), which
contains at least two alternatively spliced forms, GXII-1 and GXII-2.
Both murine GXII-1 (mGXII-1) PLA2 and GXII-2 (mGXII-2)
PLA2 contain a histidine-based PLA2 catalytic
domain and are cysteine-rich, although the position and spacing of the
cysteine residues are different from those in other PLA2s.
The enzymatic activity of mGXII-1 PLA2 is optimal at pH 8.0 and in low millimolar calcium. Importantly, mGXII PLA2, in
particular mGXII-2 PLA2, is preferentially expressed in Th2 cells, where it is induced upon stimulation through the T cell receptor. Thus, GXII PLA2 is novel in its 20-kDa size,
distribution of cysteine residues, and preferential expression in Th2 cells.
Cell Culture--
Murine Th2 clones, D10 and CDC35, and murine
Th1 clones, AE7, OF6, and AR5, were maintained in RPMI supplemented
with 10% fetal calf serum and 10% conditioned medium from Con
A-stimulated rat splenocytes. The murine Th1 clone D1.1 was maintained
in RPMI supplemented with 10% fetal calf serum, 5% T-STIM
(Collaborative Biomedical, Bedford, MA), and human IL-2 at 50 units/ml.
When indicated, Th clones were stimulated with plate-bound anti-CD3 for
6 h before harvesting. The BHK cell is a baby hamster kidney cell
line maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.
In Vitro Differentiation and Stimulation of CD4+Th and B
Cells--
CD4+Th cells and B cells were purified from spleens and
lymph nodes harvested from 6-8-week-old BALB/C mice by using
CD4 (L3T4) and CD45R (B220) MicroBeads (Miltenyi Biotec Inc.,
Auburn, CA) according to the manufacturer's instructions.
Purified CD4+Th cells were stimulated in vitro with
plate-bound anti-CD3 monoclonal antibody (2C11) at 1 µg/ml in the
presence of anti-IL-12 monoclonal antibody (5C3) at 20 µg/ml
(Th2-skewing conditions) or anti-IL-4 monoclonal antibody (11B11) at 5 µg/ml (Th1-skewing conditions). 24 h poststimulation, IL-2 at 50 units/ml was added to all cultures. In addition, IL-4 at 500 units/ml
or IL-12 at 50 units/ml was added into Th2 or Th1 cultures,
respectively. 7 days poststimulation, cells were harvested, washed
thoroughly, and restimulated with plate-bound anti-CD3. 24 h after
restimulation, cells were harvested for RNA preparation. All antibodies
were purchased from PharMingen (San Diego, CA). The purified B cells
were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and
ionomycin (1 µM) for 6 h before harvesting.
Preparation of Recombinant Proteins--
An
SmaI/HindIII restriction fragment encompassing
amino acid residues 11-192 of mGXII-1 PLA2 was cloned into
the EcoRV and HindIII site of pET-29c (Novagene,
Madison, WI). The resulting plasmid and the empty pET-29c,
respectively, were used to transform the BL-21 Escherichia
coli (Novagene). 100 µl of the overnight culture of the
transformed BL-21 cells was diluted with 10 ml of fresh LB medium
containing kanamycin (30 µg/ml), cultured at 37 °C for 3 h,
and induced with 1 mM
isopropyl-1-thio- Characterization of Phospholipase A2
Activity--
PLA2 activity was assessed by the hydrolysis
of
1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine
to liberate [14C]arachidonic acid using a lipsome-based
assay. 10-25 ng of recombinant mGXII-I PLA2 or an equal
volume of a concomitantly prepared insoluble fraction of empty
pET29c-transformed BL-21 cells was adjusted to a final volume of 125 µl containing 1.2 mM CaCl2, 10 mM
Tris-HCl (pH 8.0), and 3.6 µM
1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine
for 0-30 min at 37 °C. The reaction was stopped by the addition of
625 µl of Dole's reagent. Free [14C]arachidonic acid
was extracted in n-heptane and counted in a liquid
Northern Analysis and RT-PCR--
Stimulated or unstimulated
cells were harvested, and total RNA was prepared by using the Trizol
reagent (Life Technologies, Inc.) according to the
manufacturer's instructions. Total RNA derived from various organs was
purchased from Ambion (Austin, TX). For Northern analysis, 10 µg of
each RNA sample was fractionated on a 1.2% agarose gel, transferred to
a nitrocellulose membrane, and hybridized with indicated cDNA
probes in the QuickHyb buffer (Stratagene, La Jolla, CA). The cDNA
probes used were the full-length mGXII-1 PLA2, the
full-length mGV PLA2, and the full-length GFP Fusion Protein Analysis--
A partially digested
SalI/BglII fragment and a partially digested
XhoI/BglII cDNA fragment encoding nearly
full-length cDNAs of mGXII-1 PLA2 and mGXII-2
PLA2, respectively, were cloned into the
XhoI/BamHI site, in frame with the GFP cDNA,
of pEGFP-N (CLONTECH, Palo Alto, CA). The resulting
expression vectors and empty pEGFP-N vector were used to transfect BHK
cells by using Effectene (Qiagen, Valencia, CA) according to the
manufacturer's instructions. The transfected cells were replated on
glass slides 24 h later, rested overnight, fixed with 3%
paraformaldehyde for 10 min at 4 °C, stained with
4,6-diamidino-2-phenylindole (0.005% in phosphate-buffered saline) for
2 min at room temperature, and observed under fluorescence microscopy.
Immunocytochemistry--
The full-length mGXII-1
PLA2 cDNA was cloned into the SalI site of
the pCI (Promega) vector. An SphI/NotI
restriction fragment, encoding the last 12 amino acid residues of
mGXII-1 PLA2, was replaced with a double-stranded
oligonucleotide encoding an HA tag. The resulting vector and an
expression vector for HA-tagged NIP45 were used to transfect BHK cells
(42). The transfected BHK cells were replated and fixed as described
above. The fixed BHK cells were then permeabilized with acetone (2 min
at Molecular Cloning of Murine GXII PLA2, a Novel Member
of the PLA2 Family--
In the process of screening a D10
Th2 cell cDNA library, we isolated a hybrid cDNA clone that
contained the 3' untranslated region of the c-maf
protooncogene fused to an ~1.5-kilobase novel cDNA.
Sequence analysis revealed that the novel 1.5-kilobase cDNA encoded
an open reading frame (ORF) of 191 amino acid residues, containing a
signal peptide at its NH2 terminus and a conserved sequence for the catalytic domain of low molecular weight
PLA2 enzymes. In addition, the ORF contained 14 cysteine
residues, similar to the GIIA PLA2 protein. However, the
position and spacing of the cysteine residues in this novel ORF were
distinct from those of other low molecular weight PLA2s.
For instance, although this ORF contains cysteine residues that are
capable of forming disulfide bonds 27-131 and 51-102, it lacks the
appropriately spaced cysteine residues required to form disulfide bonds
29-45, 44-109, 61-95, and 85-100, which are found in all the
mammalian low molecular weight PLA2s. The position and
spacing of cysteine residues in this novel ORF are also different from
those of other histidine-based PLA2s, such as GIII, GIX,
and GXI (13). This novel ORF also lacks a canonical calcium binding
domain for low molecular weight PLA2s (Fig.
1). Furthermore, its predicted molecular mass is ~21 kDa, which is significantly larger than that of
the low molecular weight PLA2s, which are typically ~14
kDa. These results implied that the ORF represented a novel member of
the PLA2 family, henceforth called group XII-1
PLA2 (GXII-1 PLA2).
Identification of Human Group XII-1 PLA2 and an
Alternatively Spliced Form of Murine Group XII-1
PLA2--
We subsequently searched the
GenBankTM Data Bank with the mGXII-1 PLA2
sequence and identified several human expressed sequence tag clones
encoding the human GXII-1 PLA2 (hGXII-1), which is more
than 90% homologous to the mGXII-1 PLA2 (Fig. 1). A
further search of the GenBankTM Data Bank with the hGXII-1
PLA2 sequence revealed that the hGXII-1 PLA2
sequence matched a genomic sequence derived from human chromosome 4q25
(accession number AC004067). Furthermore, a Drosophila ortholog (accession number AAF49567), which contains a consensus
histidine catalytic PLA2 domain and 14 cysteine residues with spacing almost identical to that of mGXII-1 PLA2, was
present in GenBankTM. This result suggests that GXII
PLA2 is evolutionarily conserved. In addition to hGXII-1
PLA2, we also identified two murine expressed sequence tag
clones (accession numbers AA008695 and AA204520) encoding an mGXII-1
PLA2-related open reading frame, in which the first 73 amino acid residues of mGXII-1 PLA2, including the signal
peptide, were replaced with a novel amino acid sequence of 34 residues
following a distinct initial methionine (Fig. 1). This related open
reading frame, independently derived from embryo and lymph node
cDNA libraries, does not encode any obvious signal peptide and
might represent an alternatively spliced form of mGXII-1 PLA2; this open reading frame was designated murine
group XII-2 PLA2 (mGXII-2 PLA2).
Characterization of Recombinant mGXII-1 Phospholipase
A2 Activity--
To determine whether mGXII-1
PLA2 has functional phospholipase A2 catalytic
activity, we expressed an NH2-terminal S-tagged protein in
E. coli and enriched the protein to >90% purity as described under "Experimental Procedures" (Fig.
2A). The capacity of the
recombinant protein to hydrolyze arachidonic acid in the sn-2 position of phosphatidylethanolamine was determined.
Maximal PLA2 activity was observed at pH 8.0 in 1.2 mM calcium (data not shown). The kinetics of release of
arachidonic acid using 10 ng of enzyme was linear from 5 to 30 min
(Fig. 2B) and yielded a specific activity of 0.24 µmol/min
per mg of enzyme.
Subcellular Localization of mGXII PLA2
Enzymes--
Similar to other low molecular weight PLA2s,
the mGXII-1 PLA2 contains a signal peptide at its
NH2 terminus, suggesting that mGXII-1
PLA2 might be a secreted protein or a membrane-associated protein. To determine the subcellular localization of both mGXII-1 PLA2 and mGXII-2 PLA2, we fused them with GFP
and then overexpressed the fusions in BHK cells. As shown in
Fig. 3A, mGXII-1
PLA2 displayed a pattern consistent with localization to
the Golgi/endoplasmic reticulum in BHK cells. The Golgi/endoplasmic
reticulum pattern of mGXII-1 PLA2 was more obvious when an
HA-tagged mGXII-1 PLA2 was used (Fig. 3B). For
controls, overexpression of the GFP expression plasmid alone displayed
a diffuse pattern, and an HA-tagged nuclear protein, NIP45 (42), was
localized to the nuclei of BHK cells. In contrast to mGXII-1
PLA2, overexpression of GFP-mGXII-2 PLA2 gave a pattern indistinguishable from that of GFP alone. This is in
agreement with the absence of a signal peptide in mGXII-2 PLA2. These results demonstrated that mGXII-1
PLA2 and mGXII-2 PLA2 have distinct subcellular
localizations.
Group XII PLA2s and Group V PLA2 Are
Preferentially Expressed in Th2 Cells--
Because there is some
evidence to suggest that the PLA2/COX/eicosanoid cascade
regulates the differentiation and function of helper T cells, we
examined the expression of mGXII PLA2s among various Th
clones by Northern analysis using a cDNA probe common to both
mGXII-1 PLA2 and mGXII-2 PLA2. As shown in Fig.
4A, the expression of mGXII
PLA2s was easily detected in Th2 clones, such as D10 and
CDC35, and was further induced upon stimulation with anti-CD3. In
contrast, the Th1 clones expressed very low levels of mGXII
PLA2s, which were also less abundant after anti-CD3
stimulation than transcript levels in Th2 clones. Furthermore, mGXII
PLA2 was expressed at very low levels in a naïve
CD4+Th cell population and was dramatically induced in cells that had
been polarized along the Th2 pathway but not the Th1 pathway (Fig.
4B). Of note, two transcripts were detected by the common
cDNA probe, further suggesting the presence of alternative splice
forms or homologues of mGXII PLA2.
Because we were unable to distinguish mGXII-1 PLA2 from
mGXII-2 PLA2 by Northern analysis, we used
sequence-specific primers in RT-PCR to examine the expression and cell
type specificity of both forms of mGXII PLA2. As shown in
Fig. 5, the expression kinetics and cell
type specificity of both mGXII-1 PLA2 and mGXII-2 PLA2, in particular mGXII-2 PLA2, as examined
by RT-PCR, are similar to those revealed by Northern analysis.
Importantly, mGXII-2 PLA2 is almost exclusively expressed
in stimulated normal Th2, but not Th1 or B, cells (Fig.
5A).
In contrast to its subset-specific expression among the Th cell
lineages, transcripts encoding mGXII-1 PLA2 could be
detected in a variety of organs and tissues (Fig. 5B).
Interestingly, transcripts encoding mGXII-2 PLA2 were only
detected in spleen and, to a much lesser degree, in embryo (Fig.
5B). This is in agreement with the origin of the two mGXII-2
PLA2 expressed sequence tag clones described previously.
The knowledge that an arachidonic acid-selective PLA2, GIV
PLA2, could function in concert with GV PLA2
for PGE2 generation in a mouse macrophage line (18) and for
PGD2 production by mouse bone marrow-derived mast cells
(43) prompted an assessment of the expression of GV PLA2 in
Th cells. We repeated the RT-PCR analysis above using primers specific
to mGV PLA2 or mGX PLA2. As shown in Fig.
5A, the expression of mGV PLA2 was detected in stimulated Th2 cells but not in Th1 cells, whereas the expression of
mGX PLA2 was comparable between Th1 and Th2 cells. The Th2 cell-specific expression of mGV PLA2 was further confirmed
by Northern analysis using Th clones. As shown in Fig. 5C,
mGV PLA2 was expressed at a very low level in resting D10
cells (Th2 cells); however, its expression was dramatically induced
within 6 h after anti-CD3 stimulation. In contrast, no expression
of mGV PLA2 was detected in AE7 cells (Th1 cells).
Here we have described the molecular cloning and characterization
of a novel PLA2 group, designated group XII. Despite the generic characteristics of being cysteine-rich and functioning optimally at a basic pH, GXII PLA2 merits this separate
designation because of the following features. It shares no significant
amino acid sequence homology, other than the catalytic histidine motif, with other PLA2 enzymes. Although GXII PLA2 is
cysteine-rich, the spacing of cysteine residues is distinct from that
of other low molecular weight PLA2 enzymes. GXII
PLA2 also lacks the canonical calcium binding domain of the
low molecular weight PLA2 enzymes. The molecular
mass of mGXII-1 PLA2 is ~20 kDa, which is somewhat larger than the 14 kDa of the low molecular weight cysteine-rich PLA2 enzymes. These observations and the calculated
specific activity of the recombinant mGXII-1 PLA2 are in
agreement with those of Gelb et al. (44), who also described
mammalian GXII PLA2 while this manuscript was under review.
In addition, GXII PLA2 is present in more than one species,
and alternative spliced forms, mGXII-1 and mGXII-2, have been identified.
Murine GXII PLA2, in particular mGXII-2 PLA2,
and mGV PLA2 are preferentially expressed in Th2 cells
within the T cell lineage. To the best of our knowledge, this is the
first report describing the differential expression of certain
PLA2 members in Th cells. These observations suggest an as
yet unknown effector function of Th2 cells. In light of the report that
COX2 and hematopoietic prostaglandin D synthase are also preferentially
expressed in Th2 cells (40), it is intriguing to postulate that the Th2
cell-specific PLA2s, such as mGXII and mGV, might be
functionally linked to COX2 and hematopoietic prostaglandin D synthase
in Th2 cells. Thus, in Th2 cells, mGXII PLA2 and mGV
PLA2 might provide arachidonic acid to COX2 for generation of
intermediates that hematopoietic prostaglandin D synthase converts to
PGD2. This scenario is further supported by three
observations. First, mGXII PLA2 was induced in Th2 cells
after T cell receptor stimulation (Fig. 4B) in a time course
parallel to that observed for COX2 induction (40). Second, exogenous GV
PLA2 was able to induce the expression of COX2 in
macrophages (17). Third, mGXII-1 PLA2 is present in a
perinuclear location in transfected BHK cells. It is known that the
generation of eicosanoids is dependent on the location within the cell
of the enzymes of their biosynthesis, which seems to occur in a
perinuclear location. Group V PLA2 has been described in
association with the nuclear envelope in mouse mast cells and acts in a
cooperative manner with GIV cytosolic PLA2 to provide arachidonic acid for eicosanoid biosynthesis (45). Thus, the mGXII
PLA2 and mGV PLA2, two Th2 cell-specific
enzymes, are well placed for a functional interaction with COX2 induced
at the nuclear envelope and endoplasmic reticulum to generate
PGG2/PGH2, which are processed by hematopoietic
prostaglandin D synthase to provide PGD2 and its
metabolites. Interestingly, a metabolite of PGD2, 15-deoxy- An alternatively spliced form, mGXII-2 PLA2, which does not
contain any signal peptide, was also identified. Most low molecular weight PLA2 members are either secreted proteins or are
associated with cell membranes and act in situ. Murine
GXII-2 PLA2, without a signal peptide, may be the only
exception within the low molecular weight PLA2 family.
Although the GFP-mGXII-2 PLA2 fusion protein is
homogeneously distributed within BHK cells, it should be noted that
this result was obtained by overexpression of a GFP fusion protein and
might not reflect the distribution of the endogenous protein. The
actual subcellular localization of GXII PLA2s can only be
definitively determined by immunocytochemistry using antibodies that
distinguish the two alternative spliced products of GXII PLA2; such antibodies are not yet available.
Assuming, however, that the subcellular localization of the GFP-mGXII-2
PLA2 fusion protein faithfully reflects that of endogenous
mGXII-2 PLA2, this result further supports the notion that
mGXII-2 PLA2 might mediate a biological function, in
addition to releasing arachidonic acid from cell membranes. The
in vivo function of GXII PLA2 will be revealed
by the analysis of GXII PLA2-deficient mice, currently being generated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and IL-2 and are responsible for delayed type
hypersensitivity and eradication of intracellular microorganisms. Type
2 helper T (Th2) cells produce cytokines such as IL-4 and IL-13, which enhance the production of IgE antibodies involved in allergic responses, and IL-5, which promotes the maturation of eosinophils. Furthermore, IL-4 regulates Fc
RI expression of human mast cells, whereas IL-5 is comitogenic with stem cell factor for their mucosal expansion (30). There is evidence that prostaglandin E2
(PGE2), a metabolite of the cyclooxygenase pathway,
selectively promotes the differentiation of Th2 cells (31). Production
of IL-2 and interferon-
by short term peripheral blood lymphocyte
cultures and by long term Th clones is inhibited by PGE2 in
a dose-dependent fashion, whereas IL-4 and IL-5 production
is enhanced (32, 33). Naïve human CD4+ T cells stimulated with
anti-CD3 in the presence of PGE2 display a Th2 phenotype,
which is maintained upon restimulation in the absence of
PGE2 (34). Furthermore, PGE2 can directly inhibit transcription of the IL-2 gene but not the IL-4 gene in human
Jurkat T cells (35, 36). In addition to its direct effect on the
transcription of cytokine genes in T cells, PGE2 inhibits the release of IL-12, the potent Th1-promoting cytokine, from antigen-presenting cells, such as dendritic cells (37, 38), and
inhibits the expression of the IL-12 receptor in differentiating Th
cells (39). Recently, PGD2, the dominant mast cell-derived prostanoid species, has been added to the growing list of Th2 effectors. Hematopoietic prostaglandin D2
synthase is expressed in human Th2 but not Th1 cells. The
up-regulated expression of hematopoietic prostaglandin D2
synthase and de novo expression of COX-2, induced with
anti-CD3, result in the production of PGD2 by Th2 cells
(40). Of note, mice rendered deficient in the PGD2 receptor, DP, have impaired Th2 responses and allergen-induced airway hyperresponsiveness in a model of allergic asthma (41).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 2 h at
37 °C to express recombinant mGXII-1 PLA2 protein. The
induced BL-21 cells were lysed with 0.6 ml of BugBusterTM protein extraction reagent (Novagene). The insoluble fractions were resuspended with 0.6 ml of BugBusterTM reagent containing 200 µg/ml lysozyme and
incubated at room temperature for 5 min. The lysozyme-treated insoluble
fractions were washed three times with 1 ml of 1:10 diluted BugBusterTM
reagent. The washed insoluble fractions were subsequently solubilized
in 0.6 ml of 1:10 diluted BugBusterTM buffer containing 6 M
guanidine HCl. The denatured proteins were refolded by sequential
dialysis at room temperature against 1 liter of buffer containing 25 mM Tris-HCl (pH 8.0), 5 mM cysteine, and 4, 2, or 1 M guanidine HCl (8 h for each dialysis buffer). Guanidine HCl was then removed by dialysis against 2 liters of 25 mM Tris-HCl (pH 8.0) for 24 h at room temperature. The
resulting recombinant mGXII-1 PLA2 (at ~50 µg/ml)
contains an NH2-terminal S tag and is more than 90% pure
as examined by Coomassie Brilliant Blue staining.
-scintillation counter.
-actin. RT-PCR
was performed by using the RT-PCR kit (Promega, Madison, WI) according
to the manufacturer's suggestions. 0.5 µg of each RNA sample was
used per RT-PCR reaction. The sequences of primers used in
RT-PCR were as follows: mGXII-1 sense, 5'-GGGCAGGAACAGGACCAGACCACCG-3'; mGXII-2 sense, 5'-CCACAGTGGTCCTGGGAAGTACTGGG-3'; mGXII-1 and mGXII-2 antisense, 5'-GGTTTATATCCATAGCGTGGAACAGGCTTCG-3'; mGV sense,
5'-GTCCATGATTGAGAAGGTGACC-3'; mGV antisense,
5'-TCATAGGACTGGGTCCGAATGG-3; mGX sense, 5'-GGTCACATGTATACAAGCGTGG-3'; mGX antisense, 5'-ATGTGATGGTCCATGCACTTCC-3';
-actin sense,
5'-GTGGGCCGCTCTAGGCACCA-3';
-actin antisense,
5'-CGGTTGGCCTTAGGGTTCAGGGGGG-3'.
20 °C), stained with anti-HA antibody (1:500 dilution of 12CA5,
Roche Molecular Biochemicals), washed three times with
phosphate-buffered saline, and restained with Cy3-conjugated goat
anti-mouse IgG (H+L) antibody (1:300 dilution, Jackson ImmunoResearch
Laboratories, West Grove, PA).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparison of the amino acid sequences of
GXII PLA2s and other low molecular weight
PLA2s. All sequences listed are in single-letter amino
acid code. The numbers on the right-hand side are the
numbers of the amino acid residues of each low molecular weight
PLA2. The catalytic domain, the conserved cysteine
residues, and the calcium binding domain of low molecular weight
PLA2s are boxed with thick,
thin, and dotted lines, respectively. The
cysteine residues that are conserved among low molecular weight
PLA2s and are used to form disulfide bonds are also
numbered, according to Ref. 49, at the bottom of the boxes.
All the sequences listed are mouse protein sequences (m),
with the exception of the human GXII-1 PLA2. The alignment
was created by using the Lasergene program (DNASTAR). The
GenBankTM accession numbers for mGXII-1 PLA2
and mGXII-2 PLA2 are AY007381 and AY007382,
respectively.
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Fig. 2.
Biochemical characterization of
murine GXII-1 PLA2. A, SDS-polyacrylamide
gel electrophoresis analysis of purified recombinant mGXII-1
PLA2. 5 µl (~250 ng) of recombinant mGXII-1
PLA2 or a concomitantly prepared insoluble fraction of
empty pET29c-transformed BL-21 cells (control), as described under
"Experimental Procedures," was resolved on a 12%
SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue.
B, 10 ng ( ), 25 ng (x), or no enzyme (
) were assayed
for PLA2 activity by the kinetics of release of
[14C]arachidonic acid product from
1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine
as described under "Experimental Procedures."
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Fig. 3.
Subcellular localization of mGXII
PLA2s. A, BHK cells were transfected with
expression vectors for GFP, GFP-mGXII-1 PLA2, or
GFP-mGXII-2 PLA2, fixed with paraformaldehyde, stained with
4,6-diamidino-2-phenylindole (DAPI), and observed under
fluorescence microscopy. B, BHK cells were transfected with
expression vectors for HA-tagged NIP45 or HA-tagged mGXII-1
PLA2 and subjected to immunocytochemistry by using an
anti-HA antibody as described under "Experimental
Procedures."
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Fig. 4.
Expression of mGXII PLA2
transcripts in murine Th1 and Th2 cells. 10 µg of each indicated
total RNA sample was fractionated on 1.2% formaldehyde-agarose gels,
transferred to nitrocellulose membranes, and hybridized with the
indicated cDNA probes. A, total RNA was derived
from resting, ionomycin-stimulated (1 µM,
Ion), or anti-CD3-stimulated Th1 clones (D1.1, OF6, and AR5)
or Th2 clones (D10 and CDC35). B, naïve CD4+Th cells
were differentiated in vitro into either Th1 or Th2 cells as
described under "Experimental Procedures." At the indicated time
points, total RNA was prepared and subjected to Northern analysis.
hr and d stand for hours and days after
stimulation, respectively. Preparation of these two Northern blots was
described previously (50, 51). Both blots were stripped prior to
hybridization with the mGXII PLA2 cDNA probe.
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Fig. 5.
A and B, RT-PCR analysis of
mGXII-1, mGXII-2, mGV, and mGX PLA2s. 0.5 µg of total RNA
prepared from (A) in vitro differentiated Th1 and
Th2 cells (either resting or 6 h after anti-CD3 stimulation) and
purified primary B cells (stimulated with phorbol 12-myristate
13-acetate/ionomycin for 6 h) and (B) from various
organs was used in RT-PCR reactions as described under "Experimental
Procedures." -actin was also amplified as a control for the amount
of input RNA. C, Northern analysis of mGV PLA2
in Th clones. Total RNA was prepared from resting or
anti-CD3-stimulated D10 and AE7 cells and subjected to Northern
analysis for the expression of mGV PLA2. The same blot was
also hybridized with a
-actin cDNA probe as a control for the
amount of input RNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12,14-PGJ2, is one of the natural
ligands of the nuclear coreceptor, peroxisome proliferator-activated
receptor-
(21, 22), which was recently shown to be induced by the
Th2 cytokine IL-4 and which can regulate gene expression in both T and
non-T cells (46, 47). Furthermore, the recent report that a second
G-protein-coupled PGD2 receptor, chemoattractant
receptor-homologous molecule expressed in Th2 cells (CRTH2), resides on
Th2 cells, eosinophils, and basophils and is pharmcacologically
distinct from DP PGD2 receptor (48) introduces the
possibility of an autocrine pathway. Alternatively, mGXII
PLA2 and mGV PLA2 might affect the
differentiation and function of Th2 cells independently of their
enzymatic activities. In agreement with this hypothesis, GI and GIIA
PLA2s can initiate signaling events by binding to
lectin-like cell surface receptors (4, 19).
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ACKNOWLEDGEMENTS |
---|
We thank Christine McCall for manuscript preparation and Richard Goddeau for technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI/AG 37833 (to L. H. G.) and PO1 HL36110 (to I.-C. H., J. P. A., and K. F. A.), a grant from the American Cancer Society (to J. P. A.), and a gift from The G. Harold and Leila Y. Mathers Charitable Foundation (to L. H. G.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY007381 and AY007382.
¶ To whom correspondence should be addressed: Dept. of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Ave., Boston, MA 02115-6017. Tel.: 617-432-1158; Fax: 617-432-0084; E-mail: iho@hsph.harvard.edu.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M008837200
2 A comprehensive abbreviation system for the various mammalian low molecular weight PLA2s is used. Each low molecular weight PLA2 is abbreviated, with a lowercase letter indicating the low molecular weight PLA2 species (m and h for mouse and human, respectively), followed by uppercase letters identifying the low molecular weight PLA2 group.
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ABBREVIATIONS |
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The abbreviations used are: PLA2, phospholipase A2; G, group; COX, cyclooxygenase; Th, helper T; IL, interleukin; PG, prostaglandin; m, murine; BHK, baby hamster kidney; RT-PCR, reverse transcription-polymerase chain reaction; GFP, green fluorescent protein; HA, hemagglutinin; ORF, open reading frame; h, human.
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