From the Institut de Pharmacologie Moléculaire
et Cellulaire, CNRS-UPR 411, 660 route des Lucioles, Sophia Antipolis,
06560 Valbonne, France and the § Laboratory of Genetic
Engineering, Cancer Research Institute, Tata Memorial Centre,
Parel, Mumbai, 400012, India
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ABSTRACT |
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Snake venom and mammalian secreted phospholipases
A2 (sPLA2s) have been associated with
toxic (neurotoxicity, myotoxicity, etc.), pathological (inflammation,
cancer, etc.), and physiological (proliferation, contraction,
secretion, etc.) processes. Specific membrane receptors (M and N types)
for sPLA2s have been initially identified with snake venom
sPLA2s as ligands, and the M-type 180-kDa receptor was
cloned from different animal species. This paper addresses the problem
of the endogenous ligands of the M-type receptor. Recombinant group IB
and group IIA sPLA2s from human and mouse species have been
prepared and analyzed for their binding properties to M-type receptors
from different animal species. Both mouse group IB and
group IIA sPLA2s are high affinity ligands (in the 1-10
nM range) for the mouse M-type receptor. These
two sPLA2s are expressed in the mouse tissues where the
M-type receptor is also expressed, making it likely that both types of
sPLA2s are physiological ligands of the mouse M-type
receptor. This conclusion does not hold for human group IB and IIA
sPLA2s and the cloned human M-type receptor. The two mouse
sPLA2s have relatively high affinities for the mouse M-type
receptor, but they can have much lower affinities for receptors from
other animal species, indicating that species specificity exists for
sPLA2 binding to M-type receptors. Caution should thus be
exerted in avoiding mixing sPLA2s, cells, or tissues from
different animal species in studies of the biological roles of
mammalian sPLA2s associated with an action through their membrane receptors.
Secreted phospholipases A2
(PLA2s,1
phosphatide 2 acylhydrolase, EC 3.1.1.4) form a growing family of
Ca2+-dependent enzymes that release free fatty
acids and lysophospholipids from glycerophospholipids (1-4). To date,
five different sPLA2s referred to as group IB, IIA, IIC, V,
and X sPLA2s have been characterized in mammals. The main
common properties of these sPLA2s are their relatively low
molecular mass (13-16 kDa), the presence of many disulfide bridges in
their structure, and a low selectivity for phospholipids with different
polar head groups and fatty acid chains (5).
Group IB sPLA2 is known as the pancreatic-type
sPLA2. It was originally found in large amounts in the
pancreas and then proposed to function in the digestion of dietary
lipids (6). Later, this enzyme was identified and cloned in other
tissues such as lung, spleen, kidney, and ovary (3, 7), and it has now
been proposed to be involved in various physiological and
pathophysiological responses such as cell proliferation (8), cell
contraction (9, 10), lipid mediator release (11), acute lung injury (12), and endotoxic shock (13).
Group IIA sPLA2 is also referred to as the
inflammatory-type sPLA2, since it is highly expressed in
the plasma and synovial fluids of patients with various inflammatory
diseases such as rheumatoid arthritis, acute pancreatitis, Crohn's
disease, and endotoxic shock (3, 14-16) as well as in various cancers
(17, 18). The group IIA sPLA2 has been shown to participate
in the production of lipid mediators of inflammation (3, 19, 20) and in
the destruction of pathogenic microorganisms (21). Recent data using
group IIA sPLA2-deficient mice have suggested, however, that this sPLA2 may not play a pivotal role in the
progression and/or pathogenesis of inflammatory processes, at least in
the mouse (22, 23). The mouse group IIA sPLA2
(mGIIA)2 has also been
proposed to have a role in cell proliferation (24) and more recently to
act as a tumor suppressor gene in a mouse model of colorectal cancer
(25, 26).
Much less is known about the regulation and the biological roles of the
more recently cloned group IIC, V, and X sPLA2s. Group IIC
sPLA2 has been cloned in rat and mouse (27) but appears to
be a nonfunctional pseudogene in humans (28). Group V sPLA2 is highly expressed in heart (29) and is detected in murine macrophages
and mastocytes, where it is proposed to play an important role in lipid
mediator production in place of the group IIA sPLA2 (22,
23). Group X sPLA2 was recently cloned in human and has structural features that resemble those of group IB and group IIA
sPLA2s (30). It is expressed in the immune system,
suggesting possible roles related to inflammation or immunity.
Snake and insect venoms also contain a large diversity of
sPLA2s (31, 32). Most venom sPLA2s are potent
toxins that exert many effects including neurotoxicity and myotoxicity
(31, 32). Two main types of high affinity sPLA2 receptors
have been identified using venom sPLA2s, including
OS1 and OS2 purified from Taipan snake venom
(33). N-type sPLA2 receptors were first identified in rat
brain membranes (34). These receptors have high affinities for
neurotoxic sPLA2s such as OS2 and the bee venom
sPLA2 (bvPLA2) but not for nontoxic
sPLA2s such as OS1, suggesting that N-type receptors contribute to the neurotoxic effects (34-36). M-type sPLA2 receptors were first identified in skeletal muscle
cells (37) but are also expressed in other tissues (33). They consist of a single 180-kDa subunit and recognize with high affinity
OS2 and OS1 but not the neurotoxic
bvPLA2. The M-type receptor was later also identified by
using as ligand the group IB sPLA2 and was then proposed to
have an essential role in the various biological effects produced by
the group IB sPLA2 (8, 38). The M-type receptor has now
been cloned in different species (39-42) and found to belong to a
novel family of membrane receptors comprising the macrophage mannose
receptor, the dendritic cell receptor, and the endothelial lectin In an effort to clarify this situation, we have prepared various native
and recombinant group IB and group IIA sPLA2s and analyzed
their binding properties to M-type receptors of different animal
species. We found that mouse group IB sPLA2 and mouse
group IIA sPLA2 are recognized by the mouse M-type
receptor, indicating that both types of sPLA2s are probably
true endogenous ligands of the M-type receptor in mouse. We also
provided evidence that this binding may occur in vivo, since
sPLA2s and the M-type receptor are co-expressed in several
mouse tissues.
Preparation of Native sPLA2s--
Oxyuranus
scutellatus scutellatus sPLA2s (OS1 and
OS2) and bvPLA2 were purified as described
previously (34). pGIB was purchased from Boehringer Mannheim and
further purified on a Spherogel TSK SP-5PW HPLC column (10 µm,
75 × 7.5 mm, 3.3 ml) equilibrated in acetic acid 1% (v/v) and
eluted using a linear gradient of ammonium acetate (0-2 M,
pH 6.8 in 70 min). Human pancreatic group IB sPLA2 (hGIB)
was provided by Dr. Hubertus Verheij.
Cloning and Preparation of Recombinant Mouse Group IB
sPLA2--
A search for homology in genome data bases with
sPLA2 protein sequences led to the identification of an
I.M.A.G.E. Consortium cDNA clone (identification number 315430, 5';
GenBankTM accession no. W12659) from the house mouse with a
strong similarity to known pancreatic group IB sPLA2s. The
obtained cDNA clone was sequenced and found to contain a cDNA
insert of 555 base pairs that encodes for the full-length mouse group
IB sPLA2 (mGIB) cDNA. The full-length cDNA
including the prepropeptide sequence was subcloned into the baculovirus
transfer vector pVL 1392 and transfected into Spodoptera
frugiperda cells (Sf9; ATCC CRL 1711) using the BaculoGoldTM transfection kit (Pharmingen). After two
rounds of virus amplification into Sf9 cells, Trichoplusia
ni High Five insect cells (Tn5) were used for the production of
recombinant sPLA2s, since preliminary experiments have
shown a 3-fold higher yield of sPLA2 production compared
with Sf9 cells. Furthermore, preliminary experiments indicated
that infection of cells with mGIB baculovirus in growth medium
containing fetal bovine serum resulted in the secretion of a mixture of
proenzyme and mature forms of mGIB, since the sPLA2
activity of cell supernatant can be increased by the addition of
trypsin, while fully activated mGIB was recovered from supernatant of
cells that were cultivated in protein-free Insect-Xpress medium (BioWhittaker) (not shown). Large scale sPLA2 productions
were thus performed with Tn5-infected cells (2.106
cells/ml) grown in spinner culture bottles in protein-free
Insect-Xpress medium for 5 days. Cell-free supernatants of infected
cells (1 liter) were diluted twice in 1% (v/v) acetic acid and
incubated batchwise for 2 h, at 4 °C, and under continuous
agitation with 150 ml of SP Sephadex C-25 gel (Amersham Pharmacia
Biotech), which had been preequilibrated with 1% acetic acid. The gel
was washed with 1% acetic acid and 1% acetic acid containing 100 mM ammonium acetate. Bound proteins were then eluted
stepwise with 1% acetic acid containing 350 mM ammonium
acetate. sPLA2 containing fractions were lyophilized and
applied to a C18 Beckman reverse phase HPLC column (10 × 250 mm,
19.6 ml, 5 µm, 100 Å). Elution was performed using an acetonitrile
linear gradient in 0.1% trifluoroacetic acid, 10-60% acetonitrile
for 40 min at a flow rate of 4.5 ml/min. The peak containing
sPLA2 activity was lyophilized and applied to the Spherogel
TSK SP-5PW HPLC column as indicated above for the purification of pGIB.
The sPLA2 peak was finally applied on a C18
NucleosilTM reverse phase HPLC column (4.6 × 250 mm,
4.2 ml, 5 µm, 300 Å) that was eluted using an acetonitrile linear
gradient in 0.1% trifluoroacetic acid, 15-25% acetonitrile for 10 min followed by 25-45% acetonitrile for 100 min at 1 ml/min. The
final yield for the production of mGIB was 1.5 mg of purified
sPLA2/liter of cell medium.
Preparation of Recombinant Human Group IIA
sPLA2s--
AV12 fibroblast recombinant hGIIA (47) was
provided by Dr. Ruth Kramer (Lilly). Recombinant hGIIA from insect Tn5
cells was prepared as above for mGIB and using the full-length hGIIA cDNA (48). Membrane-bound hGIIA sPLA2 activity (3) was
extracted from pelleted baculovirus-infected Tn5 cells for 30 min with
100 ml of phosphate-buffered saline buffer containing 1 M
KCl and combined with the hGIIA sPLA2 activity of cell-free
supernatant. The pooled medium was diluted twice with water and
incubated batchwise for 1 h at 4 °C and under continuous
agitation with 40 ml of heparin-Sepharose CL-6B gel (Amersham Pharmacia
Biotech), which had been preequilibrated with 20 mM Tris,
pH 7.4, containing 140 mM NaCl. The gel was washed with the
equilibration buffer and 20 mM Tris pH 7.4 containing 200 mM NaCl. Bound proteins were then eluted stepwise with 20 mM Tris, pH 7.4, containing 1 M NaCl.
sPLA2-containing fractions were dialyzed and loaded as
described above for mGIB on a TSK SP-5PW column and C18
NucleosilTM large pore column. The final yield for the
production of hGIIA was about 0.3 mg of purified
sPLA2/liter of cell medium.
Preparation of Native and Recombinant Mouse Group IIA
sPLA2s--
Native mGIIA was acid-extracted from BALB/c
mouse intestine tissue and then loaded on a Bio-Gel P100 column as
described previously (49). The Bio-Gel P100 sPLA2 fractions
were further purified on TSK SP-5PW column and C18
NucleosilTM large pore column as described above. For
production of mGIIA in human 293 fibroblast cells, the mGIIA cDNA
(50) was cloned into the expression vector pRc/CMV (Invitrogen Corp.)
and stably transfected into 293 cells. A neomycin-resistant clone was
selected for its high level of expression and then used for cell
extraction of recombinant mGIIA as described above for intestinal
mGIIA. Recombinant mGIIA from baculovirus-infected Tn5 cells was
prepared essentially as described above for hGIIA. The final yield for the production of mGIIA in Tn5 cells was about 0.1 mg of purified sPLA2/liter of cell-infected medium.
Protein Characterization Techniques--
Protein concentrations
of sPLA2s were determined using the molar absorbance
coefficients calculated from protein sequence. Ion spray mass
spectrometry analysis was performed on a simple quadrupole mass
spectrometer equipped with an ion spray source and using polypropylene
glycol to calibrate quadrupole. N-terminal sequences were determined by
automated Edman degradation of sPLA2 with an Applied
Biosystems sequencer equipped with an on-line phenylthiohydantoin-derivative analyzer. sPLA2 activity
assays were performed using Escherichia coli membranes as
substrate (51).
Binding Studies--
Crude microsomal membranes from cells and
BALB/c adult mouse tissues were prepared as described previously (37,
52). Membrane preparations containing bovine, rat, mouse, and rabbit
M-type receptors were obtained from Madin-Darby bovine kidney cells
(ATCC CCL 22), rat aortic smooth muscle cells (A7r5; ATCC CRL 1444), mouse embryo fibroblast cells (NIH 3T3; ATCC CRL 1658), and rabbit skeletal muscle cells (37). COS cell membranes expressing the human
cloned M-type receptor were obtained as described (40). Recombinant
expression of the mouse M-type receptor was performed in COS cells
after cloning of its full-length cDNA from NIH 3T3 cells according
to published sequence (41) and transfection into COS cells as for the
human M-type receptor. All binding experiments were performed under
equilibrium binding conditions using as ligand 125I-OS1 labeled to a specific activity of
3000-3500 cpm/fmol as described by Lambeau et al. (37).
Briefly, membranes, 125I-OS1, and competitors
were incubated at 20 °C in 0.5 or 1 ml of buffer (140 mM
NaCl, 0.1 mM CaCl2, 20 mM Tris, pH
7.4, and 0.1% BSA). Incubations were started by the addition of
membranes and filtered after 90 min of incubation through GF/C glass
fiber filters presoaked in 0.5% polyethyleneimine. Cross-linking
experiments were performed with 50 µM suberic acid
bis-N-hydroxysuccinimide ester (Sigma) as described
previously (37, 52).
Northern Blot Analysis--
A homemade and a commercial mouse
Northern blot (CLONTECH Laboratories, Inc., catalog
no. 7762-1) containing RNAs from various adult mouse BALB/c tissues
were first probed with the random primed 32P-labeled
full-length mGIIA cDNA in 50% formamide, 5× SSPE (0.9 M NaCl, 50 mM sodium phosphate, pH 7.4, 5 mM EDTA), 5× Denhardt's solution, 0.1% SDS, 20 mM sodium phosphate, pH 6.5, and 250 µg/ml denatured
salmon sperm DNA at 50 °C for 18 h. Blots were washed to a
final stringency of 0.1× SSC (30 mM NaCl, 3 mM
trisodium citrate, pH 7.0) with 0.1% SDS at 55 °C and exposed to
Biomax MS Kodak films with an HE intensifying screen (Amersham
Pharmacia Biotech). Northern blots were then stripped, checked for
dehybridization, and hybridized under the same conditions as above with
the entire coding sequence of mGIB. The integrity and relative
quantities of RNAs were checked with the manufacturer's mouse
Preparation of Native and Recombinant Mammalian
sPLA2s--
Native or recombinant group IB and
group IIA sPLA2s from different species have been prepared
in order to analyze their binding properties to M-type receptors. The
human pancreatic group IB sPLA2 (hGIB) was purified to
homogeneity as described previously (53) and migrates as a single band
on SDS-polyacrylamide gel (Fig. 1).
Highly purified pGIB was obtained after further purification of the
commercially available pGIB preparation on a cation exchange HPLC
column. Six fractions with sPLA2 activity and molecular
masses close to 14 kDa as determined by gel analysis (data not shown) were resolved. N-terminal sequence and electrospray mass spectrometry indicated that the major peak (molecular mass of 13,981 Da) corresponds to the authentic isoform Binding Properties of Group IB and Group IIA sPLA2s to
M-type Receptors from Different
Species--
125I-OS1, a specific and very
high affinity ligand of the M-type receptor (33), was used to analyze
the binding properties of the various group IB and group IIA
sPLA2s to M-type receptors from different species. Fig.
2 and Table
I show the results obtained from
competition binding experiments between labeled OS1 and
unlabeled sPLA2s to M-type receptors from mouse, rat,
rabbit, human, and bovine species. OS1 has a similar and
very high affinity (100-200 pM) for all receptors except
for the human receptor, for which the observed
K0.5 value is only 4 nM (Table I). A
common pharmacological property of the different M-type receptors is
that 125I-OS1 binding is not inhibited by the
neurotoxic bvPLA2, which was previously found to be a
specific sPLA2 ligand for rat and rabbit N-type
sPLA2 receptors (33, 34, 37).
The binding profiles of pGIB and hGIB to the various M-type receptors
are very similar. Both sPLA2s display an affinity in the
nanomolar range for mouse, rat, and bovine receptors, have a
50-100-fold weaker affinity for the rabbit receptor, and bind very
weakly to the human receptor (Table I). These results are in accordance
with previous data obtained with pGIB and hGIB on mouse, rat, and
bovine M-type receptors (38). The binding profile of mGIB is slightly
different from that of pGIB and hGIB as it binds with high affinity to
mouse and rabbit receptors but with an ~10-fold lower affinity to the
rat receptor. However, as for pGIB and hGIB (40), mGIB is a very low
affinity ligand of the human M-type receptor. These data indicate that
the pancreatic-type sPLA2 mGIB is probably a natural
endogenous ligand for the mouse M-type receptor (Table I). However,
this does not hold for hGIB and the human M-type receptor (Table
I).
In contrast with the similar binding profiles observed with the group
IB sPLA2s, the group IIA sPLA2s mGIIA and hGIIA
were found to have very distinct binding properties (Fig. 2 and Table I). Recombinant hGIIA sPLA2s prepared from mammalian or
insect cells behave similarly. They do not bind to rat, bovine, and
human M-type receptors; they associate with the mouse receptor but only with a very low affinity; and they bind with a nanomolar affinity to
the rabbit receptor. Taken together, these results suggest that hGIIA
is not a physiological ligand for the human receptor. However, the
recombinant hGIIA proteins appear properly folded, since they can bind
with high affinities to the rabbit M-type receptor. Similar to hGIIA,
both native and recombinant mGIIA sPLA2s were found to
recognize the rabbit receptor with a nanomolar affinity, while they do
not bind to the human receptor. However, mGIIA was found to associate
with a relatively high affinity of ~10 nM to the mouse
M-type receptor, and it also binds to the rat and bovine receptors
(Table I), indicating that mGIIA would be a second natural ligand for
the M-type receptor in the mouse.
Altogether, both mGIB and mGIIA appear as ligands of the mouse M-type
receptor endogenously expressed in NIH 3T3 cells (Fig. 2). To confirm
this view, we investigated the binding properties of these
sPLA2s to recombinant mouse M-type receptor expressed in
transfected COS cells. The recombinant receptor was found to have the
expected binding properties for venom sPLA2s,
i.e. a high affinity for OS1
(K0.5 = 0.3 nM) and no measurable
affinity for bvPLA2, suggesting that this receptor is
successfully expressed in COS cells (Fig.
3). Furthermore, labeled OS1
was found unable to bind to mock-transfected cells (not shown),
indicating that OS1 binds specifically to the mouse M-type
receptor. Fig. 3 shows that mGIB and mGIIA bind to the recombinant
mouse M-type receptor with affinities similar to those observed in
OS1 competition assays on NIH 3T3 membranes (Fig. 2),
clearly indicating that these two sPLA2s can bind to the
cloned mouse M-type receptor.
Since the M-type receptor was found to share similarities with the
macrophage mannose receptor that belongs to the C-type lectin
superfamily, and since the rabbit M-type receptor was previously found
to bind with nanomolar affinities various glycoconjugates of BSA (39)
(i.e. to display lectin-like properties), it was of interest
to analyze the binding properties of the different M-type receptors for
the various glycoconjugated derivatives of BSA. We observed that only
the rabbit receptor has a high affinity for the various BSA
glycoconjugates, whereas the other receptors displayed either a much
weaker affinity (human and mouse) or even no measurable affinity (rat),
suggesting that lectin properties of the M-type receptor (such as
binding of glycosylated BSA) have not been conserved between mammalian
species and therefore would not be physiologically relevant. This view
is in agreement with a previous observation that mannosylated BSA is
unable to bind to the bovine M-type receptor (42).
Tissue Distribution of the Mouse M-type Receptor, mGIB, and
mGIIA--
The above binding data indicate that both mGIB and mGIIA
bind to the mouse M-type receptor and therefore may be physiological ligands in the mouse. To strengthen this view, the tissue distribution of the M-type receptor was analyzed in BALB/c mice and then compared with that of mGIB and mGIIA, with the idea that a colocalization of the
receptor with the expression sites of one or both sPLA2s would add further evidence for a physiological significance of the
binding of mGIB or mGIIA to the mouse M-type receptor. The presence of
the M-type receptor in various BALB/c mouse tissues was determined with
the labeled ligand OS1, and the binding results are
presented in Fig. 4 and Table
II. The M-type receptor is expressed in
various tissues and corresponds to a single family of binding sites
with an equilibrium binding constant (Kd) close to
50 pM for labeled OS1 (Table II and Fig. 4).
Lung, colon, kidney, and salivary glands were found to contain the
highest amounts of receptor, while binding sites for
125I-OS1 were absent in brain membranes. The
maximal number of binding sites in the various tissues remains low as
compared with that observed in the mouse fibroblast cell line NIH 3T3
(Table II), which was used as a source of mouse M-type receptor for the
competition binding assays (Fig. 2). The tissue distribution of the
M-type receptor protein shown here using
125I-OS1 binding is in agreement with the
previous analysis of the M-type receptor transcript in mouse (41). Most
notably, the highest amounts of transcripts were also found in lung and
kidney, while lower levels were observed in heart and liver (41).
The tissue distribution of mGIB and mGIIA was carried out by
successively probing BALB/c mouse tissue Northern blots at high stringency with the two different cDNAs (Fig.
5). Transcripts of 0.8 kb coding for mGIB
were detected at very high levels in pancreas and at lower levels in
liver, lung, and spleen. No transcript was detected in other tissues
such as intestine, heart, brain, skeletal muscle, kidney, and testis.
This tissue distribution is in accordance with that previously
described in mice (41) and is similar to the distribution observed in
humans, with the exception of liver where no transcript was detected
(7, 30). Also in agreement with previous data (56, 57), we found that very high amounts of the mGIIA transcript are present in intestine but
not in other analyzed tissues with the exception of liver, where a very
weak expression is observed. Taken together, these data indicate that
different mouse tissues express both the mouse M-type receptor and mGIB
or mGIIA.
Both mGIB and mGIIA sPLA2s Bind to the 180-kDa M-type
Receptor Expressed in Mouse Colon--
To finally confirm that the
binding properties of mGIB and mGIIA observed on mouse M-type receptor
endogenously expressed in the fibroblast NIH 3T3 cell line (Fig. 2) or
transiently expressed in COS cells (Fig. 3) are similar in normal mouse
tissues, we performed competition binding experiments as well as
cross-linking experiments with mGIB and mGIIA on mouse colon membranes
(Fig. 6). The K0.5
values determined for mGIB (K0.5 = 1.3 nM) and mGIIA (K0.5 = 10 nM) appeared very similar to those measured on NIH 3T3
membranes and on the recombinant M-type receptor (Figs. 6A, 2, and 3, respectively). Cross-linking experiments with labeled OS1 on mouse colon membranes resulted in the labeling of a
single band of about 180 kDa, that fits well with the previously
determined molecular mass of the M-type receptor in various species
(33, 37, 38, 58). Furthermore, the labeling displayed the expected pharmacological profile (i.e. it was totally prevented by
unlabeled OS1, mGIB, and mGIIA but not by unlabeled
bvPLA2).
The 180-kDa M-type receptor was initially identified using snake
venom sPLA2s such as OS1 (37). A first clue to
the physiological function of the M-type receptor was provided when it
was observed that the mammalian pancreatic group IB sPLA2,
but not the inflammatory group IIA sPLA2, was a ligand of
this M-type receptor with a Kd value of 1 nM (8). However, other binding experiments to the rabbit
M-type receptor suggested that both group IB and group IIA
sPLA2s may be natural endogenous ligands of this receptor (39). On the other hand, the human M-type receptor was found to have
very weak affinities for both group IB and group IIA sPLA2s (40). The situation was thus clearly confusing, and the physiological relevance of these binding data was difficult to evaluate because sPLA2s and M-type receptors from different animal species
were used in many of these binding experiments (38, 39).
This paper now presents data showing that mGIB and mGIIA
sPLA2s are high affinity ligands of the mouse M-type
receptor (Fig. 2) and therefore would be natural candidates to act as
endogenous ligands of this receptor. This view is strengthened by the
colocalization in mice of the two sPLA2s and of the M-type
receptor. In particular, both mGIIA and the M-type receptor are
expressed in small intestine and colon (Table II, Fig. 5, and Ref. 56).
Although the affinity of mGIIA for the M-type receptor is not very high
(K0.5 close to 10 nM), this binding
is likely to occur in small intestine and colon, because huge amounts
of mGIIA transcript and protein are found in these tissues (49, 56,
57). The presence of mGIB and mGIIA sPLA2s in mouse serum
or platelets is not well documented. However, since both group IB and
group IIA sPLA2 activities were detected in serum from
other animal species (14), it is likely that these sPLA2s
are also present in mouse serum and then may reach M-type receptors
that are far away from cells producing sPLA2s.
The view that both group IB and group IIA sPLA2s would
operate as endogenous ligands of the M-type receptor in the mouse would not apply to humans, since both types of human sPLA2s were
found unable to bind to the human M-type receptor (Ref. 40 and Table I). Another situation is found in the rat, since the rat M-type receptor binds rGIB but not rGIIA (38). The rabbit M-type receptor is
unique in binding all the different mammalian group IB and group IIA
sPLA2s analyzed so far (Table I). It is also the only one
that has lectin-like binding properties (Table I). Altogether, the
available data indicate that group IB sPLA2 could behave as an endogenous ligand of M-type receptors at least in mouse, rat, and
porcine (33) species. On the other hand, the group IIA
sPLA2 appears to serve as an endogenous ligand of the
M-type receptor in mice but not in rats (38) and humans (40). It is
then possible that rGIIA and hGIIA have their own receptors, distinct
from the M-type receptor. Finally, whether the more recently
characterized group IIC, group V, and group X sPLA2s are
endogenous ligands of M-type receptors remains to be determined.
The CRD5 domain of the M-type receptor is centrally involved in
sPLA2 binding (46). It is therefore likely that the
different binding properties of the M-type receptor in various animal
species have to do with differences between these receptors in the CRD5 domain. Interestingly, we previously observed that, of the eight CRDs
of the M-type receptor, the CRD5 domain is the least conserved among
rabbit, mouse, bovine, and human receptors (46). On the other hand, we
have also previously demonstrated that residues close to or within the
Ca2+-binding loop domain of sPLA2 are involved
in binding to the M-type receptor (45). We particularly suggested that,
besides glycine 30 and aspartate 49, which are perfectly conserved in
sPLA2s and which are essential for binding, the identity of
the residues at position 31 and possibly 34, which greatly varies in
sPLA2s (59), may determine whether binding to the M-type
receptor is possible or not. It is noteworthy that this view fits well
with the fact that mGIIA binds with high affinity to the mouse M-type receptor, while rGIIA and hGIIA do not bind to this receptor (Table I
and Ref. 38). Indeed, the Ca2+-binding loop domain of these
three sPLA2s is perfectly conserved except at positions 31 and 34, where rGIIA and hGIIA, but not mGIIA, have the same residues
(3, 48, 57). This observation, however, does not eliminate the
possibility that residues located elsewhere in the sPLA2
structure also contribute to the large differences in binding properties.
The physiological reason why mGIIA binds to the mouse M-type receptor
while rGIIA and hGIIA do not bind to the respective rat and human
M-type receptors is hard to understand. A tempting hypothesis would be
that mGIIA is not the ortholog of rGIIA and hGIIA, i.e. that
mGIIA has physiological functions that are distinct from those of rGIIA
and hGIIA. In addition to differences in binding properties, several
other lines of evidence would support this hypothesis. First, mGIIA,
rGIIA, and hGIIA display relatively low levels of sequence identity
compared with those observed between group IB sPLA2s. mGIIA
has only 76 and 67% of identity with rGIIA and hGIIA, while mGIB has
89 and 81% of identity with rGIB and hGIB, respectively. Second, both
hGIIA and rGIIA are expressed in many different tissues and cells (3,
30), while the tissue distribution of mGIIA is thus far essentially
restricted to intestine (where it is expressed at a very high level),
with a low expression level in liver (Fig. 5) and the skin of new born
mice (56). Furthermore, in hGIIA transgenic mice established with the
complete hGIIA gene, the tissue distribution of hGIIA resembles that of hGIIA in humans, where it is expressed in many organs (60), in marked
contrast with the endogenous expression of mGIIA (Fig. 5). In these
transgenic mice, hGIIA was not expressed in the intestinal Paneth cells
(60), whereas Paneth cells in wild-type mice are known to contain huge
amounts of mGIIA (56, 61, 62). Since the transgenic mice were
established with a transgene comprising a reasonably large 5' noncoding
sequence of 1.6 kilobase pairs that contains transcriptional regulatory
elements of the hGIIA gene (63, 64), and since one would expect a
conservation of the transcriptional regulatory elements between mGIIA
and hGIIA genes if these latter were true orthologs, the difference
observed between the endogenous expression of mGIIA and that of hGIIA
in transgenic mice suggests that the transcriptional regulatory
elements of the mGIIA and hGIIA genes are distinct and therefore
supports the idea that mGIIA and hGIIA are not true orthologs. The
third indication that mGIIA may not be the ortholog of rGIIA and hGIIA comes from physiological considerations. While there are many studies
showing that expression of rGIIA and hGIIA is dramatically increased by
proinflammatory cytokines, in many human inflammatory diseases, and in
various rat models of inflammatory diseases (3), there is no clear
evidence for an increased expression of mGIIA in inflammatory
conditions in mouse cells or tissues. Thus far, only two studies have
shown that the expression of mGIIA could be increased in intestine
after injection of lipopolysaccharide (LPS) (65, 66), but this increase
was only modest as compared with the induction observed in LPS-treated
rats (3, 67). Finally, while many studies have shown that rGIIA and
hGIIA play a role in lipid mediator release (3, 68), mGIIA-deficient mice were found to have a normal inflammatory response that is mediated
by mouse group V sPLA2 (22, 23); and while mGIIA has been
proposed as a genetic modifier of colon tumorigenesis (25, 26), the
numerous attempts to demonstrate a similar role for hGIIA in human
colorectal cancer were unsuccessful (69-75).
The M-type receptor has been associated with a myriad of biological
roles such as cell proliferation, cell contraction, cell migration,
hormone release, and lipid mediator release (38, 76). These effects are
currently believed to be mediated by group IB sPLA2s but
not by group IIA sPLA2s. However, since group IIA
sPLA2s and cells from different animal species have been
used in these previous studies, and since we now know that animal
specificity is important for the M-type receptor interaction, some of
these previous data may require reevaluation. For example, the
mitogenic effect of pGIB on mouse fibroblasts was believed to indicate
a specific action of group IB sPLA2s through binding to the
mouse M-type receptor, since rGIIA and rbGIIA were found unable to bind to this receptor (8). Since mGIIA now appears as a ligand of the mouse
M-type receptor, this suggests that group IIA sPLA2s may
also have mitogenic effects on these mouse fibroblasts that may be
linked to the mouse M-type receptor. The recent targeted disruption of
the M-type receptor gene in the mouse suggests that the M-type receptor
plays a critical role in inflammatory processes induced by LPS and
leading to endotoxic shock (13). M-type receptor-deficient mice have a
longer survival time than wild-type mice after challenge with LPS and
are also resistant to the lethal effects of pGIB after sensitization
with sublethal dose of LPS. The results of the present study indicating
that mGIB is a natural ligand of the mouse M-type receptor fit well
with the view that group IB sPLA2 would play a role in
processes leading to endotoxic shock after LPS challenge through
binding to the mouse M-type receptor (13). mGIIA, which now appears as
a second natural ligand of the mouse M-type receptor, is certainly not
implicated in this resistance to endotoxic shock, since M-type receptor
knock-out mice are also naturally deficient for mGIIA (13, 25, 65). Since several other mouse sPLA2s have now been identified
(among them the mouse group V that has been shown to play a role in the release of lipid mediators of inflammation in place of mGIIA (22, 23)),
it will be important to analyze whether they can also act as natural
ligands of the M-type receptor and whether they are involved in the
inflammatory processes leading to endotoxic shock.
Besides a possible role of mGIIA in the production of inflammatory
lipid mediators (20), mGIIA has been proposed to have bactericidal
properties to protect the small intestine crypts from microbial
invasion (62), and mice lacking mGIIA show an altered response to
microbial infection (77). Whether the M-type receptor may contribute to
these effects remains to be analyzed. mGIIA was originally discovered
as an intestinal protein called enhancing factor that increases the
binding of the epidermal growth factor and synergizes with this latter
to stimulate cell proliferation (24). The potential role of the M-type
receptor in these effects would be worth analyzing. More recently,
mGIIA was identified as a genetic modifier of tumor formation in a
mouse model of colorectal cancer (25, 26). Mice carrying a deficient
mGIIA gene were found to develop more intestinal adenomas than mice
expressing a functional mGIIA (25). In addition, transgenic mice
overexpressing mGIIA become resistant to intestinal tumorigenesis (26).
While the mechanism by which mGIIA confers protection against adenoma formation is presently unknown (26), the colocalization of the M-type
receptor with mGIIA in colon may suggest an implication of the M-type
receptor in the resistance to intestinal tumorigenesis conferred by mGIIA.
In conclusion, this work has shown that both mGIB and mGIIA are
probably physiological ligands of the mouse M-type receptor. This
observation may be important to the understanding of the physiological
and pathological roles of these sPLA2s in various processes
such as cell proliferation, inflammation, and cancer. Furthermore,
comparison of the binding properties of mouse and human
sPLA2s to receptors from different animal species has
indicated that sPLA2 binding to M-type receptors is
species-specific. Interestingly, species specificity of binding was
previously observed for cytokines such as tumor necrosis factor and
interleukin-1 (78-80). In examining the biological effects of
sPLA2s in further studies, it will be essential to use as
much as possible sPLA2s extracted from the same animal as
that used for in vitro or in vivo physiological studies. This work also suggests that mGIIA and hGIIA may not have
similar functions and that new sPLA2s corresponding to the true orthologs of mGIIa and hGIIA may exist in mice and humans.
INTRODUCTION
Top
Abstract
Introduction
References
receptor (43, 44). The protein domains involved in the
sPLA2-receptor binding have been elucidated (38, 45, 46).
Knock-out mice for the M-type receptor have now been generated (13).
The real endogenous ligands of the M-type receptor, however, remain
elusive. Available data on the binding properties of mammalian
sPLA2s to the M-type receptor appear contradictory, depending on the animal origin of both the sPLA2 and the
M-type receptor. For instance, rat and bovine M-type receptors can bind both porcine (pGIB) or rat (rGIB) group IB sPLA2s with a
high affinity (Kd values of ~1 nM) but
do not associate with rat (rGIIA) or rabbit (rbGIIA) group IIA
sPLA2s, suggesting that group IB sPLA2s, but
not group IIA sPLA2s, are the physiological ligands of the
M-type receptor (38). On the contrary, the rabbit M-type receptor
associates with high affinity with both pGIB and human group IIA
sPLA2 (hGIIA) with Kd values of 1-10 nM, suggesting that both types of sPLA2s may be
the natural ligands of the M-type receptor (39). Finally, the cloned
human M-type receptor binds with very weak affinities group IB and
group IIA sPLA2s, suggesting that neither of these two
sPLA2s are physiological ligands of this receptor (40).
EXPERIMENTAL PROCEDURES
-actin probe (not shown).
RESULTS
of the porcine pancreatic
sPLA2 (theoretical molecular mass of 13,980 Da; Refs. 7 and
54). Recombinant mGIB was prepared using the baculovirus expression
system (see "Experimental Procedures" for details) after cloning of
its cDNA (GenBankTM accession no. AF097637) by
screening public data bases with sPLA2 sequences (30). The
full-length cDNA was found to code for a protein of 146 residues
containing a signal peptide sequence of 15 residues and a propeptide of
seven residues ending with a single arginine, followed by a mature
protein of 124 residues. The mature protein contains seven disulfide
bridges located at positions that are typical of group IB
sPLA2s and a pancreatic loop of six residues and displays
89 and 81% identity with rGIB and hGIB, respectively (7, 55). Taken
together, these properties indicate that the cloned cDNA codes for
mGIB. Although SDS-polyacrylamide gel electrophoresis analysis
indicates that the electrophoretic mobility of the recombinant mGIB
protein is slightly faster than that of hGIB and pGIB (Fig. 1), the
electrospray molecular mass (14,075 Da), the N-terminal sequence, and
the amino acid composition of the recombinant mGIB protein (not shown)
were found to be identical to those predicted from the cDNA
sequence, indicating the successful recombinant expression of mGIB.
hGIIA was obtained from mammalian fibroblasts (47) and from insect
cells by using the baculovirus expression system. The two products were
identical as checked by gel analysis (Fig. 1), mass spectrometry
(13,904 Da), amino acid composition, and N-terminal sequence (not
shown). Furthermore, their molecular masses are identical to the
theoretical value calculated from the cDNA sequence, indicating
that the recombinant sPLA2 proteins are full-length
proteins and are not modified after translation, despite the presence
of a potential site of N-glycosylation in the protein
sequence (30). The mGIIA purified from BALB/c mouse intestine tissue
and the corresponding recombinant proteins produced in mammalian and
insect cells have a similar electrophoretic mobility (Fig. 1) and also
have the same molecular mass (13,958 Da), N-terminal sequence, and
amino acid composition (not shown) as those predicted from the cDNA
sequence, indicating the absence of any post-translational modification
such as glycosylation in both native and recombinant mGIIA
sPLA2s.
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Fig. 1.
SDS-polyacrylamide gel electrophoresis
analysis of the various mammalian sPLA2 preparations.
50 ng of purified sPLA2s were loaded on a 14%
SDS-polyacrylamide gel under reducing conditions. The gel was
silver-stained. Molecular mass markers (phosphorylase B, 97,400 Da;
BSA, 66,200 Da; ovalbumin, 45,000 Da; carbonic anhydrase, 31,000 Da;
soybean trypsin inhibitor, 21,500 Da; and cytochrome c,
14,400 Da) were from Bio-Rad.
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Fig. 2.
Binding properties of sPLA2s to
the mouse M-type receptor from NIH 3T3 cells. Competition
experiments between 125I-OS1 and unlabeled
venom sPLA2s (A), group IB sPLA2
from pigs, humans, and mice (B), and group IIA
sPLA2 from mice and humans (C) on mouse NIH 3T3
membranes. Membranes (50 µg of protein/ml) were incubated in the
presence of 125I-OS1 (50 pM) and
various concentrations of unlabeled sPLA2s. All results are
expressed as percentages of the specific binding measured in the
absence of unlabeled sPLA2s. 100% corresponds to a
125I-OS1 specific binding of 2.7 pM. The nonspecific binding was determined in the presence
of 30 nM unlabeled OS1 and was below 20% of
the total binding.
Binding properties of the M-type receptor from different animal species
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Fig. 3.
Binding properties of sPLA2s to
the recombinant mouse M-type receptor expressed in COS cells.
Membranes (10 µg of protein/ml) of COS cells transfected with the
full-length mouse M-type receptor were incubated in the presence of
125I-OS1 (25 pM) and various
concentrations of OS1 or recombinant mGIB and mGIIA from
Sf9 baculovirus-infected cells. All results are expressed as
percentages of the specific binding measured in the absence of
unlabeled sPLA2s. 100% corresponds to a
125I-OS1 specific binding of 1.6 pM. The nonspecific binding was determined in the presence
of 30 nM unlabeled OS1 and was below 6% of the
total binding.
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Fig. 4.
Equilibrium binding of
125I-OS1 to mouse colon membranes.
A, membranes (140 µg/ml) were incubated with increasing
concentrations of 125I-OS1 in the absence ( )
or presence (
) of 30 nM unlabeled OS1.
Specific binding (
) represents the difference between total binding
(
) and nonspecific binding (
). B, Scatchard plot
analysis of the specific binding (
).
Tissue distribution of the mouse M-type receptor
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Fig. 5.
High stringency Northern blot analysis of
mGIB and mGIIA expression in various BALB/c mouse tissues.
Northern blots containing 2 µg of poly(A+) mRNA/lane
(A) or 25 µg of total RNA/lane (B) from various
adult mouse tissues were successively hybridized at high stringency
with 32P-labeled probes for mGIIA sPLA2 and
mGIB sPLA2 as described under "Experimental
Procedures." sk. muscle, skeletal muscle. Filters were
exposed for 3 days using Biomax MS Kodak films with an HE intensifying
screen.
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Fig. 6.
Competition binding experiments between
125I-OS1 and different unlabeled
sPLA2s for binding to mouse colon membranes.
A, membranes (300 µg of protein/ml) were incubated in the
presence of 125I-OS1 (30 pM) and
various concentrations of unlabeled sPLA2s. All results are
expressed as percentages of the specific binding measured in the
absence of unlabeled sPLA2s. 100% corresponds to
125I-OS1 specific binding of 2.5 pM. The nonspecific binding was determined in the presence
of 30 nM unlabeled OS1 and was below 15% of
total binding. B, cross-linking experiments of
125I-OS1 to the mouse M-type receptor. Mouse
colon membranes (300 µg of protein/ml) were incubated with 150 pM 125I-OS1 in the absence or
presence of 300 nM of various unlabeled sPLA2s
and then cross-linked with 50 µM suberic acid
bis-N-hydroxysuccinimide ester. 100 µg of proteins were
loaded under reducing conditions on a 7% SDS-polyacrylamide gel. Only
one band was labeled with an apparent Mr of
180,000 after correction for the OS1 molecular mass
assuming that one 125I-OS1 molecule is bound
per molecule of receptor. Molecular mass markers (myosin, 200,000 Da;
-galactosidase, 116,200 Da; phosphorylase B, 97,400 Da; BSA, 66,200 Da; ovalbumin, 45,000 Da; carbonic anhydrase, 31,000 Da; soybean
trypsin inhibitor, 21,500 Da; cytochrome c, 14,400 Da) were
from Bio-Rad. The gel was exposed on Kodak X-Omat AR films for 17 days.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dr. Hubertus Verheij (Department of Enzymology and Protein Engineering, 3508 TB, Utrecht, The Netherlands) and Dr. Ruth Kramer (Lilly) for the generous gift of hGIB and hGIIA sPLA2s, respectively. We are grateful to Dr. Danielle Moinier, Dr. Mostafa Kouach, and Dr. Nicole Zylber for excellent work in the determination of N-terminal sequences, electrospray molecular masses, and amino acid compositions of sPLA2s, respectively. The photographic work of Franck Aguila and the skillful secretarial assistance of Valérie Briet are greatly appreciated and acknowledged.
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FOOTNOTES |
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* This work was supported by CNRS, the Association pour la Recherche sur le Cancer (ARC), and Ministère de la Défense Nationale Grant DGA-DRET 96/096.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) AF097637.
¶ To whom correspondence should be addressed. Tel.: 33 4 93 95 77 02 or 33 4 93 95 77 03; Fax: 33 4 93 95 77 04; E-mail: ipmc{at}ipmc.cnrs.fr.
2 A comprehensive abbreviation system for the various mammalian sPLA2s is used. Each sPLA2 is abbreviated with a first lowercase letter indicating the sPLA2 species (b, h, m, p, r, and rb representing bovine, human, mouse, porcine, rat, and rabbit species, respectively) that is followed by capital letters identifying the sPLA2 group (GIB, GIIA, GIIC, GV, and GX representing group IB, IIA, IIC, V, and X sPLA2, respectively).
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ABBREVIATIONS |
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The abbreviations used are: sPLA2, secreted phospholipase A2; OS1, O. scutellatus sPLA2-1; OS2, O. scutellatus sPLA2-2; bvPLA2, bee venom sPLA2; BSA, bovine serum albumin; CRD, carbohydrate recognition domain; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide.
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REFERENCES |
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