Both Group IB and Group IIA Secreted Phospholipases A2 Are Natural Ligands of the Mouse 180-kDa M-type Receptor*

Lionel CupillardDagger , Rita Mulherkar§, Nathalie GomezDagger , Shilpa Kadam§, Emmanuel ValentinDagger , Michel LazdunskiDagger , and Gérard LambeauDagger

From the Dagger  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

    ABSTRACT
Top
Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

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 lambda  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).

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.

    EXPERIMENTAL PROCEDURES

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 beta -actin probe (not shown).

    RESULTS

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 alpha  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.

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).


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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.

                              
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Table I
Binding properties of the M-type receptor from different animal species
Membranes containing the various M-type receptors were prepared as described under "Experimental Procedures" and incubated with 125I-OS1 in the presence of various concentrations of unlabeled sPLA2s or BSA glycoconjugates under equilibrium binding conditions such as those shown in Fig. 1. K0.5 values were determined from competition curves as the concentrations that inhibit half of the 125I-OS1 specific binding. BSA glycoconjugates were from Sigma.

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.


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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.

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).


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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 (open circle ) or presence () of 30 nM unlabeled OS1. Specific binding () represents the difference between total binding (open circle ) and nonspecific binding (). B, Scatchard plot analysis of the specific binding ().

                              
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Table II
Tissue distribution of the mouse M-type receptor
The different membrane preparations were incubated with increasing concentrations of 125I-OS1 as indicated in Fig. 1. Data are mean values from at least two independent sets of experiments on different membrane preparations.

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.


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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.

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).


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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; beta -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

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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).

    ABBREVIATIONS

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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