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Originally published In Press as doi:10.1074/jbc.M108752200 on February 5, 2002

J. Biol. Chem., Vol. 277, Issue 16, 13583-13588, April 19, 2002
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Identification of a Soluble Form Phospholipase A2 Receptor as a Circulating Endogenous Inhibitor for Secretory Phospholipase A2*

Ken-ichi Higashino, Yasunori Yokota, Takashi Ono, Shigeki Kamitani, Hitoshi Arita, and Kohji HanasakiDagger

From the Shionogi Research Laboratories, Shionogi & Co., Ltd. 12-4 Sagisu, 5-Chome, Fukushima-ku, Osaka 553-0002, Japan

Received for publication, September 11, 2001, and in revised form, January 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Venomous snakes have various types of phospholipase A2 inhibitory proteins (PLIs) in their circulatory system to protect them from attack by their own phospholipase A2s (PLA2s). Here we show the first evidence for the existence of circulating PLI against secretory PLA2s (sPLA2s) in mammals. In mouse serum, we detected specific binding activities of group IB and X sPLA2s, which was in contrast with the absence of binding activities in serum prepared from mice deficient in PLA2 receptor (PLA2R), a type I transmembrane glycoprotein related to the C-type animal lectin family. Western blot analysis after partial purification with group IB sPLA2 affinity column confirmed the identity of serum sPLA2-binding protein as a soluble form of PLA2R (sPLA2R) that retained all of the extracellular domains of the membrane-bound receptor. Both purified sPLA2R and the recombinant soluble receptor having all of the extracellular portions blocked the biological functions of group X sPLA2, including its potent enzymatic activity and its binding to the membrane-bound receptor. Protease inhibitor tests with PLA2R-overexpressing Chinese hamster ovary cells suggested that sPLA2R is produced by cleavage of the membrane-bound receptor by metalloproteinases. Thus, sPLA2R is the first example of circulating PLI that acts as an endogenous inhibitor for enzymatic activities and receptor-mediated functions of sPLA2s in mice.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phospholipase A2 (PLA2)1 is an enzyme that catalyzes the hydrolysis of the sn-2 ester bond of glycerophospholipids (1, 2). Since its discovery, PLA2 has been a molecular target of extensive research because of its critical involvement in physiological and pathological events such as phospholipid turnover and production of proinflammatory lipid mediators (3). To date, a number of mammalian intracellular and extracellular PLA2s have been identified and classified into different families according to their biochemical features (4). Among them, sPLA2s have several common characteristics including a relatively low molecular mass (13-18 kDa), the presence of 6-8 disulfide bridges, and an absolute catalytic requirement for millimolar concentrations of Ca2+ (5, 6). At present, mammalian sPLA2s are classified into 10 different groups (IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII) depending on the primary structure characterized by the number and positions of cysteine residues (5, 7-12). Given their potent enzymatic activities and enhanced expression in various inflammatory conditions, sPLA2s are thought to play crucial roles in the development of various disease states. For example, high levels of group IIA sPLA2 (sPLA2-IIA) were detected in the plasma of patients with sepsis, and sPLA2-IIA plays a critical role in the hydrolysis of lung surfactant phospholipids during the progression of acute lung injury (13, 14). We and other groups have shown that group X sPLA2 (sPLA2-X) possesses stronger hydrolyzing activity toward phosphatidylcholine than sPLA2-IIA and elicits marked release of arachidonic acid linked to the production of various lipid mediators in macrophages and colon cancer cells (15, 16).

In addition to its digestive function, sPLA2 can exert various biological responses via its binding to the PLA2 receptor (PLA2R) (17). PLA2R is a type I transmembrane protein with a molecular mass of 180 kDa. Its overall molecular organization is related to a unique member of the C-type animal lectin family (subgroup VI), which includes the macrophage mannose receptor (18). PLA2R is composed of a large extracellular portion consisting of an N-terminal cystein-rich region, a fibronectin-like type II domain, a tandem repeat of eight carbohydrate-recognition domains (CRDs), and a short intracellular C-terminal region (19). In mice, group IB sPLA2 (sPLA2-IB) was identified as the first endogenous ligand of PLA2R (20). Recently, sPLA2-X was also identified as a high affinity ligand of PLA2R (21). Although sPLA2-IIA can bind PLA2R with about 10-fold lower affinity compared with sPLA2-IB and -X, some inbred mouse strains possess a natural mutation in the sPLA2-IIA gene (5, 6). We have shown that sPLA2-IB exerts various biological responses via binding to PLA2R, including cell proliferation, lipid mediator productions, and chemokinetic migration in various cell types (22-24). Furthermore, our recent analysis of PLA2R-deficient mice has revealed that PLA2R may play a role in the production of proinflammatory cytokines during the progression of endotoxic shock (25).

Venomous snakes have PLA2 inhibitory proteins (PLIs) in their blood sera to protect them from leakage of their own venom PLA2s into their circulatory system (26). Mammalian sPLA2s exhibit a wide variety of pathological functions via enzymatic activities or receptor-mediated responses; however, the existence of PLI molecules in mammalian circulation has not yet been demonstrated. In our recent analysis by sandwich enzyme-linked immunosorbent assay (ELISA), we found the presence of a soluble form of PLA2R (sPLA2R) in mouse plasma, although its biochemical features and biological significance has not been clarified yet (27). In the present study, we definitely identified the sPLA2-binding protein in mouse sera as sPLA2R, which retains all of the extracellular domains of the cell-associated receptor. We found that circulating sPLA2R can block both the enzymatic activity and receptor binding activity of sPLA2-X. Thus, we report here that sPLA2R is the first example of circulating PLI in mammals.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Sodium [125I] iodine (carrier-free, 3.7 GBq/ml) was purchased from Amersham Biosciences. Porcine pancreatic sPLA2-IB was obtained from Roche Molecular Biochemicals. Recombinant mouse sPLA2-X, recombinant soluble PLA2R (rsPLA2R), and biotin-labeled rabbit anti-sPLA2R antibody were prepared as described in the previous paper (27, 28). Lipopolysaccharide (Salmonella typhosa 0901) was purchased from Difco Laboratories. Bovine serum albumin (BSA) and bovine gamma -globulins were purchased from Sigma. 1-Palmitonyl-2-palmitonyl-sn-glycero-3-phosphocholine (DPPC) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Polyethylene glycol 6000 was purchased from Nacalai Tesque. Protease inhibitors were obtained from the Peptide Institute, Inc. and Wako Pure Chemical Industries. BB2516 was synthesized at Shionogi Research Laboratories. The generation of PLA2R-deficient mice was described in our previous paper (25), and they were backcrossed more than 11 times. In each experiment, C57BL/6J mice matched for gender and age were used as wild-type littermates.

Binding of sPLA2-IB and sPLA2-X to Mouse Serum-- Iodination of porcine sPLA2-IB or mouse sPLA2-X was performed by the chloramine-T method (29), and the specific radioactivities of 125I-sPLA2-IB and 125I-sPLA2-X were about 7000 and 4600 cpm/fmol, respectively. The serum prepared from wild-type or PLA2R-deficient mice (100 µl) was incubated with 3 nM 125I-sPLA2-IB or 125I-sPLA2-X in the binding buffer (20 mM Tris-HCl, pH 7.4, containing 2 mM EDTA and 0.1% BSA). After incubation for 1 h at 4 °C, 600 µg of bovine gamma -globulins and 20% polyethylene glycol 6000 were added. After further incubation for 30 min at 4 °C, the reaction mixture was filtered through a Whatman GF/B glass filter, and the radioactivity of the filter was measured. Specific binding was determined as the difference between the presence and absence of unlabeled 500 nM porcine sPLA2-IB.

Western Blot Analysis of Serum sPLA2R-- Serum sPLA2R was partially purified from wild-type or PLA2R-deficient mice with a sPLA2-IB affinity column as described previously (30) and then concentrated with Ultrafree (Millipore). Chinese hamster ovary (CHO) cells that stably express the membrane-bound mouse PLA2R (PLA2R-CHO cells) and their crude membrane lysates were prepared as described previously (28, 29). The serum sPLA2R materials, the membrane fractions of PLA2R-CHO or parent CHO cells, and purified rsPLA2R protein were analyzed by SDS-PAGE using a 4-20% gradient gel (Daiichi Chemical Co., Ltd.). Western blot analysis was performed according to our previous paper (27) with biotin-labeled rabbit anti-sPLA2R antibody (1 µg/ml) and streptoavidin-conjugated horseradish peroxidase (Roche Molecular Biochemicals). The blot was incubated with a chemiluminescent detection reagent (ECL Western blotting detection reagents; Amersham Biosciences) according to the manufacturer's instructions and analyzed using a Fluor-S MAX MultiImager (Bio-Rad).

Inhibition of sPLA2-X Enzymatic Activity by sPLA2R-- Mouse sPLA2-X (0.14 nM) was preincubated with various concentrations of rsPLA2R in the assay buffer (0.1 M Tris-HCl, pH 8.0, containing 0.15 M NaCl, 0.01 M CaCl2, and 1 mg/ml BSA) for 1 h at 37 °C. In separate experiments, mouse sPLA2-X was preincubated in the absence or presence of serum sPLA2R prepared from wild-type or PLA2R-deficient mice. The enzymatic activity was evaluated using 0.1 mM DPPC as a substrate. After incubation for 1 h at 37 °C, the reaction was stopped by the addition of Dole's reagent (heptane, 2-propanal, 2 N sulfuric acid = 10:40:1, v/v/v), and the released fatty acids were quantified by reverse-phase high performance liquid chromatography on a LiChroCART 75-4 Superspher 60 RP-8 column (Merck), as described by Tojo et al. (31).

Effect of rsPLA2R on sPLA2-X-induced Fatty Acid Release in Mouse Splenic Cells-- Splenic cells were prepared from male C57BL/6J mice (10 weeks) according to the method of Funk et al. (32). The prepared cells were washed with Hanks' buffered saline (pH 7.6) containing 0.1% BSA and suspended in the same buffer at a density of 9.5 × 106 cells/ml. Aliquots of cell suspension (0.4 ml) were preincubated for 10 min at 37 °C and then stimulated with 2 nM mouse sPLA2-X with or without various concentrations of rsPLA2R in a final volume of 0.5 ml. The reaction was stopped by the addition of 2 ml of Dole's reagent. The released fatty acids were extracted and quantified as described previously (31).

Inhibition of sPLA2 Binding to PLA2R by sPLA2R-- PLA2R-CHO cells were cultured in 24-well plates in 10% fetal calf serum/Dulbecco's modified Eagle's medium. At confluence, the cells were washed three times with phosphate-buffered saline and incubated with 1 nM 125I-sPLA2-X in the absence or presence of various concentrations of rsPLA2R in 0.4 ml of the binding buffer (Hanks' balanced salt solution, pH 7.4, containing 0.1% BSA) for 2 h at 4 °C. After the incubation, the cells were washed with the binding buffer three times, and the cell-bound radioactivity was measured. In separate experiments, the membrane fractions of PLA2R-CHO cells were incubated with 0.2 nM 125I-sPLA2-X in the absence or presence of serum sPLA2R prepared from wild-type or PLA2R-deficient mice or 12.5 nM rsPLA2R. After incubation for 2 h at room temperature, the reaction mixture was filtered through a Whatman GF/C glass filter. The specific binding was determined as the difference between the presence and absence of unlabeled 500 nM mouse sPLA2-X.

Effects of Protease Inhibitors on the Release of sPLA2R from PLA2R-CHO Cells-- After cultivation in 96-well plates, PLA2R-CHO cells were washed three times with PBS and then incubated with or without protease inhibitors at 37 °C for various times. After the incubation, the supernatant was collected by centrifugation at 8,000 rpm for 10 min at 4 °C, and the released sPLA2R was measured with established sandwich ELISA (27). In this assay, purified rsPLA2R was used as a conventional standard because of its molecular mass being similar to that of serum sPLA2R (the detection range from 0.1 to 100 ng). In separate experiments, the membranes of PLA2R-CHO cells were incubated in the absence or presence of protease inhibitors for 1 h at 37 °C. After centrifugation at 40,000 rpm for 1 h at 4 °C, the released sPLA2R in the supernatant was measured with sandwich ELISA.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of sPLA2s to Mouse Serum-- To explore the existence of circulating PLI in mammals, we first examined the binding potency of endogenous sPLA2s to mouse serum. We used two types of 125I-labeled sPLA2s (sPLA2-IB and sPLA2-X) and evaluated their bindings to mouse serum proteins using polyethylene glycol precipitation assay. As shown in Fig. 1, specific binding activities were detected with both ligands (10.5 and 7.3 fmol/ml of serum, respectively). The specific binding linearly increased depending on the serum volume added up to 100 µl (data not shown). We have previously shown that sPLA2-IB and sPLA2-X can specifically bind to the PLA2R expressed in alveolar type II epithelial cells and splenic lymphocytes in mice (21). In addition, our recent analysis with sandwich ELISA suggested the presence of sPLA2R in mouse plasma (27). To examine the relationships between serum sPLA2-binding proteins and putative sPLA2R, we next examined the binding potencies of 125I-labeled sPLA2s to serum prepared from PLA2R-deficient mice, in which there was no positive signal for sPLA2R in sandwich ELISA (27). As shown in Fig. 1, there were few, if any, specific binding activities of both sPLA2 ligands in the serum prepared from knockout mice. These findings suggest that serum sPLA2R protein(s) are representative of the specific binding activities of sPLA2 ligands.


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  		Fig. 1.   Binding of sPLA2-IB and sPLA2-X to mouse serum. The binding of 125I-sPLA2-IB or 125I-sPLA2-X was evaluated with serum prepared from wild-type (WT) and PLA2R-deficient mice (KO). The specific binding activity was determined as the difference between the presence and absence of 500 nM porcine sPLA2-IB. Each point represents the mean ± S.E. of triplicate measurements. The data are representative of three experiments.

Identification of Serum sPLA2R Protein-- We next performed the partial purification of serum-binding proteins with an sPLA2-IB affinity column. The specific binding activities of 125I-labeled sPLA2s were detected in the acid-eluted materials in contrast to their absence from the pass-through fractions. Conversely, there was little protein in the eluted fractions of serum prepared from PLA2R-deficient mice (data not shown), demonstrating the binding proteins to be serum sPLA2R(s). To clarify their identities, we performed Western blot analysis with anti-PLA2R antibody, which can specifically detect the membrane-bound PLA2R protein (~200 kDa) in the membrane fractions of PLA2R-CHO cells compared with those of the parent CHO cells (Fig. 2, lanes 1 and 2). rsPLA2R composed of all of the extracellular domains of the membrane-bound receptor (1-1365 amino acids) showed a slightly smaller molecular mass (~180 kDa in lane 3) than that of the membrane-bound form. Serum sPLA2R was detected as a single band at the same position with rsPLA2R (lane 4), which contrasted with no visible bands in the eluted fractions prepared from PLA2R-deficient mouse serum (lane 5). These results demonstrate that serum sPLA2R contains all of the extracellular domains of PLA2R with high binding activity to sPLA2-IB and sPLA2-X.


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  		Fig. 2.   Characterization of serum sPLA2R. Serum sPLA2R was partially purified with an sPLA2-IB affinity column, and Western blot was performed using rabbit anti-PLA2R antibody, as described under "Experimental Procedures." Lane 1, membrane fractions of CHO cells; lane 2, membrane fractions of PLA2R-CHO cells; lane 3, rsPLA2R protein; lane 4, sPLA2R materials purified from wild-type mouse serum; lane 5, sPLA2R fractions prepared from PLA2R-deficient mouse serum. Molecular mass markers are indicated on the left side of the figure.

Functions of Serum sPLA2R-- There are two biological responses elicited by sPLA2s; one is dependent on the phospholipid-hydrolyzing activity, and the other is mediated via the PLA2R binding (17). Because sPLA2-X was proven to elicit both responses in mice (28), we next examined the effects of serum sPLA2R on sPLA2-X-induced responses. First, we examined the effects of rsPLA2R and serum sPLA2R on sPLA2-X enzymatic activity toward DPPC as a substrate. In this in vitro assay system, the enzyme activity of about 0.1 nM mouse sPLA2-X can be detected, and the purified rsPLA2R markedly blocked the enzymatic activity with an IC50 value of 0.04 ± 0.01 nM (Fig. 3A). The acid-eluted materials prepared with sPLA2-IB affinity column (Fig. 2) were found to contain the sPLA2R at the concentration of 0.37 nM by sandwich ELISA using rsPLA2R as a conventional standard. As shown in Fig. 3B, the serum sPLA2R fractions effectively blocked the enzymatic activity of sPLA2-X up to 70%, which contrasted with no significant suppression with sPLA2R fractions prepared from PLA2R-deficient mouse serum. We then examined the effect on enzymatic activity of sPLA2-X toward intact cell membranes. We have previously reported that mouse sPLA2-X induced prompt and marked release of arachidonic acid from mouse spleen cells (28). However, a relatively higher concentration of sPLA2-X (over 0.5 nM) was required for detection of sufficient responses in intact cell systems. As shown in Fig. 3C, rsPLA2R dose-dependently blocked the arachidonic acid release evoked by mouse sPLA2-X (2 nM). However, significant suppression could not be observed using the serum sPLA2R fractions, possibly because of its lower quantities of sPLA2R protein (data not shown).


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  		Fig. 3.   Inhibitory potency of sPLA2R for sPLA2-X enzymatic activity. A, inhibitory potency of rsPLA2R for sPLA2-X enzymatic activity. Mouse sPLA2-X (0.14 nM) was incubated with DPPC in the absence or presence of various concentrations of rsPLA2R. B, inhibitory potency of circulating sPLA2R for sPLA2-X enzymatic activity. Mouse sPLA2-X (0.14 nM) was incubated with DPPC in the absence (none) or presence of serum sPLA2R fractions prepared from wild-type (WT) or PLA2R-deficient (KO) mice or 1 nM rsPLA2R. C, inhibitory potency of rsPLA2R on sPLA2-X-induced arachidonic acid release from spleen cells. The spleen cells prepared from male C57BL/6J mice were stimulated with mouse sPLA2-X (2 nM) with or without various concentrations of rsPLA2R, and the released arachidonic acid was quantified as described under "Experimental Procedures." The results are expressed as the percentages of sPLA2-X enzymatic activity in the absence of sPLA2R. Each point represents the mean ± S.E. of triplicate measurements. The data are representative of three experiments. The IC50 value of rsPLA2R in A was calculated to be 0.04 ± 0.01 nM from three separate experiments. The statistical significance in B was tested using Student's t test. *, p < 0.01 versus control.

Next, we examined the effects of rsPLA2R and serum sPLA2R on sPLA2-X binding activity. As shown in Fig. 4A, rsPLA2R protein blocked the sPLA2-X binding to PLA2R-CHO cells with an IC50 value of 1.2 ± 0.2 nM, which coincided with the binding affinity of sPLA2-X to the cell surface PLA2R in mouse osteoblastic MC3T3-E1 cells (Kd = 4.6 nM) (21). We next examined the effect of concentrated sPLA2R materials prepared from a sPLA2-IB affinity column, which was shown to have the sPLA2R concentration of 1.36 nM by ELISA. As can be seen from Fig. 4B, the sPLA2-X binding was blocked up to 48% by these materials, which contrasted with no significant inhibition by the eluted fractions prepared from PLA2R-deficient mouse serum. Taken together, these findings demonstrate that serum sPLA2R is functional in terms of the suppression of sPLA2-X-induced biological responses.


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  		Fig. 4.   Inhibitory potency of sPLA2R for the sPLA2-X binding activity. A, inhibitory potency of rsPLA2R for sPLA2-X binding. PLA2R-CHO cells in 24-well plate were incubated with 125I-sPLA2-X (1.0 nM) in the absence or presence of various concentrations of rsPLA2R. B, inhibitory potency of circulating sPLA2R for sPLA2-X binding activity. The membrane fractions of PLA2R-CHO cells were incubated with 125I-sPLA2-X (0.2 nM) in the absence (none) or presence of serum sPLA2R fractions from wild-type (WT) or PLA2R-deficient (KO) mice, or 12.5 nM rsPLA2R. The specific binding activity was determined as the differences between the presence and absence of 500 nM mouse sPLA2-X, and the results are expressed as the percentages of 125I-sPLA2-X specific binding in the absence of sPLA2R. Each point represents the mean ± S.E. of triplicate measurements. The data are representative of three experiments. The IC50 value of rsPLA2R in A was calculated to be 1.2 ± 0.2 nM from three separate experiments. The statistical significance in B was tested using Student's t test. *, p < 0.05 versus control.

Study of the Mechanisms Underlying sPLA2R Production-- To investigate the mechanisms underlying the production of sPLA2R, we used PLA2R-CHO cells that spontaneously release sPLA2R protein into the culture medium. We confirmed that sPLA2R protein isolated from the conditioned medium of PLA2R-CHO cells has the same molecular mass as the serum sPLA2R by Western blot analysis (data not shown). As shown in Fig. 5A, the sPLA2R levels in the supernatant were time-dependently increased during the cultivation of PLA2R-CHO cells. This release response was suppressed over 80% by the addition of BB2516, a specific broad spectrum metalloproteinase inhibitor (33), in contrast to no significant inhibition by p-APMSF, a serine protease inhibitor (34). Next, the effects of various types of protease inhibitors were evaluated for the spontaneous release of sPLA2R from the membrane fractions of PLA2R-CHO cells to eliminate their toxic effects on intact cells. The concentration used was optimized for their efficient and specific inhibition against target proteases in vitro. After incubation in the presence of inhibitors for 1 h, the released sPLA2R in the supernatant was evaluated with sandwich ELISA. As shown in Fig. 5B, EDTA, a metal chelator, and BB2516 blocked the sPLA2R release up to 78 and 69%, respectively. Especially, the IC50 value of BB2516 was evaluated to be 4.7 nM (data not shown). In contrast, there was no significant suppression by other protease inhibitors, including p-APMSF, leupeptin, and pepstatin, which are known as inhibitors for serine, cysteine, or asparate proteases (35). These findings suggest that metalloproteinases are involved, at least in part, in the proteolytic cleavage of the membrane-bound receptor in PLA2R-CHO cells.


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  		Fig. 5.   Effects of protease inhibitors on the release of sPLA2R from PLA2R-CHO cells. A, PLA2R-CHO cells were incubated with or without 1 µM BB2516 or 1 mM p-APMSF for various times at 37 °C. After centrifugation, the supernatant was collected, and the released sPLA2R was quantified by sandwich ELISA. B, the membrane fractions of PLA2R-CHO cells were incubated with or without various protease inhibitors (20 mM EDTA, 1 µM BB2516, 1 mM p-APMSF, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A) for 1 h at 37 °C. After centrifugation, the released sPLA2R in the supernatant was quantified by sandwich ELISA. The results are expressed as the percentages of the released sPLA2R in the absence of inhibitors. Each point represents the mean ± S.E. of triplicate measurements. The data are representative of three experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Venomous snakes have PLIs in their plasma to protect themselves from their own venomous PLA2s that elicit a wide variety of toxicities, such as neurotoxicity and myotoxicity (36). In mammals, there are diverse types of sPLA2s that can be released outside of the cells and exist in the tissue fluids and blood plasma (3). The present study is the first to identify mouse sPLA2R as a circulating PLI for sPLA2s in mammals. Western blot analysis revealed that serum sPLA2R retained all of the extracellular domains of the membrane-bound form of the receptor, similar to rsPLA2R (Fig. 2). We have previously shown that rsPLA2R retained the same binding dissociation constant (Kd = 0.97 nM for sPLA2-IB) and ligand specificity as the membrane-bound receptor (17, 37). Scatchard plot analysis has revealed that rsPLA2R can bind one sPLA2 ligand per molecule, like the native cell surface receptor (37). In fact, rsPLA2R protein strongly blocked the sPLA2-X binding to the membrane-bound receptor with an IC50 value of 1.2 nM (Fig. 4A), which coincided with the binding affinity of sPLA2-X to the cell surface PLA2R (21). In the present study, serum sPLA2R was found to possess binding activity for sPLA2-IB and sPLA2-X (Fig. 1). In addition, serum sPLA2R blocked the enzymatic activity and receptor binding activity of sPLA2-X (Figs. 3B and 4B). These findings demonstrate that serum sPLA2R has similar characteristics to rsPLA2R in terms of biochemical properties as well as functional activities and thus confirmed our previous estimation of the plasma sPLA2R concentration in normal mice with sandwich ELISA (0.13 nM), in which rsPLA2R was used as a conventional standard (27).

We have previously reported that sPLA2-IB can elicit various biological responses possibly via PLA2R binding, such as proliferation and chemokinetic migration of vascular smooth muscle cells and potent vasoactive actions in cerebral arteries (17, 38). In addition, we have recently demonstrated that sPLA2-X elicits marked release of arachidonic acid leading to lipid mediator productions in various cell types, including monocytes/macrophages (15, 16, 39). At the normal circulating level (0.13 nM), rsPLA2R can effectively block the enzymatic activity of sPLA2-X (0.14 nM) toward the substrate DPPC (Fig. 3A), and the prepared serum sPLA2R fractions (0.37 nM) were found to suppress the activity up to 70% (Fig. 3B). However, in mouse spleen cells, the inhibitory potency of rsPLA2R for sPLA2-X-induced arachidonic acid release was weaker compared with that in the DPPC hydrolysis, possibly because of the requirement of higher concentration of sPLA2-X for detection of the released fatty acids in intact cell systems. For the sPLA2-X binding, rsPLA2R was found to block up to 20% at the normal circulating concentration (Fig. 4A), and its inhibitory potency can be greatly enhanced at the concentration over 0.3 nM. In fact, the concentrated serum materials containing higher levels of sPLA2R (1.36 nM) could significantly block the sPLA2-X binding (Fig. 4B). It has been reported that the normal serum concentration of sPLA2-IB is 5.1 ng/ml (0.36 nM) in humans (40). Although the normal plasma level of sPLA2-IB and sPLA2-X has not yet been examined in mice, these findings suggest that the circulating sPLA2R can play one of the endogenous inhibitors that block the biological functions of these sPLA2s in the normal conditions. In patients with acute pancreatitis and renal failure, increased systemic levels of sPLA2-IB have been observed (40). We have recently shown that the plasma sPLA2R concentration is significantly elevated to up to 1.5-fold of the normal level during murine endotoxic shock (27), although this enhanced level is not enough for the effective suppression of the enzymatic activities and the receptor-mediated responses evoked by higher concentration of sPLA2s. Further analysis of the circulating sPLA2R and sPLA2s levels during various pathological states are required to establish the role of circulating sPLA2R as an endogenous inhibitor to protect the pathological functions of sPLA2s.

In the plasma of various snakes, three distinct types of PLIs (PLI-alpha , -beta , and -gamma ) have been identified. PLIalpha is a 75-kDa glycoprotein composed of a trimer of 20-kDa subunits having sequence homology to the CRD of C-type lectins and preferentially blocks group II acidic PLA2s (41). Intriguingly, sPLA2R contains all of the extracellular domains of the membrane-bound receptor including the tandem repeat of eight CRD-like domains. Previous deletion experiments have demonstrated that three CRD-like domains (CRD3, CRD4, and CRD5) are representative of the sPLA2 binding activity (42, 43). In addition, the CRD of PLIalpha present in the blood plasma of the Habu snake Trimeresurus flavoviridis has similarities with the CRD5 of PLA2R (28%) (6). Also, the soluble lung surfactant protein SP-A has been reported to have CRD that shares sequence homology with PLIalpha and blocks the Habu snake venom sPLA2 activity (44). Notably, SP-A can also bind to guinea pig sPLA2-IIA to suppress its enzymatic activity in contrast to the absence of inhibition of porcine sPLA2-IB (45). However, the binding affinity of SP-A for sPLA2-IIA has not yet been determined, and SP-A can also interact with other molecules, such as carbohydrate ligands and its high affinity receptor present in alveolar type II cells (46). In contrast, mouse PLA2R specifically recognizes sPLA2-IB and X with a high affinity (Kd of 1.43 and 4.6 nM, respectively) and does not possess lectin activity (20). Thus, further studies are required to ascertain the biological roles of SP-A in the inhibition of sPLA2-IIA activity in the lung. Nevertheless, these findings suggest that other C-type lectin family members having CRD-like domains might also behave as physiological sPLA2 inhibitors in circulation and/or local tissue areas.

Numerous types of soluble-form membrane receptors and cell adhesion molecules are known to exist in circulation as innate host defense systems against exaggerated receptor-mediated responses (47). One possible mechanism underlying the production of soluble molecules is the alternative splicing event. In fact, the potential production of sPLA2R in human kidney was suggested based on the finding of an alternatively processed transcript encoding the ectodomains of PLA2R (48). However, the existence of a soluble form of PLA2R has not been confirmed in humans, and we have not detected the alternatively spliced transcript in any of the tissues examined by Northern blot and RT-PCR analysis in mice (data not shown). In another setting, the ectodomains of many membrane proteins are released by regulated proteolytic cleavage (49). In the present study, BB2516 and EDTA efficiently suppressed the proteolytic cleavage of membrane-bound PLA2R in both intact PLA2R-CHO cells and the membrane preparations (Fig. 5). In particular, the IC50 value of BB2516 for inhibition of the cleavage of membrane-bound PLA2R was 4.7 nM, which corresponded well with its inhibitory potency on metalloproteinases activity in vitro (50). Because BB2516 can suppress all types of metalloproteinases (33), the subtypes involved in the shedding process could not be identified in this study. Tumor necrosis factor (TNF)-alpha -converting enzyme, one of the members of a disintegrin and metalloproteinase family (51), is the first identified "sheddase" that is involved in the cleavage of TNF-alpha as well as various membrane-bound proteins including the TNF p75 receptor, L-selectin, and transforming growth factor-alpha (52). CHO cells are known to constitutively express active TNF-alpha -converting enzyme on the cell surface (53), suggesting a potential participation of TNF-alpha -converting enzyme in the cleavage of membrane-bound PLA2R, although further studies are needed to elucidate the metalloproteinases involved in vivo. A similar shedding mechanism has been observed with the mannose receptor, another member of the subgroup VI of the C-type lectin family (18), because its soluble form in mouse serum can be produced by cleavage of the membrane-bound form via a disintegrin and metalloproteinase or matrix metalloproteinases (MMPs) (54). Thus, the biological function as a soluble receptor after processing by metalloproteinases might be another critical implication for this family member. Among the metalloproteinases, MMP-14 (MT1-MMP) and MMP-9 (gelatinase B) transcripts are constitutively expressed in the spleen where PLA2R and its ligands, sPLA2-IB and X, also exist (20, 27, 28). In addition, the expressions of MMP-14, MMP-13 (collagenase 3), and MMP-11 (stromelysin 1) transcripts were elevated in mouse spleen at 1 h after lipopolysaccharide injection (55). Upon endotoxin challenge, the expression of PLA2R mRNA was also elevated in the splenic lymphocytes and alveolar type II epithelial cells after 1 h, and the circulating sPLA2R level increased after 2-3 h (27). These findings suggest a possible contribution of these MMPs to the production of sPLA2R in the tissues and circulation under physiological and pathological states. Further studies with specific inhibitors should help identify the metalloproteinase types involved.

In conclusion, we have identified circulating sPLA2R as an endogenous inhibitor of sPLA2s in mice. In this study, the use of PLA2R-deficient mice enabled us to clearly show the existence of the receptor in serum. Because there is strict species specificity in the relationships between sPLA2 ligands and PLA2R, further studies on the identification of sPLA2R and its ligand in humans are required for elucidation of their physiological and pathological roles.

    ACKNOWLEDGEMENTS

We are grateful to Kazumi Nakano and Satomi Shinonome for excellent technical assistance, Fumihiko Watanabe for the synthesis of BB2516, and Dr. Hiroyuki Okamoto and Dr. Jun Ishizaki for fruitful discussions and support in the preparation of the manuscript.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed. Tel.: 81-6-6455-2104; Fax: 81-6-6458-0987; E-mail: kohji.hanasaki@shionogi.co.jp.

Published, JBC Papers in Press, February 5, 2002, DOI 10.1074/jbc.M108752200

    ABBREVIATIONS

The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; sPLA2-IB, -IIA, and -X, group IB, IIA, and X, sPLA2, respectively; PLA2R, PLA2 receptor; sPLA2R, soluble form of PLA2R; rsPLA2R, recombinant sPLA2R; PLI, PLA2 inhibitory protein; CRD, carbohydrate recognition domain; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; DPPC, 1-palmitonyl-2-palmitonyl-sn-glycero-3-phosphocholine; p-APMSF, (p-amidinophenyl)methanesulfonyl fluoride; TNF, tumor necrosis factor; MMP, matrix metalloproteinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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