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J Biol Chem, Vol. 274, Issue 37, 26315-26320, September 10, 1999

Identification and Purification of a Novel Receptor for Secretory Phospholipase A₂ in Porcine Cerebral Cortex^*

Alenka opi, Nataa Vuemilo, Franc Gubenek^§, and Igor Kriaj^¶

From the  Department of Biochemistry and Molecular Biology, Joef Stefan Institute, Jamova 39 and ^§ Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, Akereva 5, University of Ljubljana, 1000 Ljubljana, Slovenia

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A specific phospholipase A₂ receptor from porcine cerebral cortex has been characterized (K_d = 145 nM, B_max = 0.4 pmol/mg membrane protein) by using a radioiodinated derivative of ammodytoxin C (AtxC), a snake venom presynaptically neurotoxic group IIA phospholipase A₂. After the receptor was solubilized in a ligand-binding form, it was approximately 14,000-fold enriched by chromatography on wheat germ lectin-Sepharose and AtxC-Affi-Gel 10. The receptor is a single chain glycoprotein with an apparent molecular mass of 180 kDa and binds toxic and non-toxic phospholipases A₂ of either group I or II. It also recognizes conjugates of bovine serum albumin with mannose, N-acetylglucosamine, and galactose. In its molecular mass and pharmacological profile, the AtxC receptor resembles the M-type receptor for secretory phospholipases A₂ from rabbit skeletal muscle (a C-type multilectin, homologous to macrophage mannose receptor), yet in terms of relative abundance in brain and antigenicity, these two receptors are completely different. A further AtxC receptor of approximately 200 kDa discovered in porcine liver was, however, recognized by anti-rabbit M-type phospholipase A₂ receptor antibodies. There are, therefore, two immunologically distinct secretory phospholipase A₂ receptors of about 200 kDa in the same species. Although the liver receptor is related to the M-type secretory phospholipase A₂ receptors, the brain receptor is not and belongs to a novel group of secretory phospholipase A₂ receptors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Secretory phospholipases A₂ (sPLA₂s,¹ EC 3.1.1.4) hydrolyze 1,2-diacyl-3-sn-phosphoglycerides at the sn-2 position, releasing free fatty acids and lysophospholipids (1, 2). The growing family of these enzymes, characterized by the molecular mass of 13-18 kDa, the presence of many disulfide bonds in their structure, a requirement of millimolar amounts of Ca²⁺ for catalytic activity, and a low selectivity for phospholipids with different polar head groups, already comprises nine different groups, IA, IB, IIA, IIB, IIC, III, V, IX, and X (1, 3-5). Secretory PLA₂s were considered primarily as digestive enzymes, but it is now known that they are involved in a number of other important physiological processes such as cell contraction (6), lipid mediator release (7), cell proliferation (8), endotoxic shock (9), acute lung injury (10), rheumatoid arthritis (11), Crohn's disease (12), acute pancreatitis (13), various cancers (14), and the destruction of pathogenic microorganisms (15). In addition, many sPLA₂s found in venom of snakes, insects, lizards, and scorpions (16) display diverse modes of toxicity (reviewed in Ref. 17). At least some of these (patho)physiological activities of sPLA₂s are receptor-mediated. The correlation between the binding of mammalian group I sPLA₂ (sPLA₂-I) to a specific 200-kDa membrane receptor on Swiss 3T3 fibroblasts and DNA synthesis by the cells has been shown (8). The enhancement of migration of rat embryonic thoracic aorta smooth muscle cells (A7r5) paralleled the binding of sPLA₂-I to a specific cell receptor (18). Similarly, the contraction of guinea pig lung parenchyma (19) and prostaglandin synthesis in the mouse osteoblastic MC3T3-E1 cell line were stimulated by the binding of sPLA₂-I to the cell receptor in a concentration-dependent manner (20). A receptor for mammalian sPLA₂-I was purified from bovine corpus luteum (21). Its sequence identified it as a homologue of the previously characterized M-type receptor for snake venom sPLA₂s in rabbit skeletal muscle (22-24). The M-type receptor has been cloned in different species (23-26) and found to constitute a new family of Ca²⁺-dependent (C-type) multilectin receptors, which includes the macrophage mannose receptor, the dendritic cell receptor DEC 205 (27), and the endothelial lectin  receptor (see Ref. 28 and reviewed in Ref. 29). The neurotoxic action of two potent presynaptically acting snake venom sPLA₂s, -bungarotoxin (-Butx) and taipoxin, was found to depend on their specific interaction with proteins, which are, however, completely different from the M-type receptors, namely certain voltage-dependent K⁺ channels (30) and proteins of the pentraxin family (31, 32). Apparently, one of the reasons for so many different (patho)physiological activities attributed to sPLA₂ lies in the versatility of receptors where they selectively bind and, probably, in their specific tissue distribution (reviewed in Ref. 5).

In the study of the molecular basis of neurotoxicity of ammodytoxin C (AtxC), a presynaptically acting group IIA sPLA₂ from the long-nosed viper venom (Vipera ammodytes ammodytes) (33, 34), we identified a novel high affinity binding protein for sPLA₂s in porcine cerebral cortex and purified it. The receptor shares some similarity (molecular mass and ligand binding characteristics) with the M-type sPLA₂ receptors, but it is not antigenically related to them. The parallel discovery, reported here, of an M-type sPLA₂ receptor in the liver of the same animal demonstrates for the first time that more than one sPLA₂ receptor of ~200 kDa exists in the same species and additionally confirms the exclusivity of the receptor for sPLA₂ in porcine brain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Ammodytoxins, ammodytin I₂ and AtnL, were purified from V. ammodytes ammodytes venom as described previously (33, 35). Crotoxin (from Crotalus durissus terrificus) and agkistrodotoxin (Agtx, from Agkistrodon blomhoffii brevicaudus) were a gift from Dr. Cassian Bon, Institut Pasteur, Paris, France. OS₂ (from Oxyranus s. scutellatus), membranes from a primary culture of rabbit skeletal muscle cells and guinea pig polyclonal antibodies against the rabbit M-type sPLA₂ receptor were a gift from Dr. Gerard Lambeau, Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France. Taipoxin (Oxyranus s. scutellatus), -bungarotoxin (Bungarus multicinctus), mannosylated BSA, N-acetylglucosaminated BSA, galactosylated BSA, mannan, and invertase were from Sigma. Porcine pancreatic PLA₂ (ppPLA₂) and bee venom PLA₂ (bvPLA₂) were purchased from Roche Molecular Biochemicals. Na¹²⁵I (carrier-free) was from NEN Life Science Products. Disuccinimidyl suberate was from Pierce. Affi-Gel 10 and protein molecular mass standards were obtained from Bio-Rad. Triton X-100 was purchased from Roche Molecular Biochemicals, and concanavalin A-Sepharose, lentil lectin-Sepharose 4B, and wheat germ lectin-Sepharose 6MB were from Amersham Pharmacia Biotech. All other reagents and chemicals were of analytical grade.

Radioiodination of AtxC-- Radiolabeled AtxC (¹²⁵I-AtxC) was prepared as described (36). ¹²⁵I-AtxC was identical to native AtxC in enzymatic, neurotoxic, and immunological characteristics. The specific radioactivity of the preparation was routinely about 300 Ci/mmol.

Membrane Preparation from Porcine Cerebral Cortex-- A demyelinated P2 fraction of porcine cerebral cortex was prepared using a slight modification of the method of Bennett et al. (37). All manipulations were carried out on ice or at 4 °C in the presence of the following inhibitors: 2 mM EDTA, 25 µg/ml bacitracin, 2 µg/ml aprotinin, 1.4 µg/ml pepstatin, 1 µg/ml leupeptin, 0.2 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride. Protein content in the membrane preparation was determined using the method of Markwell et al. (38). Bovine serum albumin (BSA) was used as a standard. Membranes were stored at 70 °C.

Preparation of Porcine Liver Membrane Fractions-- Porcine liver (60 g) was cut into small pieces and homogenized in 240 ml of 10 mM Hepes, pH 7.4, containing 0.25 M sucrose and 1 mM EDTA. The homogenate was centrifuged at 1,000 × g for 10 min and the pellet (P1 fraction) discarded. The supernatant was further centrifuged at 3,000 × g for 10 min to give the P2 pellet, and the supernatant was spun once again (10,000 × g for 20 min) to give the P3 pellet. The pellets were resuspended in the homogenizing buffer and stored at 20 °C until used.

Solubilization of AtxC-binding Proteins-- Membranes from porcine cerebral cortex or liver (2.6 mg of membrane protein/ml) were extracted for 1 h by gentle agitation at 4 °C in 75 mM Hepes, pH 8.2, containing 150 mM NaCl, 10 mM SrCl₂, 0.5 mM EGTA, and 4% (w/v) Triton X-100 and afterward centrifuged at 106,200 × g for 1 h. The detergent extract thus obtained was usually diluted 2-fold with cold deionized water before the next purification step.

Cross-linking of ¹²⁵I-AtxC to the Solubilized AtxC-binding Proteins-- The membrane extract or the fractions containing solubilized receptor were incubated for 30 min at room temperature with ¹²⁵I-AtxC (10 nM final concentration) in the presence or absence of an unlabeled competitor. Disuccinimidyl suberate, dissolved in 5 µl of dimethyl sulfoxide, was added to a final concentration of 100 µM. The cross-linking reaction was stopped by the addition of SDS-PAGE sample buffer. Samples were analyzed by SDS-PAGE under reducing conditions (50 mM dithiothreitol) (39). Gels were dried and autoradiographed at 70 °C using Kodak X-Omat AR films and two intensifying screens.

Chromatography on Wheat Germ Lectin-Sepharose 6MB-- 9 ml of wheat germ lectin-Sepharose 6MB were equilibrated in 50 mM Hepes, pH 8.2, containing 140 mM NaCl and 2 mM CaCl₂. The diluted detergent extract was incubated with the gel for 4 h at 4 °C under moderate agitation. Two washing steps followed, the first with 80 ml of the equilibration buffer containing 500 mM NaCl and 0.1% (w/v) Triton X-100, and the second with 40 ml of equilibration buffer with 0.3% (w/v) Triton X-100. The bound material was eluted with 10 ml of the equilibration buffer containing 0.1% (w/v) Triton X-100 and 0.6 M N-acetylglucosamine.

Coupling of AtxC to Affi-Gel 10-- 6 ml of Affi-Gel 10 were incubated under agitation for 4 h at 4 °C with 6 ml of 100 mM MES, pH 6.5, 5 mM CaCl₂, containing 1 mg/ml AtxC. The amount of toxin bound to the gel was determined by measuring A₂₈₀ of the supernatant. Routinely more than 90% of AtxC was bound to the matrix. The resin was washed with 100 ml of 50 mM Hepes, pH 8.2, containing 140 mM NaCl, 2 mM CaCl₂, and 0.1% (w/v) Triton X-100 and stored in the same buffer at 4 °C.

Chromatography on AtxC-Affi-Gel 10-- The gel was equilibrated with 100 ml of 50 mM Hepes, pH 8.2, containing 140 mM NaCl, 2 mM CaCl₂, and 0.1% (w/v) Triton X-100. The eluate from wheat germ lectin-Sepharose 6MB was incubated with 6 ml of the gel at 4 °C for 4 h under gentle agitation. After washing with 100 ml of equilibration buffer, the resin was transferred to the column and washed with the equilibration buffer containing 0.3% (w/v) Triton X-100. The receptors were eluted with 70 mM MES, pH 5.0, containing 100 mM NaCl, 2 mM CaCl₂, and 0.1% (w/v) Triton X-100. 1-ml fractions were collected and analyzed for the presence of receptor proteins by affinity labeling with ¹²⁵I-AtxC as described. The protein composition of each fraction was assessed by SDS-PAGE and silver staining (40).

Electroblotting and Immuno-chemiluminescence Detection-- Samples were run on SDS-PAGE (7.5% acrylamide gels) and transferred for 100 min at 60 V to a nitrocellulose membrane (Serva). The transfer buffer was 25 mM Tris, 200 mM glycine. After transfer, nonspecific binding sites on the membrane were blocked with 1% (w/v) non-fat dried milk in PBS. The membrane was then incubated with guinea pig polyclonal antibodies raised against the rabbit M-type sPLA₂ receptor diluted 1:5,000 in PBS and subsequently with the peroxidase-conjugated goat anti-guinea pig IgG diluted 1:10,000 in PBS (Cappel Research Products). After extensive washing in PBS containing 0.1% (w/v) Tween 20, the secondary antibodies were detected by the BM chemiluminescent Western blotting system (Roche Molecular Biochemicals) following the manufacturer's instructions.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Membrane Extracts-- In order to solubilize AtxC-binding proteins from the demyelinated P2 fraction of porcine cerebral cortex, various detergents were tested. Judged on the basis of the lowest AtxC-specific binding site content in the pellet after extraction, the detergent with the highest efficiency was Triton X-100 (data not shown). The ligand binding activity of solubilized receptors was assessed by affinity labeling of the membrane extracts with ¹²⁵I-AtxC. Specific adducts shown in Fig. 1A proved that AtxC-binding proteins retain toxin binding activity after solubilization in 4% (w/v) Triton X-100. The addition of proteinase inhibitors to the extraction buffer was not critical because no proteolytic inactivation of the receptors was observed in their absence. In the case of porcine liver the same extraction conditions were successful.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Inhibition by unlabeled AtxC of cross-linking of 125I-AtxC to its receptors solubilized from porcine cerebral cortex under equilibrium conditions. A, autoradiogram of the SDS-PAGE gel (10%) of Triton X-100 extracts of porcine cerebral cortex demyelinated P2 fraction (100 µg of protein/lane) labeled with 125I-AtxC (10 nM final concentration) in the presence of the indicated concentrations of unlabeled AtxC. The molecular masses of the specific adducts (arrows) were determined using molecular mass standard as follows: myosin (204 kDa), -galactosidase (121 kDa), bovine serum albumin (78 kDa), carbonic anhydrase (39.5 kDa), soybean trypsin inhibitor (30.7 kDa). B, dose-response curve relating the extent of cross-linking of 125I-AtxC with the 180-kDa receptor to the concentration of unlabeled AtxC present during the incubation. The data were obtained by quantifying the intensity of the specific 125I-AtxC adducts from the autoradiogram in A, using QuantiScan. The nonspecific binding (Bns) is the value obtained in the presence of the largest excess of unlabeled AtxC used in the experiment (A, 8th lane). The value for Bns was subtracted from each value measured (Bt) to obtain the specific binding (Bsp = Bt  Bns). The data are displayed as Bsp relative to the maximum specific binding (Bsp,0) obtained in the absence of the unlabeled AtxC (Bsp,0 = Bt,0  Bns). An IC50 of 155 nM was obtained using the nonlinear curve fitting program GraFit 3.0 (46).

AtxC-binding Proteins in Detergent Extract of Porcine Cerebral Cortex-- As shown in Fig. 1A, two specific adducts were observed after cross-linking ¹²⁵I-AtxC with the detergent extract of porcine cerebral cortex P2 fraction. Besides the 39-kDa adduct, already identified by affinity labeling experiments on membranes (41), a second specific adduct, slightly smaller than 200 kDa, was observed. Assuming one to one stoichiometry of binding between the receptors and 14-kDa AtxC, there are thus two specific receptors for AtxC in porcine cerebral cortex with apparent molecular masses of 25 (R25) and 180 kDa (R180), respectively.

Characterization of R180 in Solution-- The affinity of AtxC for its solubilized receptors was estimated in the cross-linking competition experiment shown in Fig. 1. The native AtxC displaced ¹²⁵I-AtxC from 50% of R180 (IC₅₀) at a concentration of 155 nM, which corresponds to a dissociation constant (K_d) of 145 nM (42). The K_d for the interaction of AtxC with R25 was lower than 25 nM, consistent with the value of 15 nM obtained using the membrane preparation (41). If Sr²⁺ and EGTA were replaced with EDTA in the extraction/cross-linking buffer, ¹²⁵I-AtxC still bound to R180 but not to R25 (data not shown).

Several toxic and non-toxic PLA₂s as well as some other molecules were examined for their ability to inhibit the ¹²⁵I-AtxC-R180 adduct formation (Table I). Binding of ¹²⁵I-AtxC to R180 was most potently inhibited by neurotoxic OS₂ (IC₅₀ = 2 nM) and taipoxin (IC₅₀ = 5 nM) and also by non-toxic ppPLA₂ (IC₅₀ = 78 nM). AtxA, a 17-fold more toxic homologue of AtxC, inhibited the specific adduct formation 10-fold better than AtxC, which suggests the importance of R180 in the neurotoxic action of ammodytoxins in porcine cerebral cortex. Inhibition was obtained also with the myotoxic PLA₂ homologue AtnL, which is enzymatically inactive (IC₅₀ = 39 nM). On the other hand, Agtx, crotoxin, and -Butx, which are potent neurotoxins, and bvPLA₂ were not inhibitory at 10 µM concentration. Glycoconjugates of BSA with mannose, N-acetylglucosamine, and galactose also prevented the binding of ¹²⁵I-AtxC to R180 although at much higher concentrations (IC₅₀ values = 570, 335 and 240 nM, respectively), whereas invertase, mannan, and monosaccharides, D-mannose, D-glucose, D-galactose, L-fucose, and N-acetylglucosamine, did not influence the interaction.

R180 was found to be very stable in solution. Its ¹²⁵I-AtxC binding ability was unchanged after prolonged storage at 20 °C or repeated freezing and thawing of the sample. Even after overnight incubation at room temperature the activity of R180 was not affected. R180 lost its affinity for AtxC at pH 5.0, but the affinity was completely restored when the pH was again raised to 7.4.

R180 was completely retained by concanavalin A, wheat germ lectin, and lentil lectin-Sepharose and subsequently eluted by the respective specific eluants. This indicates that R180 is glycosylated.

Purification of R180 from the Demyelinated P2 Fraction of Porcine Cerebral Cortex-- 40 ml of the membrane preparation (83 pmol of R180) were extracted with 4% (w/v) Triton X-100. The extract was incubated with wheat germ lectin-Sepharose since we have shown that R180 binds with high affinity and reversibly to this lectin. ¹²⁵I-AtxC affinity labeling was used to follow R180 during the isolation. The analysis of the solution after the incubation showed that all the R180 was retained on the resin. Following washing of the gel, elution of R180 was achieved with 0.6 M N-acetylglucosamine. Reduction of Triton X-100 concentration to 0.1% (w/v) in the eluate did not affect the ability of R180 to bind AtxC.

The final purification of R180 was achieved by toxin affinity chromatography. Affi-Gel 10 was chosen as a matrix to which AtxC was attached. The affinity resin with the highest receptor binding activity was synthesized at pH 6.5. Initially, the affinity column was able to bind R25 and R180; however, its capacity for R25 declined very quickly. After using three times, the resin completely lost the R25-retaining ability, whereas its binding of R180 remained unaffected; it became R180-specific. As established by cross-linking experiments, R180 completely lost its toxin binding activity at pH 5.0 and regained it on returning the pH to 7.4. We therefore eluted R180 from the AtxC-Affi-Gel 10 at pH 5.0 and subsequently raised the pH to 7.4, collecting fractions to trace the receptor by ¹²⁵I-AtxC affinity labeling. In Fig. 2A, the total protein composition at each purification step is shown using silver-stained SDS-PAGE. A homogenous product with an apparent molecular mass of 180 kDa was obtained after the AtxC-affinity chromatography step (Fig. 2A, lane 7). Under non-reducing conditions only one band with the same molecular mass was visible (data not shown), showing that R180 is not composed of subunits linked by disulfide bonds. The final product exhibited AtxC binding activity, as shown in Fig. 2B, confirming that the isolated protein is indeed the AtxC receptor. About 2 µg of pure R180 was obtained as determined by semiquantitative densitometric analysis of the silver stained SDS-PAGE band. Purification from the membrane fraction was therefore about 14,000-fold and recovery about 13%. The purification yield was essentially the same whether prepared from fresh or frozen brain cortexes.

View larger version (78K): [in this window] [in a new window]   Fig. 2.   Purification of R180 from porcine cerebral cortex. A, aliquots of the samples obtained in different steps of the purification procedure were analyzed by 10% SDS-PAGE under reducing conditions. The gel was silver-stained. Lane 1, molecular mass standards (-galactosidase, 139 kDa; bovine serum albumin, 80 kDa; carbonic anhydrase, 42.9 kDa; soybean trypsin inhibitor, 32.5 kDa); lane 2, crude membrane extract, 5 µg of protein; lane 3, breakthrough from wheat germ lectin-Sepharose 6MB, 4 µg of protein; lane 4, eluate from wheat germ lectin-Sepharose 6MB, 4 µl out of 8 ml; lane 5, breakthrough from AtxC-Affi-Gel 10, 4 µl out of 8 ml; lane 6, AtxC-Affi-Gel 10 Triton X-100 (0.3% (w/v)) washing, 4 µl out of 40 ml; lane 7, eluate from AtxC-Affi-Gel 10, 100 µl out of 4.2 ml. The position of pure R180 in lane 7 is indicated by the arrow. B, the final product (lane 7) specifically reacted with 125I-AtxC. An aliquot of the final product was incubated with 125I-AtxC in the absence (T) or presence (C) of 200-fold excess of unlabeled AtxC over the labeled toxin.

Immunological Comparison of R180 and Rabbit M-type sPLA₂ Receptor-- To characterize R180, Western blot analysis was made using guinea pig polyclonal antibodies raised against the rabbit M-type sPLA₂ receptor. Besides rabbit, these antibodies are able to recognize mouse, rat, and human M-type sPLA₂ receptors.² Neither the extract of the demyelinated P2 fraction of porcine cerebral cortex (Fig. 3A, lane 2) nor purified R180 (data not shown) reacted with these antibodies.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Comparison of AtxC receptors from porcine cerebral cortex (R180) and porcine liver. A, immunoblotting of detergent extracts of membrane preparations from porcine cerebral cortex and liver with polyclonal antibodies against rabbit M-type sPLA2 receptor. Lane 1, extract of crude membrane preparation from porcine liver, 225 µg of protein; lane 2, extract of demyelinated P2 membrane preparation from porcine cerebral cortex, 43.5 µg of protein; lane 3, membranes from rabbit skeletal muscle cells in primary culture, 24 µg of protein. B, 125I-AtxC affinity labeling of detergent extracts of brain cortex and liver membrane preparations. Extracts were incubated with 125I-AtxC in the absence (T) or presence (C) of 200-fold excess of unlabeled AtxC over the labeled toxin. Detergent extracts of demyelinated P2 membrane fraction from porcine cerebral cortex contained 116 µg of protein, and detergent extracts of crude membrane preparation from porcine liver contained 600 µg of protein. Molecular mass standards were myosin (208 kDa), -galactosidase (127 kDa), bovine serum albumin (85 kDa), and carbonic anhydrase (45 kDa). The arrows indicate the specific adducts. Note the difference in molecular mass between specific adducts from brain and liver.

An M-type sPLA₂ Receptor Is Present in Pig-- We examined porcine liver as a non-neuronal tissue source for potential AtxC receptors. ¹²⁵I-AtxC affinity labeled a specific receptor with a slightly higher molecular mass than that of R180 (Fig. 3B). The liver membrane extract (Fig. 3A, lane 1) cross-reacted with polyclonal anti-rabbit M-type sPLA₂ receptor antibodies, showing that the two sPLA₂ receptors are related. The rabbit M-type sPLA₂ receptor and the porcine liver sPLA₂ receptor were also similar in their low recovery of ligand binding activity following the pH shift from 7.4 to 5.0 and back, and in their instability at room temperature, neither of which was observed in the case of R180. On the contrary, we have found no essential difference in ligand binding specificity between porcine brain (R180) and liver AtxC receptors with the substances tested (Table I). This is not surprising given that we tested only exogenous molecules (except for ppPLA₂), which cannot be physiological ligands for these receptors (43).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Radiolabeled AtxC was found to interact specifically with the demyelinated P2 membrane fraction of porcine cerebral cortex. The specific binding of ¹²⁵I-AtxC was reduced when divalent cations (Ca²⁺, Sr²⁺, or Ba²⁺) were removed from the solution by EDTA, consistent with the fact that R25 failed to form an adduct with ¹²⁵I-AtxC in the presence of chelator (41). By studying the detergent extract of the membranes, we showed that the remaining EDTA-independent specific binding of AtxC is associated with a 180-kDa membrane protein. The equivalent experiments using intact membranes failed to show the presence of this component, although re-examination of the ¹²⁵I-AtxC cross-linking patterns, especially those that were overexposed, confirmed the formation of the specific adduct at 200 kDa on the membrane preparations also. The reason for the large difference in intensity of the specific signal near 200 kDa between the membranes and the membrane extract most probably indicates that the topology of R180 in the membrane is unsuitable for efficient cross-linking.

R180 and R25 differ considerably in their ligand binding specificities. Whereas R25 binds exclusively neurotoxic ammodytoxins (41), R180 binds both toxic and non-toxic sPLA₂s with high affinity (Table I). R180 recognizes sPLA₂s of both mammalian group I (ppPLA₂) and non-mammalian group I (OS₂ and taipoxin), as well as group II sPLA₂s (ammodytoxins and AtnL). The specific binding of the enzymatically inactive sPLA₂ homologue AtnL to R180 confirmed that the ability of sPLA₂ to bind to R180 is independent of the enzymatic activity.

A two-step procedure has been developed to isolate R180. The wheat germ lectin-Sepharose chromatography step was important to decrease the concentration of Triton X-100 from 2% (w/v) in the extract to 0.1% (w/v) in the eluate without diluting the sample. The decreased concentration of the detergent and increased concentration of R180 in the eluate were crucial for the success of the final purification step, AtxC affinity chromatography. Freshly synthesized AtxC-Affi-Gel 10 resin retained both AtxC-binding proteins, R25 and R180, but after the third application, the resin became only R180-specific. From the competition studies it is clear that AtxC has different receptor interaction sites for R25 and R180 (41). A possible explanation for the observed binding ability of AtxC-Affi-Gel 10 column could be in a minor conformational change of immobilized AtxC, which would lead to a loss of affinity for one receptor but not for the other. The AtxC affinity step gave rise to highly purified R180 as judged by SDS-PAGE analysis.

As a high affinity binding receptor for snake venom PLA₂s, R180 resembles the M-type sPLA₂ receptor, which has been purified from rabbit skeletal muscle (22). Their ligand binding specificities are very similar. Both avidly bind OS₂, taipoxin, and ppPLA₂ but not bvPLA₂, crotoxin, and -Butx. Invertase, mannan, D-mannose, D-glucose, D-galactose, L-fucose, and N-acetylglucosamine, which are the ligands of macrophage mannose receptor (44), do not affect ¹²⁵I-AtxC binding to R180, just as they also failed to influence the interaction of ¹²⁵I-OS₂ with the rabbit M-type sPLA₂ receptor (22). R180 also displays lectin-like properties, indicating the presence of carbohydrate recognition domains in its structure. Its much lower affinity for BSA glycoconjugates compared with the rabbit M-type sPLA₂ and macrophage mannose receptors (24, 44) suggests, however, that the lectin properties of R180 are not physiologically relevant. The molecular masses of R180 and rabbit M-type sPLA₂ receptor are practically identical, and both proteins are glycosylated. Nevertheless, the two receptors differ substantially in their abundance in brain. Although we detected 400 fmol of R180 per mg of membrane protein in porcine brain membrane preparation, only 12 fmol of M-type sPLA₂ receptor per mg of protein was found in rabbit brain using ¹²⁵I-OS₁, an M-type receptor-specific ligand (45). The mouse, rat, and human M-type sPLA₂ receptor homologues were not detected at all in brain by RNA blotting analysis using cDNA probes derived from respective animals and ¹²⁵I-OS₁ binding studies (25, 26, 45).

The relation between R180 and the M-type sPLA₂ receptor from rabbit skeletal muscle was further investigated by Western blotting with guinea pig polyclonal antibodies against the latter. The antibodies, which cross-react also with mouse, rat, and human M-type sPLA₂ receptors,² failed to recognize R180, but they cross-reacted with the AtxC receptor discovered in porcine liver. In pig, therefore, two immunologically different sPLA₂ receptors of ~200 kDa are present. One of them, found in liver, is a homologue of the M-type sPLA₂ receptors, whereas the other, purified from brain, is quite different. Based on primary structure comparison between sPLA₂ binding regions in the M-type sPLA₂ receptors from different animals, and on the analysis of the human genome for the M-type sPLA₂ receptor genes, it was concluded that M-type sPLA₂ receptors from different animals are probably encoded by homologous, single copy genes (25). This finding further emphasizes the distinction between R180 and M-type sPLA₂ receptors.

Cupillard et al. (43) showed that M-type sPLA₂ receptors differ substantially in affinity for the respective endogenous group I and II sPLA₂s and suggested the existence of other types of sPLA₂ receptors distinct from the M-type. According to the molecular mass and some ligand binding characteristics, the porcine brain sPLA₂ receptor probably belongs to the multilectin mannose receptor family (29). However, it cannot be a member of the same group of receptors as the M-type sPLA₂ receptors, which are all immunologically related, and it evidently represents (one of) the "missing" new type sPLA₂ receptor(s).

The physiological role of the new sPLA₂ receptor and its involvement in the neurotoxic action of AtxC remain important questions. In view of the relatively high abundance of R180 in the brain, its function should be very specific, although its natural ligands still have to be identified. Recently, besides endogenous group I and II sPLA₂s, group IIC, V, and X sPLA₂s have been described which may also be potential candidates, but their receptor binding properties have not yet been determined.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Cassian Bon, Institut Pasteur, Paris, France, who kindly provided crotoxin and agkistrodotoxin and Dr. Gerard Lambeau, Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France, who provided OS₂, membranes from a primary culture of rabbit skeletal muscle cells, and guinea pig polyclonal antibodies against the rabbit M-type sPLA₂ receptor. We also thank Dr. Roger H. Pain and Dr. Joe Pungerar for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by the Ministry of Science and Technology of Slovenia Grant J1-7261-0106.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 386 61 177 3626; Fax: 386 61 273 594; E-mail: igor.krizaj@ijs.si.

² G. Lambeau, personal communication.

	ABBREVIATIONS

The abbreviations used are: sPLA₂, secretory phospholipase A₂; Agtx, agkistrodotoxin; AtnL, ammodytin L; AtxC, ammodytoxin C; BSA, bovine serum albumin; bvPLA₂, bee venom phospholipase A₂; -Butx, -bungarotoxin; MES, 2-(N-morpholino)ethanesulfonic acid; OS₂, Oxyranus scutellatus phospholipase A₂; PLA₂, phospholipase A₂; ppPLA₂, porcine pancreatic PLA₂; R25 and R180, receptors for AtxC in porcine cerebral cortex of 25 and 180 kDa, respectively; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.

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