THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 16, Issue of June 5, pp. 9526-9532,199O Printed in U.S.A.

Identification and Purification of a Very High Affinity Binding Protein for Toxic Phospholipases A2 in Skeletal Muscle*

(Received for publication, November 16, 1989)

G&ard Lambeau, Annie Schmid-Alliana, Michel Lazdunski, and Jacques Barhanin From the Institut de Pharmacologic Mokculaire et Celluluire, UPR 411 du Centre National de la Recherche Scientifiique, 660 route des Lucioles, Sophia Antipolis 06560 Valbonne, France

Oxyuranus scuteZlratus toxin 1 (OS1) and toxin 2 (OSZ) are two monochain phospholipases AZ isolated from the venom of Taipan. Their iodinated derivatives have been used to characterize phospholipase AZ recep- tors on rabbit skeletal muscle cells in culture. Both ligands recognize one family of binding sites on myo- tube membranes with a B,,, of 1.9 to 2.2 pmol/mg of protein and dissociation constant values of 7.4 pM for ‘251-OS2 and 38 pM for 125I-OS1. Other snake venom phospholipases A2 are able to inhibit 1251-OS2 binding to the muscle receptor. Competition experiments with these unlabeled phospholipases AZ define a pharmaco- logical profile of the muscle receptor very different from the previously described pharmacological profile of the neuronal phospholipase AZ receptors.

The number of ‘251-OS2 receptors in skeletal muscle cells increases during in vitro cell maturation but there is no clear relation between the increase of B,,, and the fusion of myoblasts into myotubes.

The phospholipase A2 binding protein from myotubes has been identified both by cross-linking experiments and by purification studies. It is composed of only one subunit of M, 180,000.

Phospholipases AX (PLAJ,’ EC 3.1.1.4, are very abundant components of snakes, scorpions and bee venoms. Venom PLAz are often very toxic to mammals (1). They induce a wide variety of pathological symptoms including neurotoxicity (2-4) and myotoxicity (4). Despite a large number of studies which include detailed sequence information on many PLA*s (for a recent review see Ref. 5), the precise structural features determining any of the pharmacological properties are still unknown. For example, the contribution of the catalytic ac- tivity of PLA2s for a given toxic effect such as the anticoag- ulant activity remains unclear and reports in the literature are contradictory (5-7).

Using an iodinated derivative of a newly purified PLA, from the Taipan venom, O& we have recently identified high affinity specific binding sites for PLA, in neuronal mem-

* This work was supported by the Association des Myopatbes de France, the Centre National de la Recherche Scientifique, and Min- &t&e de la Defense Nationale Grant DRET 88/054. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The abbreviations used are: PLAs, phospholipase As; CHAPS, 3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate; DSS, suberic acid bis-N-hydroxysuccinimide ester; Hepes, 4-(Z-hydroxy- ethyl)-1-piperazineethanesulfonic acid; MES, 2-[N-morpholinoleth- anesulfonic acid; Mops, 3-morpholinopropanesulfonic acid; OS,, 0. scutellatus toxin 1; OS,, 0. scutellatus toxin 2; SDS, sodium dodecyl sulfate; WGA, wheat germ agglutinin.

branes (8). These receptors are of protein nature and recog- nized by a large number of PLAzs of different origins. Affin- ities of the studied PLA2s were comprised between 2 pM and several micromolar and were in good correlation with their neurotoxicity, indicating that the binding component is di- rectly involved in the neurotoxic activity of these PLA2s.

Kini and Evans (5) recently proposed an hypothetical model to describe the specificity of pharmacological effects induced by venom PLAns. This model postulates a “pharmacological site,” located on the surface of the PLA, itself, and the existence of specific target sites which would distinguish a target cell from a non-target cell. Our previous work on neuronal membranes (8) is a direct demonstration of this model. The model also suggests that different PLA9 receptors exist in different tissues to explain the tissue specificity of some of the PLA2 actions.

In this report, we make use of iodinated derivatives of two monochain PLA2s is from Taipan, OS1 and OSn, to character- ize specific PLA2 binding sites in rabbit muscle cells in culture and affinity label and purify to homogeneity the PLA2 binding protein. These newly identified muscle receptors are phar- macologically and structurally different from those found in neuronal membranes.

EXPERIMENTAL PROCEDURES

Materials-Oxyuranw scutellutus scutetiutus toxins, PLA,s from Nuja mossambica mossambicn (8), from Apis mellifera (91, and from Crotalus atrox (10) venoms were purified as described previously. Crotoxin subunits (from Crotalus durissus terrificus) were a gift from Dr. C. Bon (Institut Pasteur, Paris, France), and notexine (from Notechis scutatus scutatw), pseudexin (from Pseudechis porphyria- cus) and textilotoxin (from Pseudonaja textilis) were gifts from Dr. J. Middlebrook (Fort Detrick, Frederick, MD). 8-Bungarotoxin, PLA, from porcine pancreas, and L-cu-phosphatidylcholine type V-E were from Sigma. Affi-Gel 10 and Bio-beads SM.2 were from Bio-Rad. CHAPS and bovine serum albumin (fraction V) were from Boehringer Mannheim. Na”“I (IMS 30) was from Amersham Corp. All other reagents were of analytical grade.

Cell Culture-Primary culture of skeletal myogenic cells was car- ried out using 3-S-day-old neonatal rabbit muscles. Muscles from the hind limbs and backs from two to four animals were removed asep- tically free of their skin and paws, minced and rinsed in a Ca’+/Mg’+- free solution containing 8 mM NaH,P04, 22.6 mM NaHCO.1, 116 mM NaCl, 5.3 mM KCl, 5.5 mM glucose, and 10 mg/ml phenol red, pH 7.4, prewarmed at 37 “C. The tissue was dissociated in this buffer in the presence of 1% (w/v) trypsin (Seromed) with continuous stirring for 20 min at 37 “C. Cells were collected after serial trypsinizations until all the tissue was dispersed. The suspensions were pooled and centrifuged for 10 min at 800 X g. The resulting pellet was resus- pended in growth medium composed of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Gibco), 10% horse serum (Gibco), and antibiotics (200 units/ml penicillin, 50 pg/ml streptomycin). Cells were then filtered through sterile nylon gauze (diameter 80 pm), plated in the growth medium at a density of 2 X 10” cells in 140-mm diameter Petri dish (Nunc tissue culture dish) and incubated in a humidified 37 “C incubator with a 5% CO, at.mos-

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Receptors for Toxic Phospholipase A2 in Skeletal Muscle

phere. Under these conditions, myoblasts proliferate and the cultures reach confluency within a few days (4-7 days). Cultures were fed with fresh medium every 2-3 days. At confluency, the growth medium was replaced by the differentiation medium (Dulbecco’s modified Eagle’s medium supplemented with 2% fetal calf serum and 3% horse serum). Under these new conditions, myoblasts differentiate rapidly into multinucleated myotubes.

Preparation of Rabbit Skeletal Muscle Cell Membranes-Cells grown on 140-mm plates were washed twice with 25 ml of 140 mM NaCl, 5 mM KCl, 2 mM CaC12, 20 mM Tris, pH 7.4. All the following manipulations were carried out at 4 “C. The cells were scraped from the plate with a rubber policeman in a buffer consisting of 20 mM Tris, pH 7.4,1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride. The cell suspension was homogenized with a cell disrupter (Vibra cell 72434, Bioblock Scientific) set at a potency of 5 watts. The resultant homogenate was centrifuged 5 min at 1,000 x g. The supernatant was centrifuged at 100,000 x g for 30 min and the pellet obtained was resuspended at a concentration near to 10 mg of protein/ml and kept frozen at -70 “C. Protein concentrations were determined according to Bradford (11) after digestion of membranes in 0.1 N NaOH and using bovine serum albumin as a standard.

Iodination of ‘2”I-OS, and ““I-O&-These two derivatives were obtained as previously described for ‘*“I-OS2 (8). Retention times for OS1 and ““I-OS1 on the TSK SP-5PW analytical column under the conditions described for I” I-OS, preparation were 300 and 340 mM ammonium acetate, respectively. The specific radioactivity obtained was 3000-4000 dpm/fmol for both toxins.

Binding Studies-All binding experiments were performed at 20 “C in 140 mM NaCl. 0.1 mM CaCl,. 20 mM Tris, pH 7.4, and 0.1% bovine serum albumin (buffer 1). Preliminary kinetic experiments indicated that, in the binding conditions used in this work, the plateau for association of both ““I-OS, and “‘I-OS, to rabbit skeletal muscle cell membranes was reached after 90 min and was stable for at least 2 h. Therefore, membranes were incubated with radiolabeled ligands for 90 min before filtration (8). Except when specified, in all binding experiments, the incubation volumes were 0.3 ml for ““I-OS, and 2 ml for ““I-OS, assays and the filtration volumes were 0.25 and 1.8 ml, respectively. Dilution of unlabeled toxins assayed were done in buffer 1.

Cross-linking Experiments of ““Z-OS, and ““I-OS, to Rabbit Brain Synaptosomes and Myotube Membranes-Using ““I-OS2 as ligand, cross-linking experiments were achieved either under stoichiometric conditions, i.e. at a receptor concentration of 0.5 nM which is very high as compared to the Kd value for the formation of the complex toxin-recept.or, or under equilibrium conditions with a receptor con- centration of 5.4 PM. Under stoichiometric conditions, membranes (0.3 mg of protein/ml) were incubated in 1 ml of buffer 1 with 0.3 nM ““I-OS, in the absence or presence of 30 nM OS,. After 1 h at 20 “C, incubation mixtures were centrifuged at 12,000 X g for 10 min and resuspended in 1 ml of 20 mM Hepes, pH 8.2, 140 mM NaCl, 0.1 mM CaCls. Under equilibrium conditions, the amount of membranes into the incubation mixture and the ““I-OS, concentration were 3 pg of protein/ml and 8 pM, respectively. The volume of the reaction was increased to 10 ml and the protection was achieved with increasing concentrations of unlabeled OS,. After centrifugation and resuspen- sion of membranes, reactions with the cross-linking agent were iden- tical for both conditions. Following addition of 50 uM suberic acid bis-N-hydroxysuccinimide ester (DSS, dissolved in 10 ~1 of dimethyl sulfoxide) for an incubation time of 5 min at 20 “C, the reactions were stopped by addition of 1 M Tris, pH 8.0 (0.1 M final) and centrifugation for 10 min at 12,000 x g. The resulting pellets were solubilized with a SDS sample buffer (12) under reducing conditions (4% 8-mercaptoethanol) and analyzed by SDS-polvacrylamide gel electrophoresis (12). Gels were stained with Coomassie Brilliant Blue, dried, and autoradioarauhed at -70 “C using Kodak (X-Omat AR) film and intensifying-screen Du Pont-Crone; Hi-plus. ‘Cross-linking experiments of ““I-OS1 to myotube membranes and of ““I-OS to rabbit brain synaptosomes were performed exactly as described for ““I-OS, under stoichiometric conditions.

Coupling of OS, to Affi-Gel 1 O-6 ml of N-hydroxysuccinimide ester of cross-linked succinylaminoalkyl Bio-Gel (Affi-Gel 10) were incu- bated overnight, at 4 “C under agitation with 9 ml of 0.1 M Mops, pH 7.6,80 mM CaCl,! containing 2 mg of OSJml. The gel was then briefly centrifuged and the optical density (A,,) of the supernatant (adjusted to pH 2.0) was measured to determine the amount of toxin coupled (generally more than 95%). Any free reactive N-hydroxysuccinimide groups remaining were reacted with 7 ml of 1 M Tris, pH 8.0, at 4 “C for 1 h. The resin was washed sequentially on a sintered glass funnel

with 50 ml of 140 mM NaCl, 2% Triton X-100, 20 mM Tris, pH 7.4, and 50 ml of 140 mM NaCl, 6 M urea, 0.1% CHAPS, 20 mM Tris, pH 7.4. The OS,-Affi-Gel 10 was resuspended in storage buffer (140 mM NaCl, 1 mM CaCl*, 0.02% NaNa, 0.1% CHAPS, 20 mM Tris, pH 7.4).

Preparation of Detergent Extract-A soluble extract was prepared from 500 ma of membrane protein corresponding to about 800 pmol of i2’I-OS, receptors. Membranes (2 mg of protein/ml) were incubated with 1.5% (w/v) of CHAPS in 20 mM Tris/Cl. pH 7.4.140 mM NaCl, 1 mM EDTA and a mixture of protease inhibitors: 6.1 mM phenyl- methylsulfonyl fluoride, 1 mM iodoacetamide, 1 kg/ml soybean tryp- sin inhibitor, 1 FM pepstatin A for 30 min at 4 “C under continuous agitation. The solution was centrifuged at 100,000 X g for 30 min. The supernatant thus obtained is referred to as detergent extract. It contained 75-85% of the myotube membrane proteins.

Chromatography on WGA-Affi-Gel IO-The detergent extract (250 ml) was diluted 2-fold in 20 mM Tris/Cl, pH 7.4, 140 mM NaCl, 1 mM CaC12, in the presence of phenylmethylsulfonyl fluoride, iodoaceta- mide, soybean trypsin inhibitor and pepstatin A and incubated batch- wise for 4 h at 4 “C under continuous agitation with 30 ml of wheat germ agglutinin-Affi-Gel 10 (WGA-Affi-Gel 10, 7 mg of WGA/ml of gel) equilibrated with the above buffer. The gel was first washed on a sintered glass funnel with 300 ml of 20 mM Tris/Cl, pH 7.4, 500 mM NaCl, 1 mM CaClp, 0.1% CHAPS and then with 60 ml of 20 mM Tris/Cl, pH 7.4, 140 mM NaCl, 1 mM CaC12, 0.3% CHAPS. The gel was finally poured into a column (1.5 x 20 cm) and eluted with a buffer containing 20 mM Tris/Cl, pH 7.4,140 mM NaCl, 1 mM CaCl,, 0.1% CHAPS, and 200 mM N-acetylglucosamine at a flow rate of 0.5 ml/min. The bound proteins (about 2-5% of the detergent extract) were eluted with approximately 5 column volumes.

Chromatography on O&-Affi-Gel IO-The WGA-Affi-Gel 10 eluate (140 ml) was directly incubated batchwise with 6 ml of OS,-Affi-Gel 10, previously equilibrated in 140 mM NaCl, 1 mM CaCl*, 0.1% CHAPS, 20 mM Tris, pH 7.4, for 15 h at 4 “C. The gel was extensively washed on a sintered glass funnel with 300 ml of equilibration buffer and then poured onto a column (1 X 8 cm). The OS,-Affi-Gel 10 column was washed again with 20 ml of 1% deionized Triton X-100, 1 mM EDTA, 20 mM Tris, pH 7.4, and finally eluted with 25 ml of 0.1% Triton X-100, 1 mM EDTA, 50 mM NaCl, 50 mM MES, pH 4.9. Protein content of affinity purified receptor samples was routinely assayed by comparison to known amounts of standard proteins (Bio- Rad) on SDS-polyacrylamide gels that were silver-stained (13) and scanned with a laser densitometer (Ultroscan XL, LKB).

Reconstitution of 0%Affi-Gel IO Eluate into Livosomes and Assays of Radioligand B&ding-The O&-Affi-Gel 10 eluate was mixed with a solution containing 3.4% deionized Triton X-100, 0.75 M Tris, pH 8.0, and 52 mg/ml L-a-phosphatidylcholine with a ratio of 5 volumes of eluate for 1 volume of phospholipids solution. For the formation of proteoliposomes, the detergent was removed by incubation of the above mixture with Bio-beads SM.2 (150 mg/ml) for 1 h at 4 “C! (this procedure was repeated one time to ensure complete removal of detergent which is known to inhibit binding at very low concentra- tions). For ‘““I-OS, binding assays, 10 ~1 of proteoliposomes were added to 990 ~1 of buffer 1 (with 1 mM CaC12) containing increasing concentrations of ““I-OS, in the absence or the presence of 10 nM unlabeled OS,. Samples were incubated for 90 min at 20 “C. Incuba- tions were stopped by a rapid filtration procedure through GF/C glass fiber filters (Whatman) presoaked in 0.5% polyetheleneimine, 10 mM Tris, pH 7.4, followed by two washes with 5 ml of 140 mM NaCl, 20 mM Tris, pH 7.4. As a control, binding experiments were also carried out with liposomes formed in the absence of purified receptor.

RESULTS

Specific Binding of ‘25I-OS2 and ‘251-OS1 to Rabbit Myotubes in Culture-A crude membrane preparation of differentiated myotubes was used to demonstrate the presence of ‘251-OSz binding sites in cultured rabbit skeletal muscle cells. Fig. 1A shows results obtained from a typical direct binding experi- ment. Only one family of binding sites was found with an equilibrium binding constant (&) value of 7 pM and a maxi- mum binding capacity (B,,,) of 2.4 pmol/mg of protein. To confirm the absence of an eventual low affinity binding site on myotubes, the same kind of direct binding experiments were carried out with higher labeled ligand concentrations (up to 5 nM). No difference was seen in the Scatchard plot in these conditions (not shown). From 10 independent similar

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9528 Receptors for Toxic Phospholipase A2 in Skeletal Muscle

FIG. 1. Equilibrium binding of ‘*61-OSz to rabbit myotube membranes. A, main panel, membranes (1.8 fig of protein/ml) were incubated with increasing concentrations of ‘*51-OSz in the absence (0) or in the presence (0) of 2 nM unlabeled OSz. Specific binding (0) represents the difference between total (0) and nonspecific bind- ing (Cl). Inset, Scatchard plot of the specific binding (0). B, bound ““I-OS, in pmol/mg of protein; F, free ‘251-OS2 in nM. B, competition experiments with ““I-OS, and different unlabeled PLA, for the bind- ing to myotube membranes. Membranes (2 pg of protein/ml) were incubated with 4.5 pM ““I-OS, and various concentrations of unla- beled OS1 (W), OSz (Cl), taipoxin (O), crotoxin AB (O), P-bungarotoxin (C), and bee venom PLA2 (+). Results are expressed as percentage of the maximal specific binding measured in the absence of competitor. 100% corresponded to 1.4 PM of “’ I-OS, specifically bound. Nonspe- cific binding was measured in the presence of 2 nM unlabeled OSz and accounted for 12% of the total binding.

binding experiments, the mean values for the ‘251-OS, binding parameters to myotube membranes were Kd = 7.4 f 0.9 pM and B,,, = 1.9 f 0.2 pmol/mg of protein.

The relative inhibitory effects of various concentrations of unlabeled OS, on ‘2”I-OS2 equilibrium binding to myotubes are illustrated in Fig. 1B. The concentration of OSz which inhibits half of the 1251-OS2 specific binding (K& was 10 pM corresponding to a Kd of 6.9 pM. Other toxic phospholipases also inhibited ‘251-OS2 specific binding to myotubes. Inhibition curves are shown in Fig. 1B for OS, taipoxin, crotoxin, p- bungarotoxin, and bee venom PLAz. K0.5 values for 12 differ- ent phospholipases are listed in Table I and compared with corresponding values obtained in similar experiments per- formed on rabbit brain synaptosomes. It clearly appears that pharmacological profiles in muscle and brain membranes are different. Two phospholipases are of special interest. OS1 is a very potent inhibitor of ‘251-OS, binding to myotubes but it is a very poor one on brain. The bee venom phospholipase is an example of the reverse situation with a good affinity for brain membranes and a very bad one for myotubes.

In order to study in more details the binding properties of OS1 to myotube membranes, this toxin was iodinated to a high specific radioactivity to perform direct binding experi- ments. Results of one of these experiments are shown in Fig. 2A. The Scatchard plot (Fig. 2A, inset) again indicates the presence of one family of binding sites. The Kd value for the interaction of ‘25I-OS1 with its myotube receptor is 38 pM and the B,,, value is 2.2 pmol/mg of protein. This B,,, value is

TABLE I Inhibition of ‘25I-OS, specific binding to rabbit brain synaptosomes

and myotube membranes by some PLAz For inhibition of Y-OS, specific binding to myotube membranes,

experimental conditions are described in Fig. 1B. Competition exper- iments on rabbit brain synaptosomes were determined by incubating the membranes (4 pg of protein/ml) in 2 ml of buffer 1 in the presence OftipM ‘*“I-OS, and various concentrations of unlabeled PLA, for 1 h at 20 “C. The specific binding obtained was 1.4 pM whereas non- specific binding accounted for 10% of total binding. Equilibrium binding experiments performed on that rabbit brain synaptosomes preparation indicated the presence of two families of binding sites with the following parameters: Kdl = 1.1 PM/B,,,~.~ = 0.31 pmol/mg of protein, K&2 = 50 PM/B,,,~.~ = 1.17 pmol/mg of protein. These Y- OS, binding characteristics were very similar to those found on rat brain synaptic membranes. Ko.5 value is the PLA? concentration which inhibit 50% of the maximum inhibition observed.

PLA, KO5

Brain synap- Myotube tosomes membranes

0s 0s OS.3 0s Taipoxin Pseudexin Notexin Textilotoxin N. mossambica mossambica P-Bungarotoxin Crotoxin

-Component A -Component B

c. atrox Bee venom

PM

1,500,000 34

6 10 48 40 28 20 68 180 18 700

190 10,000 74 700

115 >300,000

320,000 320,000 9 36,000

>>3,000,000 x=-3,000,000 30 40,000

20,800 22,000 80 x=-300,000

not significantly different from the one found with 1’5I-OSz on the same preparation. No binding sites for iz51-OS1 were detected in brain synaptosomes (not shown). Unlabeled OS1 and OSz inhibit ‘251-OS1 specific binding to myotube mem- branes with Ko.5 values of 88 pM (corresponding to a Kd of 21 PM, after correction for the experimental conditions) and 10 pM, respectively (Fig. 2B). The bee venom phospholipase has no significant effect on the specific binding of ‘251-OS1 to myotube membranes.

The Phospholipase Receptor in Rabbit Skeletal Muscle Cells at Different Stages of in Vitro Development-The 1251-OS2 receptor is present in the early undifferentiated myoblasts 24 h after plating, at a culture time where cells are not confluent and are actively growing. However, the amount of receptor sites at this stage is only 0.3 f 0.1 (n = 4) pmol/mg of protein as compared to 1.9 f 0.2 (n = 10) pmol/mg of protein on fully differentiated cells, 14 days after the shift to the differentia- tion medium (Fig. 3). Under our culture conditions, the fusion of myoblasts into myotubes is a process which takes place between days 1 and 4 of culture in the differentiation medium and reaches a maximum extent of 4050%. The time course of increase of ‘251-OSz binding sites density in these cells is shown in Fig. 3. It does not follow the onset of fusion, the maximum level being reached only after 13-14 days. All along this period, the equilibrium dissociation constant for the interaction ‘251-OS2 receptor remains at a stable value of 5.3 + 0.1 pM (Fig. 3).

Affinity Labeling of the “51-0S2 Binding Protein in Myotube Membranes-The bifunctional reagent DSS was used to cross-link ‘251-OS2 to its receptor. Preliminary experiments have shown that a DSS concentration as low as 50 pM is sufficient to obtain a specific covalent labeling of the receptor

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Receptors for Toxic Phospholipase AS in Skeletal Muscle

1 2 3456 Mr

-0 100 200 300 125I-OS1 free, pM

log WA21 , M

Frc. 2. Equilibrium binding of ‘““I-OS, to rabbit myotube membranes. A, main panel, membranes (5 pg of protein/ml) were incubated with increasing concentrations of ‘“I-OS, in the absence (0, total binding) or in the presence (0, nonspecific binding) of 30 nM unlabeled OS,. Inset. Scatchard nlot of the specific binding (0). H. hound ““I-OS,. in pmol/mg of protein; F, free ““I-OS, in ni; g, competition experiments involving ““I-OS, and unlabeled PLA, for binding to myotuhe membranes. Membranes (5 pg of protein/ml) were incubated in the presence of 130 pM ““I-OS, and various concentrations of OS, (H), OS, (Cl), or bee venom PLA? (+). Results are expressed as the percentage of the maximal specific binding (8.5 PM). Nonspecific binding was measured in the presence of 30 nM unlabeled OS, and represented 20% of the total binding.

IO 20 days of culture

FIG. 3. Appearance of ‘251-OS4 binding sites during the in vitro differentiation of rabbit skeletal muscle cells. Cells were grown in the proliferation medium up to confluency which was reached at day 5 of the culture. Differentiation was then triggered by shifting the cells to the differentiation medium (arrow). The density of “‘I-OS2 binding sites (0) and the dissociation constant of the toxin-receptor complex (W) were measured on cell homogenates pre- pared at varying ages of the culture. The binding conditions were as described in Fig. 1A.

without appreciable modification of the protein gel pattern (not shown). Fig. 4 shows results of an affinity labeling experiment performed under stoichiometric conditions, i.e. a toxin receptor concentration very high (0.5 nM) in front of the Kd of the interaction (7 PM). These conditions are made possible because of the very high affinity of the toxins to their receptor. Only one band was labeled with an apparent M, of 180,000 after correction assuming that one ““I-OS, molecule (M, 14,000) is bound per molecule of receptor. The amount of

9529

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I

(x10 -3)

200

116

96.5

66.2

45

FIG. 4. Comparison of autoradiogram patterns of SDS- polyacrylamide gels of rabbit brain synaptosomes and my- otube membranes labeled under stoichiometric conditions with ‘““I-OSI and ‘““I-OSa. Lanes I and 2, rabbit brain membranes labeled with ““I-OS,. Lanes 3-6. mvotube membranes labeled with “‘I-OSr (lanes 3 and-4) or “‘I-OS, (&es 5 and 6). In lanes 2, 4, and 6, the cross-linking experiments were done in the presence of 30 nM of the homologous unlabeled toxin (protected samples). 100 pg of protein were loaded in each track. The position of the M, markers are indicated (myosin, 200,000; p-galactosidase, 116,000; phosphoryl- ase b, 96,500; bovine serum albumin, 66,200; ovalhumin, 45,000; carbonic anhydrase, 31,000; soybean trypsin inhibitor, 21,500; cyto- chrome c, 14,400 from Bio-Rad). Gels (4-14%) were exposed on Kodak X-Omat AR films for 48 h (rabbit brain) or 13 h (myotuhe mem- branes).

counts/min specifically incorporated into this band was de- termined by counting the sliced band detected by autoradi- ography and the band at the corresponding position in the protection lane. It indicated that 20 fmol of toxin/100 pg were covalently incorporated corresponding to 22% of the total amount of ‘““I-OS2 specifically bound to the receptor. The cross-linking efficiency is therefore very high and it is unlikely that the labeling could be of a nonspecific nature. When SDS- polyacrylamide gel was carried out under nonreducing condi- tions, the same component was labeled. The labeling was specific since it was totally protected when the reaction was done in the presence of unlabeled OS, (Fig. 4). The protein of M, 180,000 was also specifically labeled by photoaffinity using an ““I-azido derivative of OS, (8) (not shown). Finally, the same labeling was also obtained in ““I-OS1 cross-linking experiments to myotube membranes (Fig. 4). Fig. 4 also compares the ““I-OS, labeling profile obtained on myotube membranes with the one found on rabbit brain synaptosomes. It is clear that the two patterns are very different. As previ- ously found with rat brain membranes (8), a complex labeling was found with rabbit brain membranes with M, of 45,000, 37,000, and 33,000 for the most labeled components (after subtraction of the OS, molecular weight contribution). In addition, in order to further demonstrate that the protein of M, 180,000 is indeed the high affinity binding component of ‘““I-O& the cross-linking experiments was also performed under equilibrium conditions. Fig. 5A shows that in these conditions the same band is labeled as under stoichiometric conditions and that incorporation of the label can be inhibited in a dose-dependent manner by nonradioactive OS,. Quanti- fication of the ‘““I-toxin specifically bound was achieved by counting the sliced bands. It allows a calculation of a K0.5 of 34 pM for unlabeled OS corresponding to a Kd value of 17 pM

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origin *

180 -)

q Mr (x10-3)

l

200 116 96.5 66.2

dye front-c-‘-- I- 31

-log [OS,], M - 12 11.5 11 10.5 10 9.5 9

FIG;. 5. Protection against cross-linking of ““I-OS2 to my- otube membranes by unlabeled OS2 under equilibrium condi- tions. Membranes (3 pg of protein/ml) were incubated with 8 pM “‘I-OS, in the presence of the indicated concentrations of unlabeled OS1. A, autoradiogram patterns of the SDS-polyacrylamide gel (‘i.5%) of myotuhe memhranes labeled with ““I-OS,. 1 pg of protein were loaded in each track. The position of the markers are indicated. Autoradiography for 110 h. The dark labeling at the front of the gel is due to the presence of the noncovalently hound “‘I-OS?. The decrease of intensity of this hand, from left to right, results from the washing steps hefore loading (see “Experimental Procedures”). H, dose-response curve relating the extent of covalent incorporation of ““I-OS, to the concentration of unlabeled OS? used in the protection. Data are determined by counting the sliced bands in each track and are expressed in femtomoles of ““I-OS, covalently bound to the OS, receptor/jq of protein.

after correction for the experimental conditions (Fig. 5B). The very good correlation between the Kd calculated in Fig. 5B and the one measured in Fig. 1 together with the fact that, even with very high toxin and receptor concentrations (stoi- chiometric conditions, Fig. 4, lane 3), the only specifically labeled band is the one of M, 180,000, clearly indicate that this polypeptide is part of the phospholipase A, binding protein in rabbit skeletal muscle cells.

Purification of the ““I-OS, Binding Protein from Myotube Membranes-Different detergents were assayed in order to solubilize the ““I-OS, binding sites from myotube membranes. All the binding activity was lost upon treatment of membranes with Triton X-100, Lubrol, Nonidet P-40, or deoxycholate. A very small binding activity was retained when membranes were solubilized with digitonin or cholate. The best results were obtained with CHAPS as solubilizing agent, even though the binding activity of the CHAPS extract was lower than the one present in the starting membranes. ““I-OS2 binding pa- rameters to the CHAPS-solubilized receptor were not deter- mined for the following reasons: (i) because of the unstability of the solubilized receptor at 20 “C, binding experiments should have to be done at 4 “C, a temperature at which the association of the toxin to the receptor is very slow even on membranes; (ii) considerable technical problems to reproduc- ibly separate the unbound from the bound ligand were en- countered. The large size of the toxin prevented the use of filtration techniques using small sieve chromatography col- umns and the presence of CHAPS in the medium results in a high nonspecific binding of ‘““I-OS, to polyethyleneimine- treated filters themselves. Others methods such as polyeth-

ylene glycol precipitation or ion-exchange chromatography were found to he inadequate.

Initial experiments with photoaffinity-labeled, solubilized ““I-OS, receptor showed that it was retained on a wheat germ agglutinin column. Approximately 80% of the receptor loaded was bound to the column while about 95% of the proteins were washed out of the column. The specific elution with 0.2 M N-acetylglucosamine resulted in the recovery of 30-50% of the labeled receptor giving rise to a purification factor of about 10 for this purification step. Therefore, this WGA affinity chromatography was chosen as the first step of the purification of the myotube OSr receptor.

The second and final purification step involved a specific affinity chromatography on a O&Affi-Gel 10 matrix. The final OS? content of the gel matrix was close to 3 mg of OSJ ml of gel, i.e. a toxin concentration high enough to retain the receptor protein, even if it was in a lower affinity state for the toxin which seems to be the case for the solubilized receptor. After extensive washing of the gel, the elution of the bound receptor was obtained by lowering the pH value to 4.9. At this pH value, it was previously shown that the association of the toxin to its receptor site was totally abolished, even in the membrane-bound form of the receptor. The protein pattern obtained on silver-stained SDS-polyacrylamide gel (13) at each purification step is shown on Fig. 6. The WGA eluate has a protein composition considerably simpler than that of the starting material but still complex. However, only one band with a M, of 180,000 was revealed by silver staining of the OSr column eluate. In order to demonstrate the specificity of the OS,-Affi-Gel 10 purification step, protection experi- ments were carried out. The WGA eluate was first incubated with 2 pM OSr for 2 h and then incubated with the OS,-Affi- Gel 10 matrix. In these conditions, absolutely no protein was detected on the silver-stained SDS-polyacrylamide gel of the O$Affi-Gel 10 column eluate.

Immediately after elution from the OS, affinity column, the pH of the eluting solution was readjusted to 7.4 and the purified receptor was reconstituted into proteoliposomes by

Mr 1 2 3 4 S 6 7

(x10-3 ) + origin

200 - 1 + 180

116 --

96.5 -

+ dye front

Frc. 6. Purification of the ‘““I-OS2 receptor from rabbit skeletal muscle myotubes. Aliquots of the fractions obtained at each purification step were loaded on a 6.5% SDS-polyacrylamide gel which was silver-stained. Lane 1, molecular weight markers (myosin, 200,000; /Cgalactosidase, 116,000; phosphorylase b, 96,500; bovine serum alhumin, 66,200; ovalhumin, 45,000; 30 ng of each); lane 2. detergent extract, 4.6 g of prot.ein; lane 3, breakthrough of WGA- Aff’i-Gel 10, 4.1 pg; lane 4, WGA-Affi-Gel 10 eluate, 0.56 pg; lane 5. O$Aff’-Gel 10 breakthrough, 0.56 ~(p; lane 6, OS,-Affi-Gel 10 Triton X-100 washing, 150 ~1 out of 20 ml; lane 7, O&Affi-Gel 10 eluate, 150 ~1 out of 25 ml (see “Experimental Procedures”). Note that the predominant polypeptide components of high M, seen in lanes 2-5 (i) are not retained on WGA and OS?-Affi-Gel 10 columns, (ii) have a mobility significantly higher than that of the OS? binding protein (lane 7). It is therefore clear that these proteins are not related to the purified OS, binding protein.

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Receptors for Toxic Phospholipase A2 in Skeletal Muscle 9531

200 400 125I-OS2 free, pM

FIG. 7. Equilibrium binding of ‘261-OSz to purified and re- constituted OS2 receptor from rabbit skeletal muscle myo- tubes. Mainpanel, proteoliposomes (0,O) or receptor-free liposomes (V, v) (10 &ml, see “Experimental Procedures”) were incubated with increasing concentrations of 12”I-OS2 in absence (0, V) or pres- ence (0, v) of 10 nM unlabeled OS,. Specific binding (H) was the difference between total (0) and nonspecific binding (0). Inset, Scatchard plot of the specific binding. The concentrations indicated in the inset correspond to the ‘2”I-OS2 bound in the original purified and reconstituted receptor preparation after correction for the dilu- tion in the incubation medium.

the Bio-beads technique. This reconstitution procedure re- sulted in the obtention of a pure receptor able to specifically bind the 1251-OSZ toxin with a Kd of 33 PM, close to the value found on the myotube membranes, and a B,., value of 0.42 nM (Fig. 7). Control liposomes formed in the absence of purified receptor were devoid of any 1251-OSZ specific binding (Fig. 7). The quantification of the protein concentration in the purified soluble or reconstituted sample was not possible because of the lack of sensitivity of all the techniques avail- able. This protein concentration is probably lower than 1 pg of protein/ml since this value is in our hands the lower limit for the very sensitive assay described by Peterson (14). There- fore, the protein content in the eluate was estimated by densitometric scanning of polyacrylamide gels loaded with different volume of the eluates. By this semiquantitative method, a concentration of 0.7 pg of protein/ml was estimated for the purified extract shown in Fig. 6, lane 7, and of 0.35 pg of protein/ml for the reconstituted eluate (not shown). Hence, a specific activity of approximately 1.2 nmol of OSZ receptor/ mg of protein was calculated for the purified, reconstituted receptor.

DISCUSSION

The highly radiolabeled derivative of OS, a toxic phospho- lipase AP isolated from the venom of Taipan (8) has allowed a detailed characterization of iz51-OS receptors in rabbit skeletal muscle cells in culture. There are marked differences between results described above for ‘*“I-OSZ binding to my- otube membranes and those previously obtained for ‘251-OS2 binding to rat brain synaptosomes (8). (i) Only one family of 1’51-OS, binding sites is found in myotube membranes (Kd = 7.4 PM, B,., = 1.9 pmol/mg of protein) while two families of ‘251-OSZ binding sites were found in rat brain membranes (Kdl = 1.5 PM, &ax1 = 1.0 pmol/mg of protein, Kd2 = 45 PM, Bmaxl = 3.0 pmol/mg of protein) (8) and also in rabbit brain syn- aptosomes (see legend to Table I). (ii) Different unlabeled PLAzs have different efficacies to inhibit ‘251-OS2 binding to the two tissues (Table I). In this respect, bee venom PLA, and OSl, another PLA, from Taipan venom, seem to be very tissue-specific, the former for neuronal receptors and the latter for muscle receptors. ‘251-OS1 binds perfectly well to myotube membranes with a high specificity. The B,., (2.2 pmol/mg of protein) is very similar to the B,,, values for lZ51-

OSZ and the Kd (38 PM) is in agreement with the Ko.5 of 34 PM found from OS1 inhibition of ‘251-OSZ binding. However, no 1251-OSl binding sites could be detected on rabbit brain synaptosomes and OS1 inhibits only very weakly ‘251-OS~ binding to its neuronal binding sites. (iii) Myotube and neu- ronal PLAz binding sites are composed of different binding proteins as indicated by cross-linking experiments. Very sim- ilar patterns of proteins specifically labeled by ‘25I-OS2 are obtained in rat brain membranes (8) and in rabbit brain membranes (Fig. 4) using DSS as cross-linking agent with three proteins of M, 45,000, 37,000, and 33,000 being most heavily labeled. In the case of muscle membranes, the 1251- OSZ labeling pattern is considerably simpler with a single band of M, 180,000 being revealed. In addition, the same polypeptide chain is labeled with ‘25I-OS1 instead of ‘251-OS2. The apparent mobility of this band in SDS-polyacrylamide gel is independent of the presence of a reducing agent but varies depending upon the polyacrylamide concentration in the gel (from 170,000 in 6% of polyacrylamide to 205,000 in 8%). This mobility variation is probably due to the glycopro- tein nature of the receptor (see below).

The expression of a number of receptors, of enzymes or of ionic channels in muscle cells is often in close relation with the differentiation state of the cells. For example, it is well known that the expression of the nicotinic acetylcholine receptor (15), of the acetylcholineesterase activity (16), of the (Na+,K’)-ATPase activity (17), of the Na+ channels (18) or of the L-type Ca2+ channels (19-21) is not the same in myoblasts and in myotubes.

A marked increase in density of ‘251-OS2 receptors was observed during cell maturation (Fig. 3). Myoblasts have a low number of binding sites. This number is increased three to 5-fold in 20-day-old myotubes. This increase is blocked when cycloheximide (4 pg/ml), a protein synthesis inhibitor, is added to the culture (not shown). The augmentation of iz51- OS2 receptor density does not temporally correlate with the fusion process of myoblasts into myotubes. Fusion is usually maximum at day 10 in our culture conditions while the full expression of ‘251-OS2 receptor needs 10 more days. Moreover, the fusion index, i.e. the percentage of nuclei found in polyn- uclear cells, is somewhat variable from one culture to another, varying from 30 to 70%. No correlation has been observed between the fusion index and the amount of ‘251-OS2 binding sites. Contaminant fibroblasts are in very low amount in our culture conditions. However, it was checked that almost no binding sites were found in cultures very enriched with fibro- blasts.

A simple two-step procedure has been designed for the purification of the myotube PLAz receptor. The first step consists in a chromatography on WGA-Affi-Gel 10 column which removes approximately 95% of the proteins from the solubilized extract but retains 30-50% of the receptor. The retention of the receptor on the lectin column indicates its glycoprotein nature. At this step, the reduction of the CHAPS concentration from 1.5% in the extract to 0.1% in the eluate without any dilution is of crucial importance for the success of the second purification step. This second step makes use of an OS-affinity matrix which binds the receptor only in the presence of low CHAPS concentrations (a too high dilution of the solubilized extract to reduce detergent concentration also results in a loss of the ability of the receptor to bind to the immobilized toxin). The OS,-affinity chromatography provides a highly purified receptor as judged by the SDS-gel analysis. Lowering the pH value of the elution buffer for desorption was prefered to a specific elution with the unla- beled toxin because of the very slow dissociation of OSZ from

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Receptors for Toxic Phospholipase A, in Skeletal Muscle

its receptor which would make it very difficult to remove the unlabeled toxin in view of subsequent binding experiments. The specificity of the affinity chromatography step was dem- onstrated by the fact that unlabeled OSz added to the WGA extract before loading on the OSz column prevented the purification of the OS, binding component. In addition, the M, of 180,000 found for the purified receptor is exactly the same as the M, determined by cross-linking experiments involving the membrane-bound receptor.

Reconstitution of the purified protein into artificial lipo- somes allowed the recovery of the very high affinity ‘251-OS2 binding sites (Fig. 7). The specific binding capacity of the purified reconstituted receptor was 1.2-1.5 nmol/mg of pro- tein, lower than the theoretical value of 5.5 nmol/mg of protein calculated assuming a M, of 180,000 for the receptor protein. Nevertheless, the fact that no other protein than the one with a M, of 180,000 is detected after our purification procedure indicates that the protein is pure. The relatively low specific activity is probably due to different reasons: (i) there is a marked denaturation of the receptor retained on the OS, affinity column due to the pH shift used for its elution. Experiments on the membrane-bound receptor have shown that such a pH shift results in a 40% decrease of the B max value after the pH had been lowered and returned to the initial value of 7.4; (ii) if there is no preferential orientation of the receptor in liposomes, one could expect 50% of the receptor being inside-out and thus unable to bind the toxin; (iii) integration of the receptor into phospholipids could have allowed the recovery of the high affinity state for OSz binding but a proportion of the receptor could have remained in the low affinity state and would not have been detected in our binding experiments; (iv) the estimation of the protein con- tent of the reconstituted material by scanning of silver-stained SDS gels could have been overestimated.

An important question which still remains to be answered concerns the function of this phospholipases Az receptor in skeletal muscle cells. The same question also holds for neu- ronal PLA2 receptors. Previous work with vipoxin, a toxic PLA2 isolated from Russell’s viper venom, has suggested that this toxin could act at biogenic amine receptors (22) and acetylcholine receptors (23). Since P-bungarotoxin, a toxic PLA2, is known to block a family of voltage-dependent K+ channels (24-28), one could also expect that some special classes of K’ channels will be the targets of PLA2s in different tissues. The demonstration of the existence of PLA, receptors in muscle cells will probably help to verify this hypothesis since electrophysiological work is possible on such cells as well as on liposomes containing the pure PLA, receptor. Finally, the availability of a pure receptor preparation will permit to obtain sequence information necessary to clone the cDNA corresponding to the OS* binding protein. Knowledge of its complete sequence deduced from the cDNA sequence could indicate analogies with other important families of receptors or ionic channels.

Acknowledgments-We thank Dr. J. Middlebrook for the gift of notexin pseudexin and textilotoxin, Dr. C. Bon for the gift of crotoxin A and B components, and Dr. H. Schweitz for purifying phospholi- pases A, from A. mellifera, C. atron and N. mossambica mossambica venoms. We thank M. Bordes for a program of fitting and represen- tation of the data. The excellent technical assistance of M.-M. Lar- roque is greatly appreciated and acknowledged, as well as the expert secretarial assistance of C. Roulinat-Bettelheim and the photograph- ical work of F. Aguila.

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Identification and purification of a very high affinity binding protein for toxic

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