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• W2083901291 abstract "One of the high affinity binding proteins for ammodytoxin C, a snake venom presynaptically neurotoxic phospholipase A2, has been purified from porcine cerebral cortex and characterized. After extraction from the membranes, the toxin-binding protein was isolated in a homogenous form using wheat germ lectin-Sepharose, Q-Sepharose, and ammodytoxin-CH-Sepharose chromatography. The specific binding of 125I-ammodytoxin C to the isolated acceptor was inhibited to different extents by some neurotoxic phospholipases A2, ammodytoxins, bee venom phospholipase A2, agkistrodotoxin, and crotoxin; but not by nontoxic phospholipases A2, ammodytin I2, porcine pancreatic phospholipase A2, and human type IIA phospholipase A2; suggesting the significance of the acceptor in the mechanism of phospholipase A2neurotoxicity. The isolated acceptor was identified as calmodulin by tandem mass spectrometry. Since calmodulin is generally considered as an intracellular protein, the identity of this acceptor supports the view that secretory phospholipase A2 neurotoxins have to be internalized to exert their toxic effect. Moreover, since ammodytoxin is known to block synaptic transmission, its interaction with calmodulin as an acceptor may constitute a valuable probe for further investigation of the role of the latter in this Ca2+-regulated process. One of the high affinity binding proteins for ammodytoxin C, a snake venom presynaptically neurotoxic phospholipase A2, has been purified from porcine cerebral cortex and characterized. After extraction from the membranes, the toxin-binding protein was isolated in a homogenous form using wheat germ lectin-Sepharose, Q-Sepharose, and ammodytoxin-CH-Sepharose chromatography. The specific binding of 125I-ammodytoxin C to the isolated acceptor was inhibited to different extents by some neurotoxic phospholipases A2, ammodytoxins, bee venom phospholipase A2, agkistrodotoxin, and crotoxin; but not by nontoxic phospholipases A2, ammodytin I2, porcine pancreatic phospholipase A2, and human type IIA phospholipase A2; suggesting the significance of the acceptor in the mechanism of phospholipase A2neurotoxicity. The isolated acceptor was identified as calmodulin by tandem mass spectrometry. Since calmodulin is generally considered as an intracellular protein, the identity of this acceptor supports the view that secretory phospholipase A2 neurotoxins have to be internalized to exert their toxic effect. Moreover, since ammodytoxin is known to block synaptic transmission, its interaction with calmodulin as an acceptor may constitute a valuable probe for further investigation of the role of the latter in this Ca2+-regulated process. Phospholipases A2(PLA2, 1The abbreviations used are:PLA2phospholipase(s) A2AtnammodytinAtxammodytoxinCaMcalmodulinDSSdisuccinimidyl suberateLCliquid chromatographyMSmass spectrometryOS2Oxyuranus scutellatus PLA2R16R25, and R180, receptors for AtxC in porcine cerebral cortex of 16, 25, and 180 kDa, respectivelysPLA2secretory PLA2PAGEpolyacrylamide gel electrophoresisMES4-morpholineethanesulfonic acidPVDFpolyvinylidene difluoride EC3.1.1.4) form an expanding superfamily of enzymes, which catalyze hydrolysis of the ester bond at the sn-2 position of 1,2-diacyl-sn-3-phosphoglycerides. Intracellular and secretory PLA2s (sPLA2s) are currently classified under 12 structurally different groups (1Dennis E.A. Trends Biol. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 2Gelb M.H. Valentin E. Ghomashchi F. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 39823-39826Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Secretory PLA2s are enzymes of 13–18 kDa containing five to eight disulfide bonds. They show much higher affinity for aggregated substrates (interfacial catalysis), and millimolar Ca2+ is essential for their catalytic activity (reviewed in Ref. 3Scott D.L. Kini R.M. Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism. John Wiley & Sons, Chichester, UK1997: 97-128Google Scholar). Secretory PLA2s have been associated with many physiological and pathophysiological processes, in certain cases not only the enzymatic activity of sPLA2 but also its interaction with a specific target protein is required (reviewed in Refs. 4Hanasaki K. Arita H. Arch. Biochem. Biophys. 1999; 372: 215-223Crossref PubMed Scopus (69) Google Scholar, 5Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 6Valentin E. Lambeau G. Biochemie. 2000; 82: 815-831Crossref PubMed Scopus (125) Google Scholar). Different membrane and soluble proteins have been identified as selective and high affinity acceptors for sPLA2s. Secretory PLA2s have been shown to bind to voltage-dependent K+channels (7Scott V.E.S. Parcej D.N. Keen J.N. Findlay J.B.C. Dolly J.O. J. Biol. Chem. 1990; 265: 20094-20097Abstract Full Text PDF PubMed Google Scholar), pentraxins (8Schlimgen A.K. Helms J.A. Vogel H. Perin M.S. Neuron. 1995; 14: 519-526Abstract Full Text PDF PubMed Scopus (142) Google Scholar, 9Dodds D.C. Omeis I.A. Cushman S.J. Helms J.A. Perin M.S. J. Biol. Chem. 1997; 272: 21488-21494Crossref PubMed Scopus (132) Google Scholar), reticulocalbins (10Dodds D. Schlimgen A.K. Lu S.-Y. Perin M.S. J. Neurochem. 1995; 64: 2339-2344Crossref PubMed Scopus (46) Google Scholar, 11Hseu M.J. Yen C.-Y. Tzeng M.-C. FEBS Lett. 1999; 445: 440-444Crossref PubMed Scopus (47) Google Scholar), C-type multilectins (12Lambeau G. Schmid-Alliana A. Lazdunski M. Barhanin J. J. Biol. Chem. 1990; 265: 9526-9532Abstract Full Text PDF PubMed Google Scholar, 13Lambeau G. Ancian P. Barhanin J. Lazdunski M. J. Biol. Chem. 1994; 269: 1575-1578Abstract Full Text PDF PubMed Google Scholar, 14Fisher A.B. Dodia C. Chander A. Beers M.F. Bates S.R. Biochim. Biophys. Acta. 1994; 1211: 256-262Crossref PubMed Scopus (30) Google Scholar), factor Xa (15Mounier C. Hackeng T.M. Schaeffer C. Faure G. Bon C. Griffin J.H. J. Biol. Chem. 1998; 273: 23764-23772Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and proteoglycan glypican (16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Inhibitors for sPLA2s, which belong to three distinct structural types, C-type lectins, three-finger proteins, and proteins containing leucine-rich repeats, have also been identified in sera of various animals (reviewed in Ref. 5Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). The increasing number of endogenous sPLA2s identified in mammals and the versatility of their acceptors suggest that many biological roles for the different sPLA2s are yet to be discovered. phospholipase(s) A2 ammodytin ammodytoxin calmodulin disuccinimidyl suberate liquid chromatography mass spectrometry Oxyuranus scutellatus PLA2 R25, and R180, receptors for AtxC in porcine cerebral cortex of 16, 25, and 180 kDa, respectively secretory PLA2 polyacrylamide gel electrophoresis 4-morpholineethanesulfonic acid polyvinylidene difluoride The inhibition of neurotransmission by some sPLA2s from snake venoms has also been found to depend on the interaction of toxic sPLA2 with specific receptor(s) in the nerve terminal of the victim (reviewed in Ref. 17Kriz̆aj I. Gubenšek F. Biochemie. 2000; 82: 807-814Crossref PubMed Scopus (50) Google Scholar). Despite numerous studies, the molecular basis of this process is still largely unknown. To learn more about the molecular mechanism of PLA2 neurotoxicity and about the physiological processes that are affected by these toxins, we have used the presynaptically neurotoxic group IIA PLA2ammodytoxin C (AtxC) from Vipera ammodytes ammodytes venom (18Gubenšek F. Ritonja A. Zupan J. Turk V. Period. Biol. 1980; 82: 443-447Google Scholar, 19Kriz̆aj I. Turk D. Ritonja A. Gubenšek F. Biochim. Biophys. Acta. 1989; 999: 198-202Crossref PubMed Scopus (62) Google Scholar). Two high affinity binding proteins for AtxC have been detected in porcine cerebral cortex, which are potentially implicated in the neurotoxicity of this PLA2 (20Vučemilo N. C̆opic̆ A. Gubenšek F. Kriz̆aj I. Biochem. Biophys. Res. Commun. 1998; 251: 209-212Crossref PubMed Scopus (35) Google Scholar, 21C̆opic̆ A. Vučemilo N. Gubenšek F. Kriz̆aj I. J. Biol. Chem. 1999; 274: 26315-26320Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and the purification and characterization of the high molecular mass AtxC-binding protein has been described previously (21C̆opic̆ A. Vučemilo N. Gubenšek F. Kriz̆aj I. J. Biol. Chem. 1999; 274: 26315-26320Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In this communication we report the purification of the other, 16-kDa, AtxC-binding protein (R16). This protein is identified as calmodulin (CaM), a very important and highly conserved EF-hand Ca2+-binding protein that participates in signaling pathways that regulate many physiological processes (reviewed in Ref.22Chin D. Means A.R. Trends Cell Biol. 2000; 10: 322-328Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). Ammodytoxins, ammodytin I2(AtnI2) and AtnL, were purified from V. ammodytes ammodytes venom as described previously (18Gubenšek F. Ritonja A. Zupan J. Turk V. Period. Biol. 1980; 82: 443-447Google Scholar, 23Kriz̆aj I. Liang N.-S. Pungerčar J. S̆trukelj B. Ritonja A. Gubenšek F. Eur. J. Biochem. 1992; 204: 1057-1062Crossref PubMed Scopus (43) Google Scholar). Crotoxin (from Crotalus durissus terrificus) and agkistrodotoxin (from Agkistrodon blomhoffii brevicaudus) were gifts from Dr. Cassian Bon, Institut Pasteur, Paris, France. OS2(from Oxyuranus scutellatus scutellatus) was a gift from Dr. Gerard Lambeau, Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France. Taipoxin (O. scutellatus scutellatus) and β-bungarotoxin (Bungarus multicinctus) were from Sigma. Porcine pancreatic PLA2, bee venom PLA2, hog brain CaM, and Triton X-100 were from Roche Molecular Biochemicals. Mouse monoclonal anti-CaM antibodies were from Upstate Biotechnology. Na125I (carrier-free) was from PerkinElmer Life Sciences. Disuccinimidyl suberate (DSS) was from Pierce. Affi-Gel 10 and protein molecular mass standards were from Bio-Rad. Q-Sepharose and wheat germ lectin-Sepharose 6MB were from Amersham Pharmacia Biotech. All other reagents and chemicals were of analytical grade. AtxC was radioiodinated as described previously (24Kriz̆aj I. Dolly J.O. Gubenšek F. Biochemistry. 1994; 33: 13938-13945Crossref PubMed Scopus (46) Google Scholar) to specific radioactivity around 300 Ci/mmol. 125I-AtxC was identical to the native AtxC in enzymatic, neurotoxic, and immunological properties. A demyelinated P2 fraction of porcine cerebral cortex was prepared and the protein content in the membrane preparation determined as described previously (21C̆opic̆ A. Vučemilo N. Gubenšek F. Kriz̆aj I. J. Biol. Chem. 1999; 274: 26315-26320Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Membranes from porcine cerebral cortex (7.2 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, 2.5 mmCaCl2, and 2.5% (w/v) Triton X-100. The extract was centrifuged at 106,200 × g for 1 h and cold deionized water added to the supernatant to give a final detergent concentration of 2.0% (w/v). Samples were incubated for 30 min at room temperature with 125I-AtxC in the presence or absence of an unlabeled competitor. Disuccinimidyl suberate (DSS) was added (100 μm final concentration), and after 5 min the cross-linking reaction was stopped by the addition of SDS-PAGE sample buffer. Samples were analyzed by SDS-PAGE and gels dried and autoradiographed at −70 °C using Kodak X-Omat AR films (21C̆opic̆ A. Vučemilo N. Gubenšek F. Kriz̆aj I. J. Biol. Chem. 1999; 274: 26315-26320Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). CH-Sepharose 4B was swollen according to the manufacturer's recommendation. AtxC (2.4 mg/ml gel) was dissolved in 100 mm MES, pH 6.5, 5 mm CaCl2, 0.5 m NaCl; added to the activated Sepharose to a final concentration of 0.8 mg/ml; and incubated with agitation at 4 °C. After 4 h the gel was washed and its remaining active groups blocked with 1 methanolamine, pH 8.0, for 1 h. Routinely about 90% of AtxC was bound to the matrix. The resin was washed as recommended by the producer; equilibrated in 75 mm Hepes, pH 8.2, 150 mm NaCl, 2.5 mm CaCl2, and 0.1% (w/v) Triton X-100; and stored at 4 °C. 9 ml of wheat germ lectin-Sepharose 6MB was equilibrated with 50 mmHepes, pH 8.2, containing 140 mm NaCl and 2 mmCaCl2. The detergent extract was incubated with the gel for 4 h at 4 °C with moderate agitation. The supernatant was separated from the gel on a sintered glass funnel. 5 ml of Q-Sepharose was equilibrated in 75 mm Hepes, pH 8.2, containing 150 mm NaCl, 2.5 mm CaCl2, and 0.1% (w/v) Triton X-100. 25 ml of the lectin-Sepharose supernatant were incubated with the gel for 1 h at 4 °C with slight agitation. The gel was extensively washed with the equilibration buffer. The bound material was eluted with 20 ml of the equilibration buffer supplemented with 0.5 m NaCl. The gel was equilibrated with 100 ml of 75 mm Hepes, pH 8.2, containing 150 mm NaCl, 2.5 mm CaCl2, and 0.1% (w/v) Triton X-100. The eluate from Q-Sepharose was incubated with 5 ml of the gel at 4 °C for 4 h with gentle agitation. The resin was transferred to the column and washed extensively with the equilibration buffer. The AtxC-binding protein was eluted at 10 ml/min with 140 mm MES, pH 5.0, containing 200 mmNaCl, 4 mm CaCl2, and 0.2% (w/v) Triton X-100. 1-ml fractions were collected directly into 0.4 ml of 0.5 mtriethanolamine, pH 8.2, 150 mm NaCl and analyzed by affinity labeling with 125I-AtxC as described. The protein composition of the samples was analyzed by SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar) under nonreducing conditions (0.5% (m/v) SDS, 10% (v/v) glycerol, 30 mm Tris/HCl, pH 6.8) followed by silver staining (26Merril C.R. Goldman D. Sedman S.A. Ebert M.A. Science. 1981; 211: 1437-1438Crossref PubMed Scopus (2105) Google Scholar). Samples were run on SDS-PAGE (12.5% acrylamide gels) and transferred (90 min at 250 mA) to a PVDF membrane (Bio-Rad). The transfer buffer was 25 mmKH2PO4/K2HPO4, pH 7.0. After transfer, the membrane was incubated with mouse monoclonal anti-CaM antibodies at the concentration of 1 μg/ml. Immunodetection was performed by the BM chemiluminescence Western blotting detection system (Roche Molecular Biochemicals) following the manufacturer's instructions. The sample was analyzed by mass spectrometry as described previously (27Hanna S.L. Sherman N.E. Kinter M.T. Goldberg J.B. Microbiology. 2000; 146: 2495-2508Crossref PubMed Scopus (143) Google Scholar). Briefly, 1 μg of the R16 sample was separated on SDS-PAGE (12% acrylamide gels). The gel was stained with silver and the protein band excised and transferred to a siliconized tube. The gel piece was destained overnight then reduced with dithiothreitol, alkylated with iodoacetamide, and digested withPromega modified trypsin for 16 h. The peptides were extracted from the gel with 50% acetonitrile/5% formic acid and concentrated for LC-MS and MS/MS analysis on a Finnigan LCQ ion trap mass spectrometer. The spectra obtained were analyzed by data base searching using the Sequest algorithm against the NCBI nonredundant data base. Those peptides not matched were interpreted manually. AtxC-binding proteins, solubilized from the demyelinated P2 fraction of porcine cerebral cortex with Triton X-100, retained their toxin binding activity (Fig.1B, lane 1). Two specific adducts were clearly observed after affinity labeling of the extract with 125I-AtxC. The 200-kDa adduct resulted from the interaction of the toxin with the already characterized 180-kDa M-type PLA2 receptor-like protein (R180) (21C̆opic̆ A. Vučemilo N. Gubenšek F. Kriz̆aj I. J. Biol. Chem. 1999; 274: 26315-26320Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The 39 kDa adduct was the product of specific cross-linking of125I-AtxC and a yet unidentified protein with an apparent molecular mass of 25 kDa (R25) (20Vučemilo N. C̆opic̆ A. Gubenšek F. Kriz̆aj I. Biochem. Biophys. Res. Commun. 1998; 251: 209-212Crossref PubMed Scopus (35) Google Scholar). We devised a strategy for purifying R25 based on the following observations. 1) The interaction between AtxC and R25 depends on Ca2+ ions. 2) R25 loses affinity for AtxC below pH 5.5 and regains it completely when the pH is returned to 7.4. 3. In contrast to R180, R25 is not retained by concanavalin A, wheat germ lectin, or lentil lectin-Sepharose (20Vučemilo N. C̆opic̆ A. Gubenšek F. Kriz̆aj I. Biochem. Biophys. Res. Commun. 1998; 251: 209-212Crossref PubMed Scopus (35) Google Scholar, 21C̆opic̆ A. Vučemilo N. Gubenšek F. Kriz̆aj I. J. Biol. Chem. 1999; 274: 26315-26320Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In addition we found that Q-Sepharose, an anion exchanger, binds most of the R25 at pH 7.4. Starting from 10 ml of the membrane preparation, the detergent extract was incubated with wheat germ lectin-Sepharose to remove R180 before being applied to Q-Sepharose at pH 7.4. The Q-Sepharose-retained Atx-binding protein was eluted batchwise with a high concentration of NaCl. This step was important to reduce the concentration of Triton X-100 in the preparation and so enable efficient subsequent purification steps. 125I-AtxC affinity labeling of the eluate from ion exchange chromatography revealed, however, not the expected 39-kDa adduct but only a specific adduct of about 30 kDa (Fig.1B, lane 3). Such a specific adduct was sometimes also visible in the crude membrane extract after storage for a longer time at −20 °C. Since the specific adduct at 39 kDa disappeared at the same time as the specific adduct at 30 kDa appeared following the Q-Sepharose step, it appears that R25 is an oligomeric protein in which the 16-kDa subunit (R16) carries the toxin-binding site. AtxC-Affi-Gel 10, successfully used in purification of R180, did not give satisfactory results in the purification of R16 (21C̆opic̆ A. Vučemilo N. Gubenšek F. Kriz̆aj I. J. Biol. Chem. 1999; 274: 26315-26320Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Using activated CH-Sepharose, we prepared an AtxC-affinity resin, which was much more efficient. Analysis of the sample after the toxin-affinity chromatography is shown in lanes 6 of Fig. 1, Aand B. From 72 mg of membrane protein in the starting preparation we obtained about 2 μg of pure R16, as judged by semiquantitative densitometric analysis of the silver-stained SDS-PAGE band of the final product. The affinity of AtxC for the isolated R16 was estimated in the cross-linking competition experiment (21C̆opic̆ A. Vučemilo N. Gubenšek F. Kriz̆aj I. J. Biol. Chem. 1999; 274: 26315-26320Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Native AtxC displaced 125I-AtxC from R16 with a dissociation constant of 11 nm. Several toxic and nontoxic sPLA2s were tested for their ability to inhibit the formation of the specific adduct between125I-AtxC and the isolated acceptor. Only the neurotoxic ammodytoxins and bee venom PLA2 were able to completely prevent the binding of 125I-AtxC to R16 under the experimental conditions used. Agkistrodotoxin, crotoxin, and myotoxic ammodytin L were weaker inhibitors, while OS2, taipoxin, and β-bungarotoxin as well as the nontoxic AtnI2, porcine pancreatic PLA2, and human type IIA PLA2 did not inhibit the binding (Fig. 2). The isolated AtxC-binding protein was identified by tandem MS analysis. R16 was reduced, alkylated, and digested with trypsin in the gel. The resulting peptides were extracted from the polyacrylamide and their molecular weights and amino acid sequences determined using an LC-MS system. Analysis of the data gave the result shown in Fig.3A. The partial sequence of R16 matches completely with that of CaM, a protein essential to many fundamental physiological processes (reviewed in Ref. 22Chin D. Means A.R. Trends Cell Biol. 2000; 10: 322-328Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). Comparing the sequence of CaM with the primary structures of other sPLA2-binding proteins, some similarity has been found only to TCBP-49 (taipoxin-associated calcium-binding protein of 49 kDa) (10Dodds D. Schlimgen A.K. Lu S.-Y. Perin M.S. J. Neurochem. 1995; 64: 2339-2344Crossref PubMed Scopus (46) Google Scholar) and crocalbin (11Hseu M.J. Yen C.-Y. Tzeng M.-C. FEBS Lett. 1999; 445: 440-444Crossref PubMed Scopus (47) Google Scholar). These two proteins belong to reticulocalbins (28Ozawa M. Muramatsu T. J. Biol. Chem. 1993; 268: 699-705Abstract Full Text PDF PubMed Google Scholar) and are, like CaM, EF-hand Ca2+-binding proteins (29Lewit-Bentley A. Réty S. Curr. Opin. Struct. Biol. 2000; 10: 637-643Crossref PubMed Scopus (421) Google Scholar). PLA2 neurotoxins interfere with membrane trafficking (reviewed in Ref. 30Montecucco C. Rossetto O. Trends Biochem. Sci. 2000; 25: 266-270Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The implication of CaM in different modes of membrane trafficking: transcytosis (31Apodaca G. Enrich C. Mostov K.E. J. Biol. Chem. 1994; 269: 19005-19013Abstract Full Text PDF PubMed Google Scholar), endosome fusion (32Kübler E. Schimmöller F. Riezman H. EMBO J. 1994; 13: 5539-5546Crossref PubMed Scopus (70) Google Scholar, 33Colombo M.I. Beron W. Stahl P.D. J. Biol. Chem. 1997; 272: 7707-7712Crossref PubMed Scopus (116) Google Scholar), vacuole fusion (Refs. 34Peters C. Mayer A. Nature. 1998; 396: 575-580Crossref PubMed Scopus (325) Google Scholar and 35Ungermann C. Wickner W. Xu Z.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11194-11199Crossref PubMed Scopus (90) Google Scholar and reviewed in Ref. 36Wickner W. Haas A. Annu. Rev. Biochem. 2000; 69: 247-275Crossref PubMed Scopus (171) Google Scholar), intra-Golgi membrane transport (37Porat A. Elazar Z. J. Biol. Chem. 2000; 275: 29233-29237Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and rapid endocytosis in adrenal chromaffin cells (38Artalejo C.R. Elhamdani A. Palfrey H.C. Neuron. 1996; 16: 195-205Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) is well documented. It may also be involved as one of the Ca2+ sensors in regulated exocytosis (39Chen Y.A. Duvvuri V. Schulman H. Scheller R.H. J. Biol. Chem. 1999; 274: 24476-26469Google Scholar, 40Quetglas S. Leveque C. Miquelis R. Sato K. Seagar M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9695-9700Crossref PubMed Scopus (86) Google Scholar). CaM would be therefore a perfect target for the toxin, which is known to block synaptic transmission. To confirm that the target for neurotoxic PLA2 is indeed a CaM, the R16 preparation was incubated with 125I-AtxC in the absence and presence of 2 μm native AtxC. Following the cross-linking, reaction mixtures were separated on SDS-PAGE and electro-blotted onto PVDF membrane. Immunodetection with anti-CaM Ab revealed a positive band at 30 kDa (Fig. 3B). The same PVDF membrane was, after the chemiluminescence had been completely quenched, autoradiographed to visualize the 125I-AtxC. As is evident from Fig. 3B,125I-AtxC was specifically present in the band having the same molecular mass as the band labeled with anti-CaM Ab. In addition, the interaction between 125I-AtxC and commercially available porcine brain CaM was demonstrated (not shown). CaM is a soluble protein, so the fact that a high concentration of the detergent was necessary to extract R16 from the P2 membranes suggests that R16 is part of an oligomeric membrane-anchored protein complex (R25). An acceptor for sPLA2, ammodytoxin, is shown to be a calmodulin. Since CaM is generally regarded as an intracellular protein, this finding strengthens the proposition that the neurotoxic sPLA2 has to enter the neuron to produce the arrest of synaptic vesicle cycling. In addition, the observed specific and high affinity interaction between CaM and neurotoxic AtxC could be used to investigate the role of CaM in this process. 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. We thank Dr. Roger H. Pain for critical reading of the manuscript." @default.
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• W2083901291 date "2001-04-01" @default.
• W2083901291 modified "2023-10-18" @default.
• W2083901291 title "A High Affinity Acceptor for Phospholipase A2 with Neurotoxic Activity Is a Calmodulin" @default.
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• W2083901291 doi "https://doi.org/10.1074/jbc.c100048200" @default.
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