From the Department of Biochemistry and Molecular
Biology, Jo
ef Stefan Institute, Jamova 39, Slovenia, the
Department of Chemistry and Biochemistry, Faculty of Chemistry
and Chemical Technology, A
ker
eva 5, University of
Ljubljana, 1000 Ljubljana, Slovenia, and the ¶ W. M. Keck
Biomedical Mass Spectrometry Laboratory and the University of Virginia
Biomedical Research Facility, University of Virginia Medical School,
Charlottesville, Virginia 22908
Received for publication, January 30, 2001, and in revised form, February 26, 2001
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ABSTRACT |
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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 A2
neurotoxicity. 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
(PLA2,1 EC
3.1.1.4) 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 (1, 2). 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. 3). 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. 4-6). 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 (7), pentraxins (8, 9), reticulocalbins (10, 11), C-type
multilectins (12-14), factor Xa (15), and proteoglycan glypican (16).
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. 5). 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.
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. 17). 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 PLA2
ammodytoxin C (AtxC) from Vipera ammodytes ammodytes venom
(18, 19). Two high affinity binding proteins for AtxC have been
detected in porcine cerebral cortex, which are potentially implicated
in the neurotoxicity of this PLA2 (20, 21), and the
purification and characterization of the high molecular mass
AtxC-binding protein has been described previously (21). 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.
22).
Materials--
Ammodytoxins, ammodytin I2
(AtnI2) and AtnL, were purified from V. ammodytes
ammodytes venom as described previously (18, 23). 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 Radioiodination of AtxC--
AtxC was radioiodinated as
described previously (24) to specific radioactivity around 300 Ci/mmol. 125I-AtxC was identical to the native AtxC in
enzymatic, neurotoxic, and immunological properties.
Membrane Preparation from Porcine Cerebral Cortex--
A
demyelinated P2 fraction of porcine cerebral cortex was prepared and
the protein content in the membrane preparation determined as described
previously (21).
Solubilization of AtxC-binding Proteins--
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 mM
CaCl2, 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).
Cross-linking of 125I-AtxC to the Solubilized
AtxC-binding Proteins--
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 Coupling of AtxC to CH-Sepharose 4B--
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 M
ethanolamine, 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.
Chromatography on Wheat Germ Lectin-Sepharose 6MB--
9 ml of
wheat germ lectin-Sepharose 6MB was equilibrated with 50 mM
Hepes, pH 8.2, containing 140 mM NaCl and 2 mM
CaCl2. 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.
Ion Exchange Chromatography--
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.
Chromatography on AtxC-CH-Sepharose 4B--
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 mM
NaCl, 4 mM CaCl2, and 0.2% (w/v) Triton X-100.
1-ml fractions were collected directly into 0.4 ml of 0.5 M
triethanolamine, 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 (25)
under nonreducing conditions (0.5% (m/v) SDS, 10% (v/v) glycerol, 30 mM Tris/HCl, pH 6.8) followed by silver staining (26).
Electroblotting and Immunochemiluminescence
Detection--
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 mM
KH2PO4/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.
Mass Spectrometry--
The sample was analyzed by mass
spectrometry as described previously (27). 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 with Promega 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) (21). The 39 kDa
adduct was the product of specific cross-linking of
125I-AtxC and a yet unidentified protein with an apparent
molecular mass of 25 kDa (R25) (20).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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.
70 °C using Kodak X-Omat AR films (21).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Purification of AtxC-binding protein (R16)
from porcine cerebral cortex. A, samples obtained at
different stages of the purification procedure were analyzed by 10%
SDS-PAGE under nonreducing conditions. The gel was silver-stained.
Lane 1, crude membrane extract (0.5 µl out of 25 ml,
i.e. 1.4 µg of protein); lane 2, breakthrough
from Q-Sepharose (0.5 µl out of 25 ml); lane 3, eluate
from Q-Sepharose (0.5 µl out of 20 ml); lane 4,
breakthrough from AtxC-CH-Sepharose (0.5 µl out of 20 ml); lane
5, AtxC-CH-Sepharose wash (21 µl out of 5.6 ml); lane
6, eluate from AtxC-CH-Sepharose (21 µl out of 5.6 ml). The
position of pure R16 in lane 6 is indicated by the
arrowhead. B, an aliquot of each sample, ordered
as under A, has been affinity-labeled with
125I-AtxC. The positions of specific adducts are indicated
by arrowheads.
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 (20, 21). 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 (21). 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, A
and 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 (21). 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 between
125I-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 -bungarotoxin as well as the nontoxic AtnI2, porcine
pancreatic PLA2, and human type IIA PLA2 did
not inhibit the binding (Fig. 2).
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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. 22). 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) (10)
and crocalbin (11). These two proteins belong to reticulocalbins (28)
and are, like CaM, EF-hand Ca2+-binding proteins (29).
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PLA2 neurotoxins interfere with membrane trafficking (reviewed in Ref. 30). The implication of CaM in different modes of membrane trafficking: transcytosis (31), endosome fusion (32, 33), vacuole fusion (Refs. 34 and 35 and reviewed in Ref. 36), intra-Golgi membrane transport (37), and rapid endocytosis in adrenal chromaffin cells (38) is well documented. It may also be involved as one of the Ca2+ sensors in regulated exocytosis (39, 40). 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* This work was supported by the Ministry of Science and Technology of Slovenia (Grant J1-7261-0106) and by a grant from the University of Virginia Pratt Committee.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.
§ Contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 386-1-477-36-26; Fax: 386-1-257-35-94; E-mail: igor.krizaj@ijs.si.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.C100048200
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ABBREVIATIONS |
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The abbreviations used are: PLA2, phospholipase(s) A2; Atn, ammodytin; Atx, ammodytoxin; CaM, calmodulin; DSS, disuccinimidyl suberate; LC, liquid chromatography; MS, mass spectrometry; OS2, Oxyuranus scutellatus PLA2; R16, R25, and R180, receptors for AtxC in porcine cerebral cortex of 16, 25, and 180 kDa, respectively; sPLA2, secretory PLA2; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; PVDF, polyvinylidene difluoride.
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