(Received for publication, September 19, 1994; and in revised form, November 15, 1994)
From the
We describe the purification and first biochemical
characterization of an enzymatic activity in venom from the marine
snail Conus magus. This enzyme, named conodipine-M, is a novel
phospholipase A with a molecular mass of 13.6 kDa and is
comprised of two polypeptide chains linked by one or more disulfide
bonds. The amino acid sequence of conodipine-M shows little if any
homology to other previously sequenced phospholipase A
enzymes (PLA
s). Conodipine-M thus represents a new
group of PLA
s. This is remarkable, since conodipine-M
displays a number of properties that are similar to those of previously
characterized 14-kDa PLA
s. The enzyme shows little, if any,
phospholipase A
, diacylglycerol lipase, triacylglycerol
lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the
presence of calcium and with specific activities that are comparable to
those of well known 14-kDa snake venom and pancreatic PLA
s.
The Conus enzyme binds tightly to vesicles of the negatively
charged phospholipid
1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the
hydrolysis of this substrate in a processive fashion. Conodipine-M does
not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or
with different polar head groups. Linoleoyl amide and a phospholipid
analog containing an alkylphosphono group at the sn-2 position
are potent inhibitors of conodipine-M. We suggest that the functional
resemblance of conodipine-M to other PLA
s might be
explained by the utilization of similar catalytic residues.
Conus are a group of predatory marine snails that prey
on fish, molluscs, and polychaete worms(1) . Venom from cone
snails are extraordinarily complex and often contain more than 100
distinct components. Most venom characterizations have focused on small
disulfide-rich peptides that target selectively to receptors and ion
channels on nerve and muscle cells. Known targets include calcium
channels (-conotoxins)(2, 3, 4) ,
sodium channels (µ- and
-conotoxins)(5, 6, 7, 8) , N-methyl-D-aspartate receptors
(conantokins)(9, 10, 11) , nicotinic
acetylcholine receptors
(
-conotoxins)(12, 13, 14, 15, 16, 17) ,
and vasopressin receptors (conopressins) (18, 19) .
Small neuroactive molecules, e.g. serotonin, have also been
found(20) .
Although larger protein components are present in cone venoms, these are generally less well characterized biochemically. Reports have included a convulsion-inducing peptide from Conus geographus, with an apparent molecular weight of 13,000(21) ; a cardiotonic glycoprotein (approximate molecular weight of 25,000) from Conus striatus, which appears to act on voltage-sensitive sodium channels(22, 23) ; a protein from Conus magus (molecular weight estimated to be between 45,000 and 65,000), which induces powerful rhythmic contractions of guinea pig vas deferens(24) ; and a 25,000 molecular weight protein from Conus distans, which inhibits neurotransmitter release in rat hippocampus(25) .
In this study, we have
characterized a 13.6-kDa component from the venom of the fish-eating
cone snail, C. magus. Novel - and
-conotoxins have
been isolated from this species previously(13, 26) .
We detail the isolation and characterization of a polypeptidic venom
component that we show to be a novel phospholipase A
(PLA
). (
)This is the first biochemical
characterization of an enzymatic activity from Conus venoms.
Low molecular mass secreted PLAs (14-18 kDa) have
been reported from a wide variety of other venoms (snakes, insects, and
lizards) as well as mammalian pancreatic and inflammation fluids (27, 28, 29, 30) . These enzymes
hydrolyze the ester at the sn-2 position of phosphoglycerides.
Snake venom and pancreatic PLA
s are particularly well
characterized(31, 32) . All require millimolar
concentration of calcium as a catalytic cofactor. In addition to
PLA
activity, some of these venom proteins show potent
neurotoxicity(33, 34, 35) . For instance
-bungarotoxin, from the Formosan banded krait (Bungarus
multicinctus) (36) and notexin isolated from the
Australian tiger snake Notechis scutatus scutatus(37) exert potent neurotoxic action by inhibiting the
release of acetylcholine from nerve terminals in neuromuscular
junctions(38) .
-Bungarotoxin consists of a PLA
chain covalently bonded by a disulfide bridge to a polypeptide
(molecular weight
20,000)(29) . The PLA
chain
of
-bungarotoxin is homologous to notexin. Enzymes isolated from
the venoms of Naja naja atra and Naja niggricollis,
however, show more potent PLA
activity, have less
presynaptic effects, and are much less lethal(39) . More than
50 proteins with PLA
activity have been isolated from snake
venoms and show substantial sequence homology with each other and with
PLA
enzymes isolated from mammalian pancreas and
inflammatory exudates(40, 41) . Sequence information
reveals that the C. magus enzyme reported here differs
substantially from any other PLA
characterized to date and
defines a new group of these enzymes.
The
preparation of
1-O-hexadecyl-2-deoxy-2-S-thiohexadecanoyl-sn-glycero-3-phosphocholine
and DMPM has been described previously(43, 44) .
[H]DPPC (50 Ci/mmol),
[
C]PAPC (50 mCi/mmol), and
[
C]SAPI (50 mCi/mmol) were from DuPont NEN.
[
H]DPPE (10 mCi/mmol),
[
H]DPPA (5 mCi/mmol), and
[
H]SAPS (400 mCi/mmol) were prepared as
described(45, 46) . [
H]DPG was
synthesized by treating 0.15 mg of [
H]DPPC (50
mCi/mmol) in 0.2 ml of 200 mM sodium phosphate, 400 µM ZnCl
, 1 mM 2-mercaptoethanol, pH 7.0, with 30
µg of phospholipase C (Bacillus cereus, Sigma P-7147) for
2 h at 37 °C with vigorous shaking. The reaction mixture was
extracted three times with ether, the extract was dried over anhydrous
Na
SO
, and the residue was dissolved in ether
and applied to a silica gel TLC plate. The plate was developed with
chloroform:ether (9:1), the product band (visualized with iodine) was
scraped from the plate, and the product was eluted with ether. The
PLA
inhibitors linoleoyl amide and MG14 were prepared as
described(47, 48, 49) . Porcine pancreatic
PLA
was obtained as a generous gift from Professor M. K.
Jain (University of Delaware) (50) , bee venom PLA
was from Boehringer Mannheim, and pancreatic lipase was from
Sigma (L-0382).
Figure 1:
HPLC purification of
conodipine-M. Panel A, extracted crude venom (see
``Experimental Procedures'') was applied to a Vydac reversed
phase C18 column (10-µm particle size; 22-mm inner diameter
25-cm length) and eluted with a gradient that began at 95% A, 5% B for
5 min and increased to 100% B over 28 min. B was held at 100% for 5 min
and then decreased to 5% over 4 min. The flow rate was 4 ml/min.
Fractions were collected every 3 min beginning at 12 min. Panel
B, material eluting at 34.3 min (arrow in panel
A) was run on a Vydac reversed phase C18 column (10-µm
particle size, 10-mm inner diameter
25-cm length) and eluted by
beginning at 70% A, 30% B for 4 min; increasing to 36% B over 6 min;
and then increasing to 56% B over 60 min. The flow rate was 3 ml/min.
Fractions were collected every 1.0-1.25 min, beginning at 3 min. Panel C, material eluting at 23.3 min (arrow in panel B) was run on a Microsorb MV reversed phase C18 column
(5-µm particle size, 4.6-mm inner diameter
25-cm length).
The gradient began at 40% C, 60% buffer D for 1 min; increased to 70% D
over 30 min; and increased to 80% D over 60 min. The flow rate was 1
ml/min. Fractions were collected according to visual inspection of the
protein absorbance. Panel D, material eluting near the peak at
47.4 min (arrow in panel C) was run again using the
same conditions as in panel C. Buffer A was 0.1%
trifluoroacetic acid; buffer B was 0.1% trifluoroacetic acid, 90%
acetonitrile in panels A and B. Buffer C was 0.1%
trifluoroacetic acid, 2% MeOH, 98% H
O; buffer D was 0.09%
trifluoroacetic acid, 90% MeOH, 10% H
O in panels C and D. It should be noted that prolonged use of
trifluoroacetic acid/methanol in the Microsorb MV column is not
recommended by the manufacturer because of potential hydrolysis. The
absorbance was monitored at 237 nm in panel A and at 214 nm in panels B-D.
Figure 2: Conodipine-M has two subunits. Native and modified conodipine-M were analyzed by HPLC. HPLC conditions are the same for each panel and are described in Experiment 1 under ``Experimental Procedures.'' Panel A, unmodified conodipine-M, when chromatographed as described below, elutes at 27.4 min. Electrospray mass spectrometry of this material shows a molecular mass of 13,602. Panel B, reduction of conodipine-M (described under ``Experimental Procedures'') produces two primary protein absorbances with migration times (24.7 and 31.9 min) which differ from unmodified material. Mass spectroscopy of these proteins shows molecular masses of 8,571 and 5,036. The arrow indicates the expected migration time of unmodified conodipine-M. The sum of the molecular masses of these two proteins approximates that of the intact protein. Panel C, reduction and alkylation of conodipine-M (described in under ``Experimental Procedures'') also results in two protein absorbances with migration times of 25.7 and 34.9 min.
Hydrolysis of
small unilamellar DMPM vesicles was carried out using a pH-stat as
described previously(43) , and 4-ml reaction mixtures contained
0.6 mM CaCl, 1 mM NaCl, 0.6 mg of DMPM,
and typically 0.3 µg of conodipine-M. For the analysis of
inhibitors, assays were carried out under conditions that measure the
initial velocity(51) , and 4-ml reaction mixtures contained 2.5
mM CaCl
, 1 mM NaCl, 20 µg of
polymyxin B sulfate (Sigma), 0.6 mg of DMPM, and typically 0.1 µg
of conodipine-M.
In some cases, radiometric assays of PLA activity were carried out. Covesicles of nonlabeled DMPM vesicles
containing radiolabeled phospholipids were prepared as described
previously(45) . The reaction mixtures were extracted with
organic solvent, and the amounts of liberated radioactive fatty acids
were analyzed as described previously(45) . In this way the
calcium dependence of PLA
activity of conodipine-M was
measured using reaction mixtures with 0.3 mg of DMPM vesicles
containing 120,000 cpm of [
H]DPPC in 2 ml of 10
mM HEPES, 100 mM NaCl, 100 mM EGTA, pH 7.4.
The mixtures also contained different amounts of 1 M K
CaEGTA solution to give the desired concentrations of
free calcium(52) . Reactions were initiated by the addition of
0.04 µg of conodipine-M and were quenched with organic solvent
after a 10-min incubation at 20 °C. Reactions for determining the
pH dependence of conodipine-M-catalyzed vesicle hydrolysis were carried
out as above except that the buffer was 10 mM HEPES, 100
mM NaCl, 1 mM CaCl
, pH 6-9.
Competitive substrate specificity studies using the double
radioisotope method were carried out as described(45) . The
reaction mixtures contained 0.6 mg of sonicated DMPM vesicles with 1
10
-8
10
cpm of
H- and
C-labeled phospholipids in 4 ml of 0.6
mM CaCl
, 1 mM NaCl, 20 µg of
polymyxin B at 21 °C. Reactions were carried out in the pH-stat
with 0.07 µg of conodipine-M (added last) and were quenched with
organic solvent after 120-130 nmol of titrant was consumed.
Reactions with DMPM vesicles containing [
C]PAPC
and [
H]DPG were quenched and extracted with
organic solvent as described above. The residue from the extract was
dissolved in low boiling petroleum ether:ether:acetic acid (70:30:1),
and nonlabeled arachidonic acid and 1,2-dioleoyl-sn-glycerol
(Sigma) were added as markers. The mixture was applied to a silica gel
TLC plate (4
12 cm), and the plate was developed with the same
solvent. Regions corresponding to fatty acid and diacylglycerol were
scraped from the plate, and the silica was soaked in scintillation
fluid for 1 h followed by scintillation counting. Relative k
/K
values for the
radioactive substrates were calculated using Equation 4
of(45) .
Studies to measure the positional specificity of
the phospholipase activity of conodipine-M were carried out with
reaction mixtures of 200 µg of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphomethanol in 4 ml of
2.5 mM CaCl, 1 mM NaCl, pH 8.0,
containing 20 µg of polymyxin B at 21 °C in the pH-stat. After
about 30% of the substrate was hydrolyzed (30 µl of 3 mM NaOH titrant), the reaction was quenched by the addition of 4 ml
of 0.1 M EDTA, 2 M NaCl, pH 7.0, followed by 0.56 ml
of 88% formic acid and 4 ml of ethyl acetate. After mixing on a vortex
mixer, the layers were separated by centrifugation, and the organic
layer was transferred to a new tube. The aqueous phase was extracted
with a second portion of ethyl acetate, the extracts were combined, and
the solvent was removed with a stream of nitrogen. To the residue were
added 0.5 ml of ether and 50 µl of methanol, and sufficient
diazomethane in ether was added until the yellow color persisted. After
30 min at room temperature, the solvent was removed with a stream of
nitrogen. The residue was taken up in ether, and a portion was analyzed
by gas chromatography on a DB-5 column (J & W Scientific). Peaks
corresponding to methyl oleate and methyl palmitate were integrated,
and the peak areas obtained from a control reaction (omission of
conodipine-M) were subtracted from those obtained in the presence of
enzyme.
Crude
venom from C. magus was extracted and initially
chromatographed using a preparative HPLC column (see Fig. 1A). Fifteen fractions were collected and tested,
and fraction 8 eluting at 34.3 min was found to inhibit
[H]isradipine binding to rat brain membranes.
This fraction was rechromatographed using a semipreparative column and
a shallower gradient. The eluant resolved into two major broad peaks,
as well as a number of minor components (see Fig. 1B).
Activity (as measured by the isradipine binding assay) was associated
with the earlier eluting major peak in Fig. 1B.
Further purification was carried out using an analytical HPLC column
(4.6-mm inner diameter 25-cm length). Resolution was difficult
because of the presence of at least three closely migrating components (Fig. 1C). The trifluoroacetic acid/acetonitrile buffer
system used in the initial steps of purification did not fully resolve
these components (data not shown). The best resolution was obtained
with a very slow gradient of trifluoroacetic acid/methanol buffer (see Fig. 1C for buffer conditions) using a reversed phase
C18 Microsorb MV column. Using these conditions, a center cut of the
middle peak reliably gave a homogeneous appearing peak on
rechromatography (see Fig. 1D). Although the material
in Fig. 1C did not appear chromatographically
homogeneous, the amount of [
H]isradipine binding
activity was proportional to the protein absorbance across all peaks,
suggesting that the active material might be present as multiple
isoforms. The major component was purified (see Fig. 1D) and was the material that was further
characterized below, which we have called conodipine-M.
Initial attempts at amino acid
sequencing revealed that the NH terminus of the
-chain
is blocked. The
-chain was reduced, alkylated (pyridylethylated),
and purified by reversed phase HPLC. The alkylated
-chain was
unblocked by digestion with pyroglutamate aminopeptidase, repurified,
and sequenced successfully through the first 34 residues. To analyze
the remainder of the chain, digests with endoproteinase Lys-C (cleaves
peptide bonds at the COOH-terminal side of Lys residues) and
endoproteinase Arg-C (cleaves peptide bonds COOH-terminally at Arg
residues) were performed. Fragments were isolated by reversed phase
HPLC and sequenced. The
-chain of conodipine-M was reduced,
pyridylethylated, and sequenced. Results of the sequence analysis are
summarized in Table 1. It is noteworthy that the sequence of the
-chain of conodipine-M is considerably shorter than the
-chain of other two-subunit PLA
s (e.g. compare
-bungarotoxin in Table 2). Mass spectrometry
measurements of conodipine-M, however, confirmed the size of the
indicated sequences of the
- and
-chains (see below).
In
the conodipine-M sequences, a few residues could not positively be
assigned (indicated by X in the tables). In several cases,
these residues appear as blanks in the sequencing run. These blanks
appeared in multiple sequencer runs, always in the same position.
Peptides from Conus contain a number of unusual amino acids
and post-translational modifications which appear as blanks during
sequencing(9) . It is likely that the unassigned residues in
the middle portions of the sequence represent such modifications (1 or
2 residues at the COOH terminus could not be assigned with certainty
because of a diminishing signal). The size of any post-translational
modification is small, however. By comparing the observed mass
(measured by mass spectrometry) with the calculated mass of the
sequence shown in Table 1, the average mass for the unassigned
residues in the -chain is 132.2 ((8,571 - 7,910)/5) and in
the
-chain 137.0 ((5,036 - 4,625)/3).
Although conodipine-M potently inhibits the binding of isradipine to L-type calcium channels, several lines of evidence suggest that this might not be the result of direct competition. As shown in Fig. 3the inhibition of isradipine binding depends on the time of preincubation of conodipine-M with cortical membranes. These results raise the possibility that the inhibition may be indirect.
Figure 3:
Time
dependence of conodipine-M inhibition of
[H]isradipine binding. Cortical rat brain
membrane was incubated in the presence or absence of 2 nM conodipine-M at 37 °C for the indicated lengths of time.
Specific binding was defined as the difference between
[
H]isradipine counts bound in the absence (total
binding) and presence (nonspecific binding) of 10 µM nifedipine (see ``Experimental Procedures''). Data were
acquired in duplicate and the results
averaged.
The larger size of
conodipine-M and the time course of isradipine binding led us to
investigate whether conodipine-M had any enzymatic activity. Since many
venoms have been reported to contain PLAs, we tested
conodipine-M for its ability to hydrolyze phospholipids. As shown in
the following section, conodipine-M is indeed a PLA
.
The
sequence of conodipine-M can be compared to other known
PLAs. The sequence of the
-chain of conodipine-M and
group I-III PLA
s is shown in Table 2. Although
conodipine-M shows little homology to any known PLA
,
certain catalytic residues may be similar (see Table 2, Table 4and ``Discussion'').
Vesicles of the anionic
phospholipid DMPM have been used extensively to detect and characterize
the kinetics of hydrolysis of the sn-2 ester of phospholipids
by PLAs(43, 56, 57) , and a brief
overview of this system is given here as it relates to the action of
conodipine-M on DMPM vesicles. Secreted PLA
s (molecular
masses of 14 kDa) bind irreversibly to DMPM vesicles and hydrolyze all
of the substrate in the outer layer in a processive fashion termed
scooting mode hydrolysis (enzyme does not leave the surface). The
reaction progress stops when all of the outer layer substrate in
enzyme-containing vesicles has been hydrolyzed. At this point the
reaction products remain in enzyme-containing vesicles, and the
vesicles remain structurally intact. As shown in Fig. 4, the
reaction ceases after a limited amount of total DMPM is hydrolyzed by
porcine pancreatic PLA
. This is because the inner layer
DMPM is not hydrolyzed, and there are more vesicles than enzyme, and
the enzyme does not hop from one vesicle to another. Thus, the total
mol of product formed is equal to the mol of catalytically active
enzyme times the mol of substrate in the outer layer of a DMPM
vesicle(58) .
Figure 4:
Reaction progress curves for the
hydrolysis of small unilamellar vesicles by 0.3 µg of porcine
pancreatic PLA (solid line) and 0.3 µg of
conodipine-M (dashed line). Other conditions are given under
``Experimental Procedures.''
As shown in Fig. 4, the addition of conodipine-M to DMPM vesicles leads to the immediate onset of lipolysis. With conodipine-M, the hydrolysis of DMPM vesicles does not stop abruptly, but a steady-state velocity is seen later in the reaction progress (Fig. 4). This continued reaction could be caused either by slow dissociation of conodipine-M from vesicles followed by rebinding to different vesicles (slow hopping) and/or by conodipine-M-promoted fusion of vesicles; such fusion would bring substrate into substrate-depleted, enzyme-containing vesicles. In any case, the fact that both conodipine-M- and porcine pancreatic-catalyzed reactions produce similar amounts of products before the reaction ceases (or slows) indicates that a large fraction, perhaps all, of the conodipine-M mass has lipolytic activity, and thus this activity is not due to a minor contaminant.
The addition of the cationic peptide
polymyxin B leads to rapid intervesicle exchange of DMPM, and this
replenishes the substrate in enzyme-containing vesicles. As a result,
the initial reaction velocity (when the mol fraction of substrate in
the vesicle is close to 1) is prolonged, and the progress curves are
linear for several min(51) . Under such conditions conodipine-M
displays a specific activity of 200 s, which is
similar to that of porcine pancreatic PLA
(320
s
)(59) . Further support that conodipine-M
has lipolytic activity comes from an additional experiment.
Conodipine-M was chromatographed on an HPLC column. The lipolytic
activity of each fraction was measured using the DMPM/polymyxin B
assay, and as shown in Fig. 5, the profile of lipolytic activity
exactly matches the peak of protein eluting from the column. Further
indication of the purity of conodipine-M is evident from HPLC analysis
of reduced and alkylated material. As shown in Fig. 2C,
when alkylated conodipine-M (43.5 µg) is chromatographed, two sharp
protein absorbances are seen (A and B chains). Contaminant peaks (if
any) represent no more than 0.1% of the total protein absorbance at 220
nM.
Figure 5:
Conodipine-M (23 µg) was run on a HPLC
column using conditions described in panel C of Fig. 1.
Fractions were collected every 30 s as indicated. Absorbance was
monitored at 220 nM (solid line). One percent of each
fraction was tested for PLA activity using the
DMPM/polymyxin B assay described under ``Experimental
Procedures.'' Activity (nmol of NaOH titrant used per min in the
pH-stat) is normalized such that the maximum activity (10.6 nmol/min)
is plotted at the same ordinate value as the UV absorbance. Note that
the PLA
activity profile (open circles) also
corresponds to the protein absorbance
profile.
Vesicles of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphomethanol were used to
test the positional specificity of the esterase activity of
conodipine-M. Such vesicles were readily hydrolyzed, and gas
chromatographic analysis of the extracted fatty acid fraction shows
that the major product is oleic acid (<5% of the fatty acids formed
is palmitic acid). Thus, conodipine-M has little, if any, PLA activity.
Figure 6:
Calcium dependence of the
conodipine-M-catalyzed hydrolysis of [H]DPPC in
DMPM vesicles. Conditions are given under ``Experimental
Procedures.''
Additional phospholipids were tested
as possible substrates of conodipine-M. The reactions were carried out
in 4 ml of 0.6 mM CaCl, 1 mM NaCl, pH
8.0, containing 0.6 mg of phospholipid, 20 µg of polymyxin B, and
0.07 µg of conodipine-M at 21 °C in the pH-stat. The enzyme
readily hydrolyzes vesicles of
1,2-dioleoyl-sn-glycero-3-phosphomethanol, but vesicles of the
D-stereoisomer were not detectably hydrolyzed. Thus, conodipine-M, like
other PLA
s, is stereospecific for phospholipids with the
natural stereochemistry at the glycerol backbone. Under the same
conditions the lysophospholipid
1-myristoyl-sn-glycero-3-phosphocholine is not a substrate.
Conodipine-M hydrolyzes vesicles of the zwitterionic phospholipid
1,2-dimyristoyl-sn-glycero-3-phosphocholine, although the
turnover number of 10 s
is considerably less than
200 s
measured with DMPM vesicles. Since fully
vesicle-bound conodipine-M does not significantly discriminate between
phospholipids with different polar head groups, the low turnover rate
on zwitterionic vesicles may be due to poor binding of enzyme to the
interface as is the case for many 14-kDa
PLA
s(45, 56, 61) , but this was
not investigated further.
Conodipine-M has no triacylglycerol lipase
activity. Adding 0.15 µg of conodipine-M to 7 mM tributyrin in 4 ml of 0.6 mM CaCl, 1 mM NaCl, 20 µg of polymyxin B, pH 8.0, at 21 °C in the
pH-stat did not result in detectable hydrolysis, whereas adding 0.25
µg of pancreatic lipase gave rise to an immediate reaction.
Conodipine-M also failed to hydrolyze 0.3 mM triolein in a
reaction mixture containing 20 mM deoxycholate, 0.6 mM CaCl
, 1 mM NaCl, pH 8.0, at 21 °C in the
pH-stat. Under such conditions, hydrolysis was detected after adding
pancreatic lipase.
Conodipine-M did not detectably hydrolyze
[H]DPG present in DMPM vesicles, although
[
C]PAPC present in the same vesicles was
hydrolyzed. Thus, conodipine-M has no diglyceride lipase activity.
Figure 7: Inhibition of conodipine-M-catalyzed hydrolysis of DMPM vesicles by linoleoyl amide (filled circles) and MG14 (open circles) present in DMPM vesicles at the indicated mol fraction. Other conditions are given under ``Experimental Procedures.''
The results demonstrate that conodipine-M is a novel
PLA; this is the first enzymatic activity extensively
characterized from cone snail venom. We have named this enzyme
conodipine-M and anticipate that this is only the first of a family of
PLA
enzymes present in these venoms. In another Conus venom, Conus caracteristicus, a related enzyme has been
partially characterized. (
)Thus, we propose that other
PLA
enzymes belonging to this family should also be
referred to as conodipines (the major enzyme from C.
caracteristicus would be referred to as conodipine-C; sequence
variants of conodipine-M from C. magus would be referred to as
conodipine-M1, conodipine-M2, etc.).
Conodipine-M was identified by
an unusual route; the assay actually used to purify this PLA activity was not enzymatic activity but rather the inhibition of
binding of a dihydropyridine drug to L-type calcium channels in rat
brain membranes. We have demonstrated that purified conodipine-M
potently inhibits [
H]isradipine binding under
standard assay conditions. There are two general possibilities. (i) The
inhibition may be relatively nonspecific, i.e. PLA
may be solubilizing the L-type calcium channel; if binding sites
can no longer be filtered, this would appear as inhibition. (ii) An
alternative explanation is that the PLA
produces a
metabolite (perhaps derived from arachidonic acid) which inhibits
binding of isradipine to L-type calcium channels. These possibilities
are presently being investigated. It is notable, however, that a
10-fold greater concentration of conodipine-M than is required to
inhibit completely the binding of [
H]isradipine
to L-type calcium channels exhibits no detectable effect on the binding
of
I-labeled
-conotoxin GVIA to N-type calcium
channels, even though both assays employ filtration of
membranes(55) .
Conodipine-M has two distinct polypeptide
chains that are apparently linked to each other through one or more
disulfide bonds; upon reduction of disulfides, conodipine-M is
dissociated in two unequally sized subunits. A similar situation is
found with several snake venom PLAs that have multiple
subunits. In some cases, at least one of the subunits appears to be
used for targeting the PLA
to a particular membrane
component (e.g. the
-chain of bungarotoxin interacts with
potassium channels)(64) . It is possible that one of the two
subunits of conodipine-M is responsible for the catalytic activity,
with the other subunit involved in targeting the enzyme to specific
sites. The physiological function of PLA
within C.
magus venom remains a matter for conjecture, but it is of interest
that this activity is apparently present in other Conus venoms, which specialize on different prey.
More than 50
PLAs from venoms have been sequenced to date, and three
sequence groups are apparent(32) . Group I is composed of
PLA
s from elapid and Old World snakes. Members of group II
include crotalids and viperids of the New World. Group I and group II
sequences are highly homologous to each other. The major differences
lie in the positioning of the one of the seven disulfides, and group II
enzymes have a short COOH-terminal extension. Group III
PLA
s, which include the enzymes from bee and lizard venoms,
are only about 20% homologous to group I and II enzyme(41) . A
novel PLA
, group IV, has recently been purified from the
cytosol of a number of proinflammatory mammalian cells. It has a
molecular weight of 85,000, and its amino acid sequence shows no
homology to group I-III
enzymes(21, 65, 66) .
The amino acid
sequence of conodipine-M does not reveal any obvious homology with
group I-IV PLAs. However, some similarity may exist
in sequences including and near amino acid residues that are known to
function in the active site. In group I-III PLA
s a
histidine residue, which functions as a general base in assisting the
attack of a water molecule on the substrate ester, is followed by an
aspartate that is one of the ligands for the active-site calcium
cofactor. This pair of residues is flanked by 3 cysteines ( Table 2and Table 4). The
-chain of conodipine-M has a
histidine-aspartate pair flanked by 2 cysteines instead of three, but
it is not known whether these residues function in a way analogous to
those in group I-III PLA
s. Group I-III
PLA
s have an aspartate that is hydrogen-bonded to the
catalytic histidine residue and is flanked by 3 cysteines ( Table 2and Table 4). The
-chain of conodipine-M has an
aspartate residue with only 1 nearby cysteine ( Table 2and Table 4). The sequence Tyr/Trp-Cys-Gly-Xaa-Gly is found in all
group I-III PLA
s. The carbonyl oxygens of the 2
glycines of this sequence are ligands to the calcium cofactor.
Conodipine-M lacks such a sequence.
Like the other enzymes,
conodipine-M requires calcium for its PLA activity,
although the concentration of calcium that gives 50% of maximal
activity (about 20 µM) is considerably lower than typical
values for group I-III enzymes (a few hundred µM).
It is thus significant that conodipine-M lacks the otherwise conserved
calcium binding residues. All sequence factors considered, conodipine-M
clearly represents a very structurally diverse enzyme, and we therefore
propose to classify it as a group V PLA
.
Given the low
homology to other PLAs, it is remarkable that conodipine-M
displays many other features of venom PLA
s as well. The
phospholipid analog MG14 is a transition state analog inhibitor of
group I-III enzymes, and the fact that it is also a potent
inhibitor of conodipine-M suggests that the catalytic mechanism of the Conus enzyme may be similar to the other venom enzymes. Also,
the pH-rate profile for conodipine-M is similar to those for group
I-III enzymes. For these latter enzymes the pH dependence
reflects, in part, that the active-site histidine is in the
nonprotonated form to function as a general base in the catalytic
reaction(67) . Conodipine-M, like other venom PLA
s,
does not significantly discriminate between phospholipids with
different polar head groups or unsaturated versus saturated
acyl chains. Further structural studies of conodipine-M will be
required to understand fully its catalytic mechanism and to compare it
with other known PLA
s. It is not known which conodipine-M
subunit contains the phospholipase activity, but as discussed above,
the
-chain may have similar catalytic sequence motifs. The amino
acids that we have postulated as key catalytic residues need to be
confirmed experimentally. Our speculation clearly predicts, however,
that catalytic activity will be carried out primarily by the
-chain.