ABSTRACT
Phospholipase activity is known to be present in the venoms of many venomous
animals, including snakes, bees, spiders, sea anemones, and one Conus species. Whole
duct venom was extracted from several specimens of Conus californicus, dissolved in
seawater, and centrifuged. The supernatant fraction was observed to possess robust
cytolytic activity. The biochemical basis of this activity was explored using two
enzymatic assays. First, proteolytic activity of the venom supernatant towards casein, a
universal protease substrate, was assayed spectrophotometrically. Following incubation
with the venom supernatant, unreacted casein was precipitated and activity assayed by
measuring any increase in absorbance of the reaction mixture as a result of digested
casein in solution. The venom showed no detectable caseinase activity. Second,
phospholipase activity of the venom supernatant toward L,a-phosphatidyl choline was
assayed. Activity was measured spectrophotometrically, based on an absorbance change
of the pH indicator phenol red due to the liberation of free fatty acids. Significant
phospholipase activity was detected. This activity increased with the addition of calcium,
suggesting that the responsible enzyme is calcium-dependent. Activity was lost
subsequent to heating, suggesting that the responsible enzyme was denatured.
INTRODUCTION
Conus californicus is a predatory marine snail found in the low intertidal and
subtidal zones along the western coast of North America, from San Francisco County in
the north to Baja California in the south (Morris et al. 1980). It preys regularly on
polychaetes, gastropods, and bivalves. As with the approximately 500 other cone snail
species (Olivera 1997), C. californicus has a highly derived radula, reduced to a set of
harpoon-like teeth, which it uses to inject neurotoxic peptides into its prey. Conus venom
is produced in a venom bulb (Maguire and Kwan 1992), which is attached to the
esophagus by a venom duct. The teeth are secreted from the radula sac and passed into
the esophagus, just anterior to the venom duct. From there, they are passed up the
proboscis and are injected into prey from the tip of the proboscis.
Significant research has focused on the small disulfide-rich peptides present in
cone snail venoms, which target calcium and sodium channels as well as various
membrane receptors (Kerr et al. 1984; Mclntosh et al. 1995; Olivera 1997; Duda 1999).
Larger protein components are present in cone snail venoms, but they are less well
described. Mclntosh et al. (1995) describe phospholipase activity in Conus magus, but
this appears to be the only published account of such activity in the genus.
Phospholipases are common in the venoms of a diverse set of venomous animal
taxa, including snakes (Nisenbom et al. 1986; Faure and Bon 1988; Ali et al. 1999), bees
(Ho and Ko 1988; Abe et al. 2000), arachnids (Conde et al. 1999), and sea anemones
(Grotendorst and Hessinger 2000). Phospholipases catalyze the hydrolysis of
phospholipids with varying specificity. Phospholipase Az, the most common
phospholipase component of venoms, catalyzes the hydrolysis of the sn-2 acyl ester of
phospholipids, releasing nonesterified fatty acids and lysophospholipids (Ali et al. 1999)
Phospholipase Az is a critical component of the cytolytic action of venoms from snakes,
bees, and sea anemones, likely facilitating the neurological effects of other venom
constituents (Grotendorst and Hessinger 1999). The presence of a phospholipase Az in a
species of cone snail (Mclntosh et al. 1995) suggests that they might employ a similar
strategy
Duct venom from C. californicus was observed to have a cytolytic effect on
several cell types, including HEK 293 cells, mouse neuroblastoma cells, and goldfish red
blood cells (unpublished data). The cytolytic effect on red blood cells of certain venom
phospholipases (Grotendorst and Hessinger 1999) suggests that a phospholipase could be
responsible for these effect. Because of their detrimental effects to cellular proteins,
proteases could also be responsible. To identify the biochemical basis of the observed
cytolytic activity of C. californicus duct venom, a proteolysis activity assay and a
phosholipase activity assay were employed.
MATERIALS AND METHODS
Venom preparation
Conus californicus snails were collected subtidally in Monterey Bay, California.
Five (5) snails were dissected, and their venom ducts, along with the venom bulbs, were
removed. Venom was extracted by squeezing the entire lengths of the 5 ducts, and the
resulting venom was dissolved in 0.5 ml of seawater. The crude whole duct venom was
centrifuged for 10 min to pellet the non-soluble fraction. The supernatant fraction was
removed and stored at -20'C. The venom was used at that concentration in subsequent
experiments.
Hemolysis assay
The cytolytic effect of the venom supernatant was assayed using red blood cells
from goldfish. The goldfish were sacrificed by rapid decapitation. One to two drops of
released blood were dissolved in 100 ul of frog Ringer solution (120 mM NaCl, 2 mM
CaClz, 5 mM HEPES at pH 7.2). The red blood cells were incubated with various
dilutions of venom supernatant for 5 min. The number of intact cells in I large grid box
of a hemacytometer to which 10 ul of cells containing Ringer had been added was
counted.
Caseinase assay
Measurements of proteolytic activity were made according to the casein digestion
method of Marsh (1971). Caseinase assays were performed using a 0.5% casein solution:
10 mg of casein (Sigma) in a reaction volume of 2 ml of 0.1 M potassium phosphate
buffer, pH 7.4. Dissolving of casein was done in an initial volume of 1 ml of phosphate
buffer in a boiling water bath for 15 min, to which the final 1 ml of phosphate buffer was
added. Venom supernatant or trypsin (Sigma) of various amounts was added to 2 ml of
casein solution to initiate potential casein digestion. Reactions were carried out for 20
min. at 25'C and were terminated by addition of 3 ml of 5% trichloroacetic acid (TCA) to
precipitate undigested casein. After 30 min the solutions were filtered. The optical
densities of the filtrates were measured at 280 nm on a Shimadzu BioSpec-1601
spectrophotometer to detect digested casein in solution.
Phospholipase assay
Phospholipase activity was assayed by the colorimetric assay of de Araujo and
Radvanyi (1987). The reaction medium contained 3.5 mM L-a-phosphatydyl choline
(Avanti Polar-Lipids), 7 mM Triton X-100 detergent (Sigma), 100 mM NaCl, 10 mM
CaCh, and 0.055 mM phenol red (Sigma). pH was adjusted to 7.6 using HCl and NaÖH.
A reaction volume of 1 ml was used. Assays were done at 25’C and lasted 5-10 min
each.
Detection of phospholipase activity is based on enzymatic release of free fatty acids,
which causes an absorbance decrease in the pH indicator phenol red. A wavelength of
558 nm was used for spectrophotometry, which corresponds to an optical density of 1.8 at
a pH equal to phenol red’s pK, which is 7.6. Optical density measurements were made
on a BioSpec-1601 from Shimadzu.
RESULT
Hemolysis assay
Several concentrations of venom were applied to red blood cells to observe its
hemolytic effect. Venom dilutions of 1:10, 1:100, 1:1000, 1:10000, and no venom were
assayed. The relative numbers of cells intact after treatment with each of these dilutions
in frog Ringer solution are given in Fig. 1. The effect of the venom on the cells after 5
min venom treatment appeared to be complete hemolysis. Cells apparently completely
disintegrated by the time the assay could be made, and no remants or debris were visible
in the field of view of the microscope.
Caseinase assay
In the caseinase assay, venom supernatant was added to 2-ml solutions of 0.5%
casein in amounts of 10 ul, 5 ul, 1 ul, 0.1 ul, and 0 ul (control). Trypsin was used as a
positive control in concentrations of 10 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.1 mg/ml, and 0
mg/ml (control). Absorbance increases due to digested casein in solution are plotted in
Fig. 2. Venom supernatant caused only an insignificant increase in the absorbance of the
filtrate: 0.072 to 0.081 absorbance units. Trypsin caused an increase in absorbance of the
filtrate from 0.012 to 1.834 absorbance units.
One unit of caseinase activity is defined as that amount of enzyme that causes an
increase of 0.001 absorbance units per minute. Absorbance change due to addition of 10
ul of venom corresponds to an activity of 0.45 units. The activity of trypsin varied from
29.6 units for 0.1 mg/ml to 91.7 units for 10 mg/ml. One unit of venom can be
extrapolated from this to be 11 ul/ml. One unit of trypsin is 0.0034 mg/ml, as calculated
by the initial reaction rate suggested by the 0.1 mg/ml sample.
Phospholipase assay
The phospholipase activity of the venom supernatant was detected in amounts of
1 ul, 10 ul, and 50 ul in a reaction volume of 1 ml. Activity increased with increasing
venom concentrations. These data are presented in Fig. 3, which shows the decrease in
optical density of phenol red at 558 nm, corresponding to a decrease in pH of the solution
due to the release of free fatty acids by phospholipase. The assay is not reliable beyond
an aborbance change of about -1 absorbance unit. That absorbance is the absorbance of
fully acidified phenol red solution, and the dye is unaffected below a certain pH. Hence,
the flattening of the 50 ul curve at an absorbance change of about -1 absorbance units is
likely not an indication of the absence of substrate or the end of the reaction but rather the
inability of the pH indicator to distinguish between increasingly acidic pHs.
To test the effect of calcium on phospholipase activity, an experiment was run
with 10 ul of venom, using the exact same conditions as before except without adding
CaCl to the reaction medium. (The normal reaction medium contained 10 mM CaCh.)
The results of this experiment are shown in Fig. 4.
To test possible heat effects on enzyme stability, samples of venom supernatant
were heated for 5 min at 65°C or 5 min at 75°C. Following heating, 10 ul of heated
venom was used in the phospholipase assay. The decrease in activity subsequent to
heating as compared to an unheated sample is shown in Fig. 5.
The phospholipase activity of venom supernatant at various dilutions and
conditions are given in Table 1. The first measure of activity given is the decrease in
absorbance per min per ul of venom. The specific activity can be calculated by
calibrating the absorbance change with known mole quantities of hydrochloric acid. A
decrease in absorbance of 0.018 was obtained with 2.5 nmol of HCl in 1 ml of reaction
medium. Hydrolysis of one L,a-phosphatydyl choline molecule yields one L,a¬
lysophosphatydyl choline molecule and one fatty acid molecule. The specific activities
in terms of moles of fatty acid released per min per ul of venom are given. One unit of
phospholipase activity is defined as that amount of phospholipase that will hydrolyze 1.0
umol of L,a-phosphatydyl choline per min. Using this standard, the number of units of
activity of each reaction has been listed in Table 1.
DISCUSSION
The hemolysis assay indicates that venom supernatant is lethal to red blood cells,
with a 1:10 dilution sufficient to kill all the red blood cells within 5 min, and even a
1:10000 dilution had a significant effect. The complete disintegration of cells suggests
that highly active membrane degradation could be taking place. The solution used for
hemolysis assays contained 2 mM CaCl, which has implications for phospholipase
activity. The pH of the solution was 7.2, close to the caseinase assay pH of 7.4 and the
phospholipase assay pH of 7.6.
In the caseinase assay, the small change in absorbance (-0.009 absorbance units)
of the filtrate after reaction with venom corresponds closely to the change in absorbance
of 2 ml of PBS upon addition of 10 ul of venom: 0.002 to 0.010 absorbance units. This
suggests that the venom has little detectable caseinase activity, or at most activity
corresponding to an increase in optical density of just 0.001. By comparison, at the
manufacturer's suggested concentration of 0.1 mg/ml, trypsin cleaved a visibly detectable
amount of casein. Increases in trypsin activity were observed at every higher
concentration used. Future experiments might explore possible caseinase activity at
different pHs and with higher concentrations of venom supernatant. Nevertheless, it
appears that caseinase activity, and presumably protease activity in general, is not
responsible for the cytolytic effect observed on red blood cells.
Duct venom from Conus californicus appears to possess robust phospholipase
activity. As would be expected, phospholipase activity increases with increasing
concentrations of venom. The loss in activity subsequent to heating the venom
supernatant suggests that a heat-labile enzyme is responsible for the acidification of the
phosphatytdyl choline solution and the associated pH and absorbance changes. This
enzyme is presumably a phospholipase.
A phospholipase could serve various purposes in C. californicus. Digestive
phospholipases are present in high concentrations in nature in intestinal fluids, bacterial
secretions, and venoms (Stryer 1995). Its presence in the venom duct, however, suggests
that it may be a component of the injected venom. Cytolytic activity of phospholipases in
venoms is important to several venomous animals (Grotendorst and Hessinger 1999), and
it may serve such a role in C. californicus. Cytolytic activity could increase exposure of
prey tissue to neurotoxic conopeptides present in the venom. Phospholipase activity
could be involved in the secretion of peptides into the venom duct or in some other part
of peptide production or processing. Phospholipases Az have also been noted to have
several myotoxic and neurotoxic effects (Ho and Ko 1988). Another major function of
phospholipases is cellular signaling.
Phopspholipases generate highly active signal
molecules and their precursors (Stryer 1995). More investigations are necessary to
determine what purpose phospholipase activity might serve in the venom.
The apparent calcium-dependence of phospholipase activity in Conus californicus
is not surprising. Most phospholipases A2 require mM concentrations of calcium for
activation (Kasurinen and Vanha-Perttula 1987; Mclntosh et al. 1995; Ali et al. 1999
Costa and Palma 2000). Calcium serves as a cofactor for these molecules, binding near
the active site. Future work should examine phospholipase activity in the presence of a
chelating agent such as EGTA or EDTA to bind and remove calcium from solution, such
as described by Kasurinen and Vanha-Perttula (1987).
If calcium is a necessary cofactor for phospholipase activity, why was there
appreciable activity in the reaction to which no CaClz was added? This activity could be
due to calcium present in the seawater in which the venom was dissolved. Seawater
contains approximately 10 mM Ca“. In a 1-ml reaction with 10 ul venom in seawater,
this would be diluted to about 0.1 mM Ca“. This is conceivably enough calcium to
permit the moderate phospholipase activity that was observed. Mclntosh et al. (1995
report no decrease in Conus magus phospholipase activity in CaClz concentrations down
to O.1 mM. Although this low concentration apparently supports phospholipase activity
in Conus californicus, the activity approximately doubles in 10 mM CaCh, suggesting
that more that 0.1 mM CaCl may be necessary for full stimulation, although 10 mM is
probably excessive.
Mclntosh et al. (1995) report a specific activity for cleavage of an sn-2 thiolester
phospholipid analog by conodipine-M, the phospholipase in C. magus, of 22 mmol per
min per mg phospholipase at a concentration of I mg enzyme per ml. It is difficult to
compare this to the maximal C. californicus activity of 1.724 x 1033 mol per min per ul
because the concentration of phospholipase in the venom supernatant is not yet known.
This information would be useful in determining the relative importance of
phospholipases in the survival of these two cone snails.
Future experiments should be run using different phospholipase assays. The
colorimetric assay of Kasurien and Vanha-Perttula (1987) should be considered to
investigate calcium-dependence, and certain fluorometric assays may be appropriate as
well. The colorimetric assay used in these experiments is not specific for phospholipases
Az because they are not the only species that catalyze the release of fatty acids.
Specificities of the various phospholipases in reactions with phospholipids are shown in
Fig. 6. More specific phospholipase assays should be employed to determine the
enzyme’s specificity.
Subsequent work on this enzyme could focus on its separation from other proteins
in the venom supernatant and on its purification. This would facilitate further
characterization of activity, pH dependence, sequencing of the enzyme, and so forth.
Additional studies directed at elucidating the biological relevance of the enzyme to
venom production or activity would also be fruitful.
ACKNOWLEDGEMENTS
I would like to thank Peter Fields for his time and sage advice, Joseph Schulz for
allowing me to figure it out for myself, Mat Brock for donating his cells, and William
Gilly for his continuous support and wisdom.
REFERENCES
Abe, T., M. Sugita, T. Fujikura, J. Hiyoshi, and M. Akasu. 2000. Giant hornet (Vespa
mandarinia) venomous phospholipases: the purification, characterization and
inhibitory properties by biscoclaurine alkaloids. Toxicon 38:1803-1816.
Ali, S. A., J. M. Alam, S. Stoeva, J. Schütz, A. Abbasi, Z. H. Zaidi, and W. Voelter.
1999. Sea snake Hydrophis cyanocinctus venom. I. Purification, characterization
and N-termincal sequence of two phospholipases A2. Toxicon 37:1505-1520.
de Araujo, A. L. and F. Radvanyi. 1987. Determination of phospholipase Az activity by a
colorimetric assay using a pH indicator. Toxicon 25:1181-1188.
Conde, R., F. Z. Zamudio, B. Becerril, and L. D. Possani. 1999. Phospholipin, a novel
heterodimeric phospholipase Az from Pandinus imperator scorpion venom. FEBS
Letters 460:447-450.
Costa, H. and M. S. Palma. 2000. Agelotoxin: a phospholipase A2 from the venom of the
neotropical social wasp cassununga (Agelaia pallipes pallipes) (Hymenoptera¬
Vespidae). Toxicon 38:1367-1379
Deems, R. A. and E. A. Dennis. 1981. Phospholipase A2 from cobra venom (Naja naja
naja). Methods Enzymol. 71:703-710.
Duda, T. F. and S. R. Palumbi. 1999. Molecular genetics of ecological diversification:
duplication and rapid evolution of toxin genes of the venomous gastropod Conus.
Proc. Natl. Acad. Sci. USA 96:6820-6823.
Faure, G. and C. Bon. 1988. Crotoxin, a phospholipase Az neurotoxin from the South
American rattlesnake Crotalus durissus terrificus: purification of several isoforms
and comparison of their molecular structure and of their biological activities.
Biochemistry 27:730-738.
Grotendorst, G. R. and D. A. Hessinger. 2000. Enzymatic characterization of the major
phospholipase Az component of sea anemone (Aiptasia pallida) nematocyst
venom. Toxicon 38:931-943.
Ho, C. L. and J. L. Ko. 1988. Purification and characterization of a lethal protein with
phospholipase Aj activity from the hornet (Vespa basalis) venom. Biochim.
Biophys. Acta 963:414-422.
Kerr, L. M. and D. Yoshikami. 1984. A venom peptide with a novel presynaptic blocking
action. Nature 308:282-284.
Kasurinen, J. and T. Vanha-Perttula. 1987. An enzymatic colorimetric assay of calcium¬
dependent phospholipases A. Anal. Biochem. 164:96-101
Maguire, D. and J. Kwan. 1992. Coneshell venoms—synthesis and packaging. pp. 11-18
in D. Watters, M. Lavin, D. Maguire, and J. Pearn, eds. Toxins and Targets:
Effects of Natural and Synthetic Poisons on Living Cells and Fragile Ecosystems,
Haywood Academic, New York.
Marsh, H. 1971. The caseinase activity of some vermivorous cone shell venoms. Toxicon
9:63-67.
Mclntosh, J. M., F. Ghomashchi, M. H. Gelb, D. J. Dooley, S. J. Stoehr, A. B. Giordani,
S. R. Naisbitt, and B. M. Olivera. 1995. Conodipine-M, a novel phospholipase A2
isolated from the venom of the marine snail Conus magus. J. Biol. Chem.
270:3518-3526.
Morris, R. H., D. P. Abbott, and E. C. Haderlie. 1980. Intertidal Invertebrates of
California, Stanford University Press, Stanford, California.
Nisenbom, H. E., C. Seki, and J. C. Vidal. 1986. Phospholipase A2 from Bothrops
alternatus venom: purification and some characteristic properties. Toxicon
24:259-272.
Olivera, B. M. 1997. Conus venom peptides, receptor and ion channel targets, and drug
design: 50 million years of neuropharmacology. Mol. Biol. Cell 8:2101-2109.
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TABLES
Table 1. Phospholipase activities of reactions with various venom concentrations and
conditions. Activities are given in change in absorbance per min per ul venom, moles
fatty acid produced per min per ul venom, and units of phospholipase activity. The
apparent drop in activity with increasing concentrations is due to normalizing the
activities according to the amount of venom used. This reflects the diminishing effect of
adding more venom due to the rate-limiting kinetics of the reaction.
Venom per 1-ml reaction
Aminsul venom
mol acid/min/ul venom
Units
1 ul venom, 10 mM Cach,
-0.124
1.724E-08
0.017
10 ul venom, 10 mM Cach
-0.0587
8.159E-09
0.082
50 ul venom, 10 mM Cach,
-0.0175
2.433E-09
0.122
10 ul venom, No Caciz
-0.0257
3.572E-09
0.036
10 ul venom, 10 mM Cachz, 75°C
-0.0093
1.293E-09
0.013
FIGURE LEGENDS
Fig. 1. Relative numbers of goldfish red blood cells intact after treatment with four
venom dilutions in frog Ringer solution after incubation for 5 min. A 1:10
dilution of venom in the presence of 2 mM CaClz is sufficient to lyse all the red
blood cells.
Fig. 2. Increase in absorbance (at 280 nm) of TCA-precipitated casein following
digestion by venom supernatant or trypsin. Venom supernatant does not
detectably degrade casein in comparison to trypsin, which had significant activity
with the lowest concentration tested (0.1 mg/ml). The slight increase in
absorbance from 0 ul venom to 10 ul venom can be attributed to the absorbance
of the added venom alone.
Fig. 3. Concentration-dependent decrease in optical density of phenol red at 558 nm,
corresponding to a decrease in pH of the solution due to the release of free fatty
acids via phospholipase activity. Reactions of 1 ul, 10 ul, and 50 ul in the
presence of 10 mM CaClz are shown.
Fig. 4. Calcium-dependence of phospholipase activity, using the same assay as in Fig. 3.
Decrease in optical density over time is shown for reactions of 10 ul venom with
and without presence of 10 mM CaCl.
Fig. 5. Decrease in phospholipase activity subsequent to heating at 65°C or 75°C for 5
min as compared to an unheated sample. The same assay was used as that used in
conjunction with Fig. 3.
Fig. 6. Specificity of phospholipases on a generic phospholipid molecule. For
phosphatydyl choline, Rz =-CHz-CH2-N(CH3);. Hydrolysis by either
phospholipase Aj or phospholipase Az causes the release of free fatty acid.
FIGURES
Fig. 1.
120
100
80
60
4
E 20
0 +


* 1:10000
° 1:1000
Venom Dilution
1:100
1.40
Fig. 2.
2
1.8

E16
314
1.2
08
506
4 04
P-O-O
4
1
9 10 11
ul venom/2 ml or mg trypsinlm
Trypsin (positive contro)
Venom
—— Linear (Venom)
ve contre
Log. (Trypsin (
20
Fig. 3.

0 %
100 200 300 400 500 600 700
-0.2 +

304

-0.6


08

-1.2 -
Time (s)
venommnl s 1Ovenomim 4S0uvenomim
21
Fig. 4.
O
„ 100 200 300 400 500 600 700
E 02 f
9 94

10
06
.

1
-1.2 L
Time (s)
e 10mM Catt, 10 ulvenomimi ANo Cat added, 10 lvenonimi
Fig 5.
100
20
o



Temperature (degrees c)

23
Fig. 6.
Ra
4.
O

COCR.
COC
O
0—
1CO



24