Intraspecific variation of venom injected by fish-hunting Conus snails
,
1 Department of Chemistry and the Beckman Institute, University of Illinois
at Urbana-Champaign, Urbana, IL 61801, USA
2 Hopkins Marine Station of Stanford University, Department of Biological
Sciences, Pacific Grove, CA 93950, USA
Authors for correspondence (e-mail:
lignje{at}stanford.edu;
sweedler{at}scs.uiuc.edu;
jschulz{at}oxy.edu)
Accepted 25 May 2005
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Summary |
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Key words: Conus striatus, Conus catus, conotoxin, injected venom, duct venom, intraspecific variation, liquid chromatography, mass spectrometry
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Introduction |
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Isolation of venom peptides from Conus snails in the vast majority
of cases has employed crude venom obtained from dissected venom ducts (duct
venom, DV), with material being pooled from multiple animals. The use of DV
enables peptide characterization based on conventional analytical and
sequencing methods that require fairly large amounts of material. However, it
is not clear how peptides in DV are related to those actually injected during
prey capture. In several studies, venom expelled through the radular tooth
during feeding behavior was collected by a simple `milking' procedure (Bingham
et al., 2005,
1996
;
Hopkins et al., 1995
;
Martinez et al., 1995
; Shon et
al., 1997
,
1995
;
Teichert et al., 2004
;
Walker et al., 1999
), and
analysis indicated that this injected venom (IV) was not identical to DV from
the same species (Bingham et al.,
1996
; Martinez et al.,
1995
). Venom milking is thus advantageous because it provides the
biologically relevant peptides used to subdue prey and does not require
sacrificing the snail. However, there have been no detailed comparisons of the
complement of peptides in DV with that in IV.
Additionally, prior research has not addressed variation in the IV
composition from snail to snail within a given species. In studies employing
IV (Bingham et al., 2005,
1996
;
Hopkins et al., 1995
;
Martinez et al., 1995
; Shon et
al., 1997
,
1995
;
Teichert et al., 2004
;
Walker et al., 1999
), the
samples were pooled from multiple snails, and therefore the degree of
intraspecific variation could not be assessed. Intraspecific variation has
been examined using DV from individual snails; however these studies have
produced conflicting results. Variation in the peptide profiles of DV between
C. textile individuals from the same reef has been reported
(Bingham et al., 1996
;
Jones et al., 1996
), whereas
DV of C. regius was found to be consistent from snail to snail,
regardless of the gender or size of the animals or season of collection
(Vianna Braga et al.,
2005
).
Genes encoding Conus toxins have evolved by a mechanism of strong
positive selection (Conticello et al.,
2001; Duda and Palumbi,
2004
,
1999
,
2000
;
Espiritu et al., 2001
), and
diversification of these peptide toxins may be responsible for success in the
acquisition of new feeding behaviors and niche expansion leading to speciation
within the genus. Although the biological relevance of these peptides is
widely recognized, it is not known how the expression, maturation and delivery
of these toxins are regulated. Thus, a detailed comparison of the peptides in
DV and IV from individual snails represents a first step in elucidating the
mechanisms involved in the delivery of toxins for prey capture.
In this study, peptides in both IV and DV were collected from individual snails and characterized using microbore high-performance liquid chromatography (HPLC) coupled with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-MS) and electrospray ionization-ion trap-mass spectrometry (ESI-MS). In many cases, these sensitive methods permit analysis of samples smaller than an IV sample from a single milking. MS is also information-rich and selective, providing a more complete profile of peptides than is possible with ultraviolet/visible (UV/Vis)-absorbance detection alone.
Here, we report the first significant intraspecific differences in the composition of venom peptides injected by individual piscivorous snails, C. striatus and C. catus. In addition, we find that the profile of peptides in IV, including known and putative novel peptides, is far less complex than the profile observed for DV.
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Materials and methods |
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Venom collection
Conus striatus L. (6.57 cm shell length: Tutuila Island,
American Samoa) and Conus catus Hwass in Bruguière 1792
(1.84.3 cm shell length: Kauai, Hawaii) were collected from limited
areas of coral reef-flats and subsequently maintained at Hopkins Marine
Station in closed tanks at 27°C under equivalent environmental conditions.
All snails were fed live fish about once per week. Conus striatus
were fed commercially procured goldfish and C. catus were fed small
marine fish, generally sculpins (Clinocottus spp. and
Oligocottus spp.), collected from local intertidal areas. Captive
snails were milked to obtain IV samples as described elsewhere
(Hopkins et al., 1995).
Briefly, a snail was induced to inject venom into a 0.5 or 1.5 ml centrifuge
tube covered by a latex membrane with a fish fin exposed on top. This
procedure was periodically carried out with identified C. striatus
and C. catus individuals for several months, after which (in the case
of the C. striatus) the animals were killed, and venom ducts were
dissected. DV samples were prepared by manually rolling out the duct contents
with further treatments as described below.
Conus striatus injected venom fractionation and mass spectrometry
In Fig. 1, single IV samples
from C. striatus (2550 µl) were fractionated on an LKB
Bromma HPLC system (Sweden) fitted with a MicrosorbTM (Rainin) column
(C18, 4.6 mmx150 mm, 5 µm particle diameter, 10 nm pore
size). Separations utilized a uniform flow rate of 0.5 ml
min1 with a solvent B gradient of 5% to 50% over 36 min and
50%80% B for an additional 9 min [Solvent A: H2O, 0.1% TFA
(v/v); Solvent B: acetonitrile, 0.08% TFA (v/v)]. Fractions were analyzed by
MALDI-MS in linear and reflectron modes as described below.
|
|
For data presented in Fig. 3D,E,F, two C. striatus IV samples were pooled for each snail, and the entire volume was fractionated using the same microbore HPLC system and solvents as for Fig. 2A,C. A separation at a uniform flow rate of 100 µl min1 using a Vydac (Hesperia, CA, USA) Reverse Phase Polymer column (2.1 mmx150 mm) with 5 µm particles and 30 nm pore size was performed. The solvent gradient began by increasing solvent B from 5% to 15% over 5 min and was completed by a gradient up to 65% B over the next 60 min. Detection was performed via a dual UV/Vis detector set at 220 nm and 280 nm as well as with online ESI-MS as described above.
|
The remaining volume (15%) of each DV sample was subjected to the same online HPLC-ESI-MS experiment as described above for the IV samples. Manual deconvolution of the ESI-MS data yielded peptide masses consistent with those obtained by MALDI-MS. Dual, complementary mass-spectrometric techniques were performed to aid in the proper mass assignment of peaks.
In order to avoid carryover of peptides from sample to sample, an extensive flushing procedure was performed between each venom sample.
MALDI mass spectrometry of venom fractions
As noted above, IV and DV fractions from C. striatus were
subjected to MALDI-MS following an HPLC separation. For the C. catus
samples, IV was collected and directly subjected to MALDI-MS. A 0.5 µl
aliquot of each sample (C. striatus venom fraction or unpurified
C. catus IV) was spotted onto a gold-plated target along with 0.5
µl of a matrix (15 mg CHCA, 600 µl acetonitrile, 400 µl water, 3
µl TFA). Positive-ion mass spectra were acquired using linear and
reflectron modes on a Voyager DE STR (Applied Biosystems, Foster City, CA,
USA) time-of-flight mass-spectrometer equipped with delayed ion extraction. A
pulsed nitrogen laser (337 nm) was used as a desorption/ionization source.
External mass calibration was performed using a peptide standard mixture
containing 60 µmol l1 of -bag cell peptide
19, 120 µmol l1 of acidic peptide, and 120 µmol
l1 of bovine insulin.
Peptide nomenclature
In the present work we use an existing scheme for naming conotoxins as
detailed elsewhere (Walker et al.,
1999). In order to be consistent with this nomenclature, the
previously reported peptide
A-SIVA
(Craig et al., 1998
) and the
reported sequence SIVB (Santos et al.,
2004
) are referred to as s4a and s4b, respectively, because the
high-affinity targets of these venom isolated peptides have not been
determined (W. P. Kelley, J. R. Schulz, J. A. Jakubowski, W. F. Gilly and J.
V. Sweedler, unpublished). The known C. striatus and C.
catus
- and
-conotoxins identified in the present study
also follow the established nomenclature. According to this naming scheme,
sequence SO5 (Lu et al., 1999
)
should be named S6.5, corresponding to venom-isolated peptide s6e; however, a
different amino acid sequence already exists that is named S6.5 that
corresponds to conotoxin
-SVIE
(Bulaj et al., 2001
).
Therefore, we retain the SO5 name for both the sequence and the putative
venom-isolated peptide (and for consistency do the same for SO4).
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Results |
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Two C. striatus individuals exhibited a radically different type
of IV peptide-profile (Fig.
1C). These IV samples contained peptides that were previously
characterized from the DV of this species, including the calcium-channel
blocker -SVIA (Ramilo et al.,
1992
) and nicotinic acetylcholine-receptor blockers
-SI and
-SII (Ramilo et al.,
1992
; Zafaralla et al.,
1988
), as confirmed by MALDI-MS. Interestingly, neither s4a nor
s4b were observed components of the IV from these snails.
Comparison of duct venom and injected venom samples in Conus striatus
In order to assess whether similar variations exist in the peptide
complement of DV, samples of DV from two C. striatus individuals with
distinct IV profiles were analyzed using HPLC-ESI-MS.
Fig. 2A,C show the IV profiles;
the corresponding DV profiles are in Fig.
2B,D, respectively. The base peak chromatograms presented closely
resemble the UV chromatogram profiles and, therefore, have been interpreted
similarly. In this case, one snail injected predominantly -SVIA,
-SI and
-SII (Fig.
2A), similar to the pattern in
Fig. 1C, whereas the other
individual showed the most commonly observed profile of mainly s4a and s4b
(Fig. 2C). Peak splitting of
s4a and s4b was observed (Fig.
2C), possibly due to cistrans
isomerization of the hydroxyproline residues
(Watson and Kenney, 1992
).
According to MS analysis, components within each peak doublet are the same
mass. The prominent peptides labeled in Figs
1C and
2A are similar except for the
last chromatographic peak, which contains peptides of masses 3649 Da and 9433
Da, respectively. Co-elution of the putative 9433 Da and 3649 Da peptides was
likely based on the ESI-MS results; however the MALDI-MS analyses of this
fraction for Fig. 1C did not
assess peptides above 6000 m/z.
The DV samples from these same individuals contain many hydrophobic
peptides in addition to the peptides observed in the IV samples. Most notably,
peptides s4a, -SVIA and
-SI as well as several putative toxins
(SO5 and masses 2556, 3431, 3335 and 2049 Da) were all present in the DV of
both snails despite the distinct nature of the IV components. It is possible
that these hydrophobic components represent precursors of mature conotoxins;
however, there are also known classes of hydrophobic conotoxins (e.g.
-conotoxins; Bulaj et al.,
2001
). Without reduction and alkylation of the disulfide bonds,
reliable sequence information was not obtained from the MS/MS scans of these
masses. While a global reductionalkylation online MS/MS procedure in
conjunction with cDNA sequencing would enable sequence determination
(Jakubowski and Sweedler,
2004
), conotoxin sequencing has not been the focus of this
comparative study.
In every case examined, there were at least 50 putative peptides in the DV,
but only the most abundant peptides are labeled in the figures. It is apparent
both by the number of peaks that appear in the chromatograms, and the masses
labeled, that IV is by no means identical to DV and contains a small subset of
DV peptides, primarily less hydrophobic ones. Identifiable DV and IV peptides
were fully processed from their propeptides and contained the known
post-translational modifications such as carboxy-terminal amidation (s4a,
-SVIB and
-SI) and proline hydroxylation (s4a and
-SVIA;
Craig et al., 1998
;
Ramilo et al., 1992
;
Zafaralla et al., 1988
).
Peptides s4a and s4b were also modified by O-glycosylation and
pyroglutamylation (Craig et al.,
1998
). Thus, the apparent simplification of DV does not reflect
differences in the modified states of the peptides.
Comparison of water-soluble duct venom and injected venom in Conus striatus
We noted that the DV is heterogeneous, containing a clear fluid as well as
white, insoluble material. Similar observations were reported in C.
californicus (Marshall et al.,
2002). In order to further assess differences in DV and IV, IV
samples from three C. striatus individuals with the most common venom
profile (mainly s4a and s4b) were analyzed by HPLC-ESI-MS
(Fig. 3A,C,E), and these data
were compared to the profiles of the water-soluble peptides in the DV from the
same individuals (Fig. 3B,D,F).
In all three comparisons, the peptide mixture comprising IV was significantly
simpler than the water-soluble components of DV, indicating that simply
partitioning the water-soluble peptides from the insoluble matter in DV does
not create IV. Although s4a was common to all DV samples
(Fig. 3B,D,F), the amount of
s4a relative to other peptides was significantly greater in the IV samples
(Fig. 3A,C,E) vs the
DV samples for each of these snails. In addition, the range of masses for the
water-soluble peptides detected in the DV (
10005200 Da) was
different than that for IV peptides from the same snail (
27007700
Da).
MALDI-MS profiling of Conus catus injected venom samples
Conus catus is another fish-hunting snail that employs the same
prey capture strategy as C. striatus. In order to determine whether
intraspecific variation in the peptide profile of IV extends to this species,
IV samples from nine C. catus individuals were analyzed by linear
MALDI-MS (see Table 1). Masses
of all putative peptides detected in at least two individuals (for
simplification) ranged from 1287 to 6910 Da. Variation between individuals
clearly exists, and IV profiles ranged in complexity from quite low (snail A)
to extreme (snail I). MALDI mass spectra of IV samples from snails A and I are
presented in Fig. S1 in the supplementary material.
|
Samples of IV from C. catus individuals A, D and I were also analyzed by reverse-phase HPLC (data not shown) to confirm the varying complexity of the IV profiles. Multiple reverse-phase HPLC runs of the IV samples of snail I also confirmed a stable profile of the IV over time (data not shown) as demonstrated for C. striatus (Fig. 1), but examination of the C. catus DV for comparison has not yet been performed to permit continuation of IV collection with these animals.
Identification of putative conopeptides in Conus striatus venom samples
While the primary goals of this study were to compare venoms between
individuals within one species, as well as within the venom apparatus of a
single snail (IV vs DV), the information-rich MS techniques enabled
us to determine mass matches to a number of identified and putative C.
striatus peptides. Identification of well-known conopeptides by such a
mass-matching approach has been used in another report
(Craig et al., 1995). Since
-SVIA,
-SVIB (Ramilo et al.,
1992
),
-SI,
-SII
(Ramilo et al., 1992
;
Zafaralla et al., 1988
) and
s4a (Craig et al., 1998
) are
well-characterized C. striatus peptides, an observed mass within 0.5
Da of the predicted value is sufficient to identify the peptides, and based on
this criterion, these peptides were identified. We also determined mass
matches based on ESI-MS and MALDI-MS data to within 0.5 Da for three putative
peptides by using the calculated molecular masses with expected
post-translational modifications predicted from published cDNA sequences.
These peptides have yet to be isolated and characterized from venom extracts
and include the predicted O-superfamily members SO4
(Lu et al., 1999
) with three
disulfide bonds and two hydroxyproline residues
(Fig. 3B) and SO5
(Lu et al., 1999
) with three
disulfide bonds (Figs 1C,
2A,B,D,
3D,F), as well as the propeller
peptide (mass 9433 Da; Ellison,
2003
), which may exist as a dimer
(Fig. 2A). The targets of these
putative peptides are thus far unknown, although bioactivity of the propeller
peptide has been reported (Ellison,
2003
). Putative matches to the y4 fragment ion in an
SO5 MS/MS spectrum and the b5 fragment ion in an s4a MS/MS spectrum
(data not shown) provide further confirmation for these peptide designations.
In all cases, further studies are required to verify these putative
assignments.
Additionally, in Fig.
3B,D,F, several different forms of putative C. striatus
contryphans were observed. Contryphan-Vn, identified in C.
ventricosus, modulates certain voltage-gated and
Ca2+-dependent K+ channels
(Massilia et al., 2003), and a
homologous peptide, contryphan-R, occurs in C. radiatus
(Jimenez et al., 1997
). In the
present case, isotopic distribution characteristic of bromination with M and
M+2 peaks in approximately a 1:1 intensity ratio were observed for peptides
with protonated monoisotopic masses of 1011 and 1068 Da. These MS data, as
well as preliminary fragmentation studies (data not shown), are consistent
with these peptides having the same sequences as [des-Gly1]
bromocontryphan-R and bromocontryphan-R, respectively, from C.
radiatus (Jimenez et al.,
1997
). We have also identified a cDNA sequence encoding a putative
contryphan in C. striatus (data not shown) that is identical to the
contryphan-R cDNA sequence from C. radiatus
(Jimenez et al., 1997
), thus
supporting this assignment. Also, peptides with protonated masses 933 and 990
Da were detected that correspond to these putative contryphan peptides without
the bromine (Jimenez et al.,
1996
). In these cases, the M+2 peaks are less than 20% of the M
peak intensity due to the loss of the bromine.
Although putative contryphans are present in the DV samples from C. striatus (Fig. 3B,D,F), they are not likely to be used for prey immobilization, at least in the snails analyzed, because they were not detected in the corresponding IV samples (Fig. 3A,C,E). Further analysis is required to determine the target of the contryphan forms of C. striatus. Table 2 summarizes the known and putative conotoxin matches proposed in this work.
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Discussion |
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Intraspecific variation of the peptides in injected venom
Our results revealed significant intraspecific variation in the peptide
composition of IV for both snail species. In the case of C. catus,
some snails displayed a relatively simple profile (six predominant peptides in
snail A; Table 1), whereas
others employed up to 14 abundant peptides (snail I;
Table 1). In C.
striatus, which was studied in more detail, we found three qualitatively
different types of IV (Fig.
1AC). One IV profile consisted predominantly of the
neuroexcitatory peptide s4a, and a second profile type contained primarily s4a
and the related peptide, s4b. Surprisingly, the third IV type lacked s4a and
s4b, but displayed the Ca2+-channel blockers, -SVIA and
-SVIB, in addition to the nicotinic acetylcholine-receptor blockers,
-SI and
-SII.
These chemically distinct venom types fall into two functional types as
well, based on the predicted effects of the peptide complement on a fish
victim. Due to the presence of excitatory toxins, s4a and s4b, the first two
venom types would undoubtedly produce the strong, convulsive tetanic paralysis
that is well-known for C. striatus
(Craig et al., 1998). In
contrast, these excitatory toxins are lacking in the third IV type, which
contains
- and
-conotoxins that are expected to block
neuromuscular transmission (Olivera,
1997
) and produce a flaccid paralysis of a fish. To our knowledge,
paralysis of prey in this manner has not been reported for C.
striatus. Observation of prey capture for snails that exhibit this third
class of IV profile would certainly be valuable. In addition, further
characterization of the putative propeller
(Ellison, 2003
) and SO5
(Lu et al., 1999
) peptides
observed in IV samples may reveal other prey capture strategies at the
biochemical level within a single species.
Biological processes or physiological mechanisms underlying the variation in IV peptide profiles are not clear. In the present study, the prominent peptides in the IV compositions of individual snails of both species remained quite constant over time in captivity based on analysis of IV samples collected at 16 month intervals. Throughout captivity, all snails within each species were housed and fed in the same manner. Moreover, there was no obvious correlation of IV profile-type with any morphological features or collection locality. It is particularly striking that the two snails with disparate IV profiles analyzed in Fig. 2 were similar in size and shell pattern, and they were both collected at the same time and location. These observations suggest that genetic differences may contribute to the observed individuality in IV profiles, although environmental factors or ontogenetic differences cannot be ruled out.
To our knowledge, such a remarkable degree of intraspecific variation in
the peptide composition of venom is novel. In the case of Conus, we
are unaware of any reports of variation in the IV profile between individuals.
Striking divergence of peptide complements when comparing venoms of different
Conus species has been noted
(Olivera, 1997). However, the
existence of multiple distinct IV profiles within the same species reveals an
even greater level of peptide diversity in Conus venoms than
heretofore realized.
Significantly less dramatic variations in the peptide complement of
injected venoms have been reported for several other organisms, including
snakes (Chippaux et al., 1991;
Creer et al., 2003
; Daltry et
al., 1996a
,
b
;
Francischetti et al., 2000
;
MacKessy et al., 2003
;
Monteiro et al., 1998a
,
b
), spiders
(Binford, 2001
;
Cristina de Oliveira et al.,
1999
; Escoubas et al.,
2002
), scorpions (El Hafny et
al., 2002
; Kalapothakis and
Chavez-Olortegui, 1997
;
Pimenta et al., 2003
) and bees
(Lai and Her, 2000
). Variation
of the peptide profiles of these other venomous animals has been associated
with sex (Binford, 2001
;
Cristina de Oliveira et al.,
1999
; Escoubas et al.,
2002
), diet (Daltry et al.,
1996b
), age (Escoubas et al.,
2002
), geography (Binford,
2001
; Creer et al.,
2003
), season (Monteiro et
al., 1998b
) and venom regeneration time
(Pimenta et al., 2003
).
Studies controlling many of these factors have still observed venom variation,
implying that intraspecific differences can be a result of genetic as well as
environmental factors (Daltry et al.,
1996a
; Francischetti et al.,
2000
; Kalapothakis and
Chavez-Olortegui, 1997
; Monteiro et al.,
1998a
,
b
). Cellular and molecular
mechanisms underlying and controlling such variation remain unknown.
Differences in composition of duct venom and injected venom
A consistent finding in this study, based on analysis of individual C.
striatus, is that the peptide composition of DV is significantly more
complex than that of IV, in agreement with previous studies
(Bingham et al., 1996;
Martinez et al., 1995
). In
general, IV appears to contain a substantially simplified subset of the DV
peptides (Fig. 2). Furthermore,
analysis of the water-soluble DV (Fig.
3) is not consistent with creation of IV by simply partitioning
the water-soluble peptides from the insoluble matter in DV. These observations
suggest that a more complex selection process exists by which certain peptides
are transported from the venom duct and utilized for injection, whereas others
are not. Moreover, many of the rejected forms are functional toxins that have
previously been identified in DV.
Mechanisms underlying the evident simplification of DV for injection are
unknown, although anatomical studies of C. californicus suggest that
a specialized epithelial zone connecting the venom duct to the pharynx may be
the site of such processing (Marshall et
al., 2002). Additionally, the pronounced intraspecific variation
observed in IV peptide profiles of both C. striatus and C.
catus suggests that selection and delivery processes, which ultimately
yield the biologically relevant venom used in prey capture, are likely to be
complex and specifically utilized by individual snails to varying degrees. The
intricacy of the venom preparation process is further exemplified by the
distinct IV complements (Fig.
2A,C) that were created from relatively similar DV profiles
(Fig. 2B,D). The
reproducibility of DV profiles from snail to snail is in qualitative agreement
with a recent study showing that the composition of DV in Conus
regius is consistent even for snails of different gender and size or
those collected in different seasons
(Vianna Braga et al.,
2005
).
Our data generally support a theory of venom processing like that discussed
above, although there are potentially additional facets of the venom
preparation process. Notably, IV may contain peptides (e.g. -SVIB in
Fig. 3) that cannot always be
detected in DV from the same snail. The apparent lack of these peptides in the
complex DV samples may be partially due to ion suppression effects. However,
we cannot rule out some long-term changes in DV composition over time in
captivity that are not reflected in IV samples obtained before the snails were
killed. Alternatively,
-SVIB (and other peptides) may be processed from
larger precursors in the venom duct, or exported out of the duct into IV more
efficiently than other peptides. Another possibility is that certain peptides
may be synthesized in some component of the venom apparatus other than the
venom duct, for example in the radular sac where the teeth are stored prior to
use. Previous work on C. californicus revealed peptides inside the
lumen of stored teeth (Marshall et al.,
2002
), but neither the identity of those peptides nor their route
of delivery is known. Similar studies have not been performed for any other
species. To further elucidate the mechanisms of conotoxin processing, it would
be informative to compare cDNA libraries created from the radular sac and
other parts of the venom apparatus with those created using venom duct tissue.
Variation in gene expression is known to exist even within the venom duct, as
shown by a recent analysis of Conus textile venom ducts
(Garrett et al., 2005
).
Studies of injected venom and conservation of Conus resources
Recently, concern over the potential over-exploitation of Conus
snails for research purposes was discussed
(Chivian et al., 2003;
Duda et al., 2004
). The
present study demonstrates that the analysis of IV samples and a limited
number of DV samples can yield a large data set from a small number of
animals. Samples of IV can be obtained from the same snails over the course of
several years for both chemical and physiological analyses. Furthermore,
knowledge of a particular snail's IV profile can facilitate isolation and
identification of peptides with novel or particularly high bioactivity, such
as in the case of the snail displaying the s4a-type of profile in
Fig. 1A. In the extreme case,
analysis of IV samples by sensitive MS-based sequencing techniques can enable
peptide discovery and characterization without the sacrifice of any animals
whatsoever.
List of abbreviations
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Acknowledgments |
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Footnotes |
---|
* Present address: GlaxoSmithKline, 709 Swedeland Road UW2930, King of
Prussia, PA 19406, USA
Present address: Occidental College, Los Angeles, CA 90041, USA
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