DTD 5 ARTICLE IN PRESS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
OOF
Mass spectrometric and high performance liquid chromatography
profiling of the venom of the Brazilian vermivorous
mollusk Conus regius: feeding behavior and identification
of one novel conotoxin
Maria Cristina Vianna Bragaa,1, Katsuhiro Konnob, Fernanda C.V. Portarob,Jose Carlos de Freitasa, Tetsuo Yamanec,
Baldomero M. Oliverad, Daniel C. Pimentab,*
aDepartment of Physiology, Bioscience Institute, University of Sao Paulo, Sao Paulo, SP, BrazilbMass Spectrometry Laboratory, CAT/CEPID, Butantan Institute, Av Vital Brasil, 1500, Sao Paulo, SP 05503900, Brazil
cMolecular Toxinology Laboratory, Butantan Institute, Sao Paulo, SP, BrazildDepartment of Biology, University of Utah, Salt Lake City, UT, USA
Received 15 April 2004; revised 27 September 2004; accepted 29 September 2004
R 8283
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
RRECTED PAbstract
Carnivorous mollusks belonging to the genus Conus paralyze their prey by injecting a rich mixture of biologically
active peptides. Conus regius is a vermivorous member of this genus that inhabits Brazilian tropical waters. Inter-, intra-
species and individual variations of cone snail venom have been previously reported. In order to investigate intra-specific
differences in C. regius venom, its feeding behavior and the correlation between these two factors, animals were pooled
according to gender, size and season of collection, and their venom composition was compared by high performance
liquid chromatography (HPLC). Both the whole venom and one specific peak were monitored by HPLC.
Chromatographic profiles revealed no significant differences in their peak areas, indicating that the venom composition,
based solely in the presence or absence of the major peaks, is stable regardless of season, gender and size. Therefore,
analysis of one given toxin, eluting in one of the major peaks, is representative among the population. Moreover, this
work presents the identification of one novel conotoxin (rg11a), which amino acid sequence was deduced by mass
spectrometry.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Conus regius; Feeding behavior; Intra-specific variation; High performance liquid chromatography; De novo sequencing; Natural
peptides; Venom; Toxin; Conotoxin
Toxicon xx (xxxx) 1–10
www.elsevier.com/locate/toxicon
UNCO
0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2004.09.018
Abbreviations: Abs, absorbance; ACN, acetonitrile; FDA, Food and Drug Administration; HPLC, high performance liquid chromatography;
RP-HPLC, reversed-phase high performance liquid chromatography; TFA, trifluoroacetic acid; LC–MS, liquid chromatography–mass
spectrometry; MS, mass spectrometry; MS/MS, tandem mass spectrometry; DTT, dithiothreitol.
* Corresponding author. Tel.: C55 11 3726 7222x2042; fax: C55 11 3721 6605.
E-mail addresses: [email protected] (M.C. Vianna Braga), [email protected] (D.C. Pimenta).1 Rua do Matao, Travessa 14, Numero 321, Sala 300, Sao Paulo, SP 05508-900, Brazil. Tel.: C55 11 3091 7522; fax: C55 11 3091 7568.
TOXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MODEL 4 – pp. 1–10
102
103
104
105
106
107
108
109
110
111
112
C
T
M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–102
DTD 5 ARTICLE IN PRESS
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
RE1. Introduction
Conus is a genus of carnivorous mollusks that uses a
cocktail of peptide toxins (from 50 to 200 different
molecules) to selectively capture prey, defend and/or escape
from predators or to deter competitors (Olivera, 1997). With
a harpoon-like modified radular tooth, each species of cone
snail delivers the complex mixture of biologically active
peptides that target specific receptors and ionic channels of
the prey, quickly and efficiently immobilizing it, through
molecules targeting the nervous system (Olivera et al.,
1990). Cone snails are successful predators that are
generally classified into three groups, depending upon
their prey. The largest class is the worm-hunting species
that prey on polychaetes worms; the second class is
molluscivorous cone snails that hunt other gastropods; and
the most remarkable group is the fish-hunting cone snails
(Terlau and Olivera, 2004).
Cone snails can be found in almost every type of tropical
marine habitat (Olivera et al., 1990). In Brazilian tropical
waters, there are approximately 18 species of cone snails.
Conus regius (Gmelin, 1791) is a species that inhabits rocky
and coral bottoms around Florida, in Central American coast
and in north, northeast and east coast of Brazil; and also in
shallow waters in Fernando de Noronha Archipelago,
Pernambuco, Brazil (Rios, 1985; Eston et al., 1986).
As each species of Conus is presumably under selective
pressure to be efficient at capturing the spectrum of prey that
it feeds on, their venom has its own distinct complement of
peptides (Olivera et al., 1999). Knowing its feeding behavior
is crucial to understand venom composition. So, in this study,
C. regius feeding behavior was investigated, as well as
variations in its venom composition. Bingham et al. (1996)
reported that there is a surprising variability in the molecular
composition of Conus toxins both between and within
species, and even within individual specimens. While
collecting C. regius at Fernando de Noronha Archipelago,
it was possible to notice two groups with different shell sizes.
During dissection, it was also possible to separate the animals
according to gender. So, specimens of C. regius were divided
into eight groups, and their venom composition, compared as
shown.
R 211212
213
214
215
216
217
218
219
220
221
222
223
224
UNCO2. Material and methods
2.1. Materials
Specimens of C. regius were collected from Fernando
de Noronha Archipelago, Pernambuco, Brazil (Eston et al.,
1986). The Brazilian Environmental Agency (IBAMA—
Instituto Brasileiro do Meio Ambiente e dos Recursos
Naturais Renovaveis) license numbers were 030/2000 and
087/2001; and the process number was 02001,000775/
00-00. The mollusks were kept alive in salt-water aquarium
for feeding behavior observation. Venom was extracted
OXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MODE
TED PROOF
from the specimens as previously described (Cruz et al.,
1976). Voucher material is deposited in the malacological
collection of Zoology Museum of University of Sao Paulo,
Sao Paulo, Brazil.
2.2. Reagents
DTT, trypsin mass spectrometry grade, a-cyano-4-
hydroxycinnamic acid, iodoacetamide and NaI were
purchase from Sigma–Aldrich (USA).
2.3. Feeding behavior observation
Prior to the dissection, the animals were placed in small
tanks and they were offered fish (Bathygobius soporator),
fire-worms (Eurythoe complanata) and other mollusks
(Tegula sp. and Astrea sp.). The feeding behavior was
observed and depicted.
2.4. Crude venom
The crude venom was obtained by dissection of
the venom duct gland and then freeze-dried and stored at
K70 8C (Cruz et al., 1976). The animals were divided into
eight groups according to their shell size (from 3.0 to 4.5 cm
and from 5.5 to 7.5 cm), gender (male and female) and
season of collection (winter and summer).
2.5. Peptide fractionation and purification
The amount of lyophilized venom from each group was
normalized to 240 mg and extracted with acetonitrile 20, 40
and 60%. Then, the crude venom extract was fractionated by
analytical HPLC using a Merck C-18 column (5m, 5!250 mm) and a two-solvent system: (A) trifluoroacetic acid
(TFA)/H2O (1:1000) and (B) TFA/acetonitrile (ACN)/H2O
(1:900:100). The column was eluted at a flow rate of
1.0 mL/min with a 0–60% gradient of solvent B over
120 min. The HPLC column eluates were monitored by their
absorbance at 220 nm. For rg11a purification two further
chromatographic steps were necessary, namely, isocratic
chromatographic elution at 40% of methanol/H2O/TFA
(900:100:1) and subsequent purification by binary gradient
at a flow rate of 1.0 mL/min with a 20–40% gradient of
solvent B over 40 min. For microLC–MS analyses, an Ettan
microLC (Amersham Biosciences, Sweden) was employed
using a mRPC C2/C18 ST 1.0/150 column (Amersham
Biosciences, Sweden) and a two-solvent system A1: formic
acid 0.1% and B1: ACN/H2O/formic acid (900:100:1).
2.6. Mass spectrometric analyses
Molecular mass analyses of the peaks and peptides
were performed on a Q-TOF Ultima API (Micromass,
Manchester, UK), under positive ionization mode
and/or by MALDI-TOF mass spectrometry on an Ettan
L 4 – pp. 1–10
TED PROOF
Fig. 1. (A) Conus regius with its extended proboscis, after prey
detection (natural shell sizeZ4.2 cm). (B) The exact moment of
venom injection in the fire-worm E. complanata. (C) After
paralyzed, the prey is engulfed by Conus regius.
M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–10 3
DTD 5 ARTICLE IN PRESS
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
MALDI-TOF/Pro system (Amersham Biosciences, Swe-
den), using a-cyano-4-hydroxycinnamic acid as matrix.
2.7. ‘De novo’ peptide sequencing
Mass spectrometric ‘de novo’ peptide sequencing was
carried out in positive ionization mode on a Q-TOF Ultima
API fitted with an electrospray ion source (Micromass,
Manchester, UK). The amounts previously lyophilized were
dissolved in 50 mM ammonium acetate, reduced with DTT,
alkylated by iodoacetamide and hydrolyzed by trypsin,
according to Westermeier and Naven (2002). The reaction
products were then lyophilized and dissolved into a mobile
phase of 50:50 aqueous formic acid 0.1%/acetonitrile and
flowing into the source at 5 ml/min by infusion pump, or
directly injected (10 ml) using a Rheodyne 7010 sample loop
coupled to a LC-10A VP Shimadzu pump at 20 ml/min,
constant flow rate. The instrument control and data
acquisition was conducted by MassLynx 4.0 data system
(Micromass, Manchester, UK) and experiments were
performed by scanning from a mass-to-charge ratio (m/z)
of 50–1800 using a scan time of 2 s applied during the whole
chromatographic process. The mass spectra corresponding
to each signal from the total ion current (TIC) chromato-
gram were averaged, allowing an accurate molecular mass
determination. External calibration of the mass scale was
performed with NaI. For the MS/MS analysis, collision
energy ranged from 18 to 45 and the precursor ions were
selected under a 1-m/z window.
2.8. Statistical analysis
Each major peak in the different groups was averaged
and the standard deviation (s) was calculated. The value of
G2s was chosen for significant difference. The area peaks
were also compared by the Z-test.
C 317318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
UNCORRE3. Results
3.1. Feeding behavior
As feeding behavior and venom composition are closed
related, C. regius’ predatory behavior was analyzed. Animals
were placed in small tanks and they were offered at least two
different options of potential prey, fish (B. soporator), fire-
worms (E. complanata) and/or other mollusks (Tegula sp.
and Astrea sp.). Thirty different specimens of C. regius were
observed and all of them preyed upon the fire-worms. This
species of polychaete, E. complanata, was chosen for being
abundant in the cone snail natural habitat and for being easily
found in all Brazilian coast, from the Northeast (where
animals were collected) to the Southeast coast (where the
feeding behavioral experiments were performed).
The predation and ingestion of the prey are illustrated in
Fig. 1. Once the prey is detected, C. regius extends its
TOXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MOD
proboscis towards the prey (Fig. 1A). It is worth it to notice
that the proboscis is extended only towards the correct prey,
the fire-worms. None of the 30 specimens observed
extended its proboscis towards the fishes or the other
mollusks. When it comes closer, the radular tooth, filled
with venom, is darted into the worm (Fig. 1B). Immediately
after stinging, the fire-worm twists its body until is
paralyzed, so the prey can be engulfed by C. regius
(Fig. 1C).
3.2. HLPC profiling
After these in vivo observations, C. regius were pooled
in eight groups according to gender (male and female), shell
size (from 3 to 4.5 cm—small; and from 5.5 to 7.5 cm—
large) and season of collection (winter and summer). The
same amount of crude venom from each group was
fractionated by RP-HPLC, as shown in Fig. 2. There were
104 detected peaks along the profile and the arrow indicates
EL 4 – pp. 1–10
UNCORRECTED PROOF
Fig. 2. Chromatography profiles of C. regius venom, fractionated in an analytical system of RP-HPLC (Abs 220 nm; flow rate 1 mL/min;
0–60% B/120 min, according to material and methods section)—(S, small; L, large; \, female; _, male). The arrow indicates the newly
characterized rg11a peptide.
TOXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MODEL 4 – pp. 1–10
M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–104
DTD 5 ARTICLE IN PRESS
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
UNCORRECTED PROOF
Table 1
Actual peak areas of the 20 major peaks observed in the HPLC profiles of C. regius toxin
Pk#a SFW area SMW area LFW Area LMW area SFS area SMS area LFS area LMS area Mean area Standard
deviation
3 –b 37,665,104c 1,850,527 1,062,098 2,954,580 – 42,462,201 33,996,615 19,998,520 19,955,477.63
14 46,169,063 39,842,112 33,788,301 37,117,717 40,981,964 45,074,980 – – 40,495,689 4,695,113.49
29 134,395,149 101,975,665 143,468,419 180,000,000d 95,186,501 73,009,489 97,241,671 106,000,000 116,409,611 34,053,520.80
35 35,801,034 20,483,096 32,050,152 39,411,666 24,256,255 31,138,845 25,943,765 28,412,504 29,687,164 6,204,651.14
42 35,221,979 15,669,158 24,664,672 40,363,760 14,734,563 19,367,578 14,174,838 16,931,023 22,640,946 10,018,453.39
46 – 8,581,919 125,236,955 83,451,973 46,128,049 66,911,338 26,705,914 21,528,653 54,077,828 40,891,946.88
47 128,566,337 52,816,502 20,404,710 14,301,719 9,472,283 15,665,270 34,211,590 23,722,768 37,395,147 39,317,259.59
50 38,214,750 19,802,285 39,309,563 38,428,212 43,003,103 32,493,857 58,408,696 6,254,984 34,489,431 15,648,450.81
54e 14,803,953 8,050,680 7,657,388 26,258,935 39,449,691 59,000,341 43,549,802 31,536,639 28,788,428 18,245,985.71
55d 53,229,091 22,136,670 58,109,424 14,295,760 – – 7,725,410 8,412,478 27,318,138 22,615,082.09
56 124,106,874 65,653,885 116,913,920 106,000,000 72,458,615 124,000,000 95,995,893 96,654,357 100,222,943 22,181,826.98
58 224,887,202 119,999,765 203,043,666 164,000,000 143,000,000 154,000,000 136,000,000 125,000,000 158,741,329 37,402,159.88
61 50,213,153 20,443,608 58,052,837 37,962,274 – – – – 41,667,968 16,387,899.44
62 – 11,168,785 30,667,363 33,810,089 34,587,752 37,063,871 36,614,509 32,434,028 30,906,628 8,985,589.87
68 14,043,920 – – 56,395,630 26,150,039 33,374,843 16,956,129 31,517,588 29,739,691 15,162,958.93
88 6,711,370 3,552,677 16,031,174 76,235,283 30,396,702 24,632,753 14,510,275 51,453,506 27,940,467 24,697,791.96
94 45,702,196 19,358,310 36,507,244 37,725,547 17,862,296 23,756,671 1,777,353 14,879,181 24,696,099 14,383,730.64
98 56,538,781 25,145,946 43,874,904 50,248,981 40,878,914 42,447,521 23,888,003 33,995,700 39,627,343 11,436,349.46
101 43,236,929 19,832,789 34,885,335 15,288,691 54,513,638 78,518,871 33,831,018 20,765,054 37,609,040 21,080,315.13
103 83,628,409 56,460,258 52,566,322 37,618,290 54,953,278 85,511,353 15,378,519 19,747,705 50,733,016 25,988,015.16
SFW: small female winter; SMW: small male winter; LFW: large female winter; LMW: large male winter; SFS: small female summer; SMS: small male summer; LFS: large female summer;
LMS: large male summer.a Peak number is used instead of retention time because of individual variation on peak composition among the profiles.b Absence of peak at the same retention time (G0.1 min).c BoldCitalics represent the criteria for selecting peaks. There must be at least three groups with peak areas larges than 3!107.d Except for peak number 55, shown as control.e Peak corresponding to the one containing the peptide rg11a.
TO
XC
ON
23
24
—2
7/1
0/2
00
4—
15
:54
—A
DM
INIS
TR
AT
OR
—1
23
24
1—
XM
LM
OD
EL
4–
pp
.1
–1
0
M.C
.V
ian
na
Bra
ga
eta
l./
To
xicon
xx(xxxx)
1–
10
5
DT
D5
AR
TIC
LE
INP
RE
SS
44
9
45
0
45
1
45
2
45
3
45
4
45
5
45
6
45
7
45
8
45
9
46
0
46
1
46
2
46
3
46
4
46
5
46
6
46
7
46
8
46
9
47
0
47
1
47
2
47
3
47
4
47
5
47
6
47
7
47
8
47
9
48
0
48
1
48
2
48
3
48
4
48
5
48
6
48
7
48
8
48
9
49
0
49
1
49
2
49
3
49
4
49
5
49
6
49
7
49
8
49
9
50
0
50
1
50
2
50
3
50
4
50
5
50
6
50
7
50
8
50
9
51
0
51
1
51
2
51
3
51
4
51
5
51
6
51
7
51
8
51
9
52
0
52
1
52
2
52
3
52
4
52
5
52
6
52
7
52
8
52
9
53
0
53
1
53
2
53
3
53
4
53
5
53
6
53
7
53
8
53
9
54
0
54
1
54
2
54
3
54
4
54
5
54
6
54
7
54
8
54
9
55
0
55
1
55
2
55
3
55
4
55
5
55
6
55
7
55
8
55
9
56
0
T
M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–106
DTD 5 ARTICLE IN PRESS
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
the peak containing the novel conotoxin rg11a (named
according to Olivera and Cruz (2001)).
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
3.3. Statistical analysis
All the peak areas were compared among the groups.
Mean values and standard deviation for each peak were
calculated. The criterion for statistical significance was
whether the peak area lies outside the range of G2s of the
mean value. Table 1 presents the actual area values for the
20 major peaks detected by absorbance at 214 nm, and also
the peak that contains rg11a (peak number 54). Peak
numbering was used, instead of retention time to simplify
the interpretation. Analyses of all individual peak areas
show that most of the individual values for one given group
are comprised in the range of mean G1s. The few values
that lied outside this range were tested for 2s and proved
themselves not to be significantly different. The area peaks
were also compared by Z-test (data not shown).
636637
638
639
640
641
642
3.4. LC–MS analysis
Fig. 3 presents a LC–MS analysis of the venom of one
group of C. regius (small, male, summer) for mass
distribution and profiling purposes. Chromatographic
UNCORREC
Fig. 3. Representative total ion count (TIC) chromatogram for small, male,
HPLC (flow rate 50 mL/min; 5–65% B1/60 min, according to material and
Table 1.
OXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MODE
conditions are slightly different from those employed in
the profiling for mass spectrometry compatibility; therefore,
a minor change in the chromatographic profile shape is
observed. Total ion current (TIC) is shown instead of
absorbance at 214 nm due to better resolution. Representa-
tive peaks were chosen along the profile (,) and the
molecular masses were measured in each peak, as presented
in Table 2. In this table, it is also possible to notice that the
LC–MS profiling conditions were successful in separating
molecules individually, for most of the peaks shown
presented one major component.
F3.5. rg11a purification
Once peak number 54 was chosen, two more
chromatographic steps were necessary to purify this
peptide, as presented in material and methods section.
Purity was assessed by HPLC (data not shown), as well as
by MALDI-TOF analysis, as in Fig. 4.
ROO3.6. ‘De novo’ peptide sequencing
One aliquot of purified rg11a was selected for ‘de novo’
sequencing and processed according to Westermeier and
Naven (2002). After cystein bridge reduction and alkylation
TED P
summer (SMS) group venom obtained after separation by microRP-
methods section). , indicates the peaks analyzed and presented in
L 4 – pp. 1–10
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
C
Table 2
Representative molecular weights measured in the major peaks of
the HPLC profile of Conus regius toxin
RTa (min) Number of majorb
components
Molecular
mass(es)c (Da)
12.57 9 LMWd
16.26 1 568.27
18.70 2 2822.34G0.48
3049.40G0.48
23.41 2 4283.69G0.69
4339.85G0.87
24.33 1 1662.57G0.01
25.42 2 2837.35G0.47
3927.45G0.01
27.44 1 3920.44G0.02
30.03 1 3311.32G0.02
31.66 3 1007.44
3077.86G0.49
3072.08G0.01
32.54 1 4730.11G0.01
36.68 1 4692.91G0.05
49.28 1 2665.66G0.51
51.94 1 1979.23G0.52
53.74 1 8682.47G0.13
70.91 1 LMWc
a Retention time regarding the profile depicted in Fig. 3.b Major component refers to a peak (or peaks) which intensity
was, at least, 10% of the full scale.c Molecular masses.d LMW—low molecular weight components: below 500 Da.
Standard deviation: calculated when there were, at least two,
different charge states of the same molecule, otherwise measured
molecular weight is presented.
M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–10 7
DTD 5 ARTICLE IN PRESS
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
REby iodoacetamide, the reaction product was digested withtrypsin. The obtained peptides (Fig. 5) were individually
selected for MS/MS analysis and fragmented by collision
with argon (CIF), yielding daughter ion spectra as presented
in Fig. 6. This routine was repeated until enough
information regarding the peptide sequence was gathered.
The MS/MS spectra were analyzed by the BioLynx software
module of MassLynx 4.0 and manually verified for accuracy
in the amino acid sequence interpretation.
R 770771
772
773
774
775
776
777
778
779
780
781
782
783
784
UNCO4. Discussion
Conus have developed a highly specialized strategy to
capture prey and their venom has evolved to selectively
paralyze fish, worm or mollusks (Endean and Rudkin,
1965). C. regius is a worm-hunter member of this genus and
they prey preferentially at night. The correlation found
between the feeding ecology of the Conidae and the animal
group specificity is expected to be due to the presence of
prey-specific neurotoxins in these venoms. As an example,
molluscivorous species of cone snails showed high specific
activities in mollusk assay, but had no effects on fish assays
TOXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MOD
TED PROOF
(Fainzilber and Zlotkin, 1992). Prey-specific morphological
adaptations of the venom apparatus have also been
described (Conticello et al., 2001).
In this work, specimens of the Brazilian vermivorous cone
snails were collected and have had their feeding behavior
analyzed under controlled environment. Fig. 1 presents one
experiment in which C. regius extends its proboscis (panel
A), harpoons its prey (panel B) and starts engulfing the fire-
worm (panel C). It is noteworthy to mention that paralysis of
the fire worm after harpooning takes longer to occur than
paralysis in fish, as observed in a parallel experiment in
which the feeding behavior of one piscivorous mollusk
(C. ermineus) was analyzed (data not shown).
As in Fig. 1C, it was observed that the prey’s body
changes color after venom inoculation, from reddish to
purple. This may lead one to speculate that there might be
some secondary effect on the fire-worm’s clotting cascade,
besides paralysis.
Moreover, the collected Conus specimens were divided
into eight groups, according to different criteria that might
represent a source or stimulus for variability, and their crude
venom extract was fractionated by HPLC (Fig. 2). One of
the major peaks on the venom was selected for character-
ization based on: (i) its homogeneity throughout the
different populations; (ii) its estimated solubility (as it
elutes in the central region of profile); (iii) its relative
abundance in the profile, being one of the major peaks; and
(iv) its molecular weight, as observed by mass spectrometry,
which is compatible to some of the already described
conotoxins.
Fig. 2 and the accompanying Table 1 summarize the
comparison of the venom composition of pooled individ-
uals, according to the criteria employed in this study.
Statistical analyses of the area of the peaks did not reveal a
significant pattern of variation, even though the visual
interpretation of chromatographic profiles may lead one to
find differences in toxin distribution among them.
One additional LC–MS analysis was performed, as
shown in Fig. 3, and in further details on Table 2, for general
assessment of the molecular masses present in this particular
venom. One can observe the presence of a higher molecular
weigh component, eluting at 53.74 min, weighing
8682.47 Da. The few already described polypeptides with
high molecular weight (O5000 Da, for classical conotox-
ins) found in Conus venom ducts present distinct biological
activities, such as enzymatic and excitotoxic (Terlau and
Olivera, 2004). Further work is in progress aiming the
characterization of this molecule.
Once the major component of peak number 54 was
purified (Fig. 4), ‘de novo’ sequencing of the peptide was
performed, as shown in Figs. 5 and 6. The deduced amino
acid sequence, CQAYGESCSAVVRCCDPNAVCCQYPE-
DAVCVTRGYCRPPATVLT, showed a peptide with a new
characterized cystein pattern (C-C-CC-CC-C-C-) from the
I-superfamily of conotoxins (Jimenez et al., 2003). Accord-
ing to the nomenclature (Olivera and Cruz, 2000), this
EL 4 – pp. 1–10
UNCORRECTED PROOF
Fig. 4. MALDI-TOF analysis of purified rg11a.
Fig. 5. ESI-Q-TOF analysis of the digested rg11a peptide. Deduced amino acid sequence is presented above the profile with corresponding
residue numbering. Arrow indicates trypsin cleavage sites. The tryptic peptides are indicated above each peak, as well as the charge state.
TOXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MODEL 4 – pp. 1–10
M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–108
DTD 5 ARTICLE IN PRESS
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
C
PROOF
Fig. 6. Representative MS/MS analysis of one of the tryptic peptides detected in Fig. 5. Deduced amino acid sequence is presented and the most
evident daughter ions are annotated.
M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–10 9
DTD 5 ARTICLE IN PRESS
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
UNCORRE
conotoxin was named rg11a. This peptide bears homology
to other conotoxins that affect vertebrate KC channels (Fan
et al., 2003; Kauferstein et al., 2003) and its physiological
action is under investigation.
Bingham et al. (1996) showed that Conus venom
composition varies among different species with same or
different feeding behavior, same species and even along
the same animal’s venom duct. They observed intra-specific
variations for specimens collected within and between
geographic regions. It was also reported that a number of
peptide peaks were only detected in one or a few specimens
of C. textile, as described.
Other inter-species variation in venom composition has
also been reported. The peptides from a snail-hunting
Conus venom contrast markedly with those from a fish-
hunting Conus venom. Among venoms of more closely
related species, some physiological overlap occurs,
although each venom posses novel components (Olivera
et al., 1990).
When they are geographically widely separated,
individuals of the same species do not exhibit unusual
peptide polymorphism (Olivera et al., 1995). There might
be some concentration differences, but the content of
peptides is constant. This was also observed with C. regius
venom composition. Comparing all the corresponding area
peaks among the groups, it was not possible to identify
TOXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MOD
TEDa statistically significant pattern of variation, even though
chromatography profiles seemed visually different.
This work represents the starting point for the charac-
terization of several new toxins from this Brazilian species
of Conus. So far, one new molecule has been identified and
characterized and several other potentially interesting
molecules have been identified in the LC–MS profiles.
rg11a is undergoing further investigation thorough
biochemical (for cystein bridging pattern) and electro-
physiological characterization; the signal sequence of the
pre-propeptide is also been investigated for the correct
characterization of this toxin in a superfamily (data not
shown). However, it is our belief that, without proper
profiling and normalization of any possible intra-specific
variation in the venom composition, one cannot state that a
novel toxin is a constitutive component of the venom, rather
than a product of gender, season or size variation. Once it
has been demonstrated the C. regius produces a fairly
homogeneous venom, regarding the HPLC profiling of the
major components, the standards are set for the quest for
new biologically active marine toxins.
5. Uncited references
Jones et al. (2001) and Olivera (2000).
EL 4 – pp. 1–10
T
M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–1010
DTD 5 ARTICLE IN PRESS
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
1022
1023
1024
1025
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
1045
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
1058
1059
1060
1061
1062
1063
1064
1065
1066
1067
1068
1069
1070
1071
1072
1073
1074
1075
1076
Acknowledgements
Dr Luiz Ricardo Lopes de Simone (Zoology Museum,
University of Sao Paulo) for critical review of
the manuscript, Fernando de Noronha National Marine
Park (PARNAMAR-FN), Instituto Brasileiro do Meio
Ambiente e dos Recursos Naturais Renovaveis (IBAMA),
Aguas Claras Dive Center, Marine Biology Center/Univer-
sity of Sao Paulo (CEBIMar/USP), Center for Applied
Toxinology/ Centers for Research, Innovation and Dis-
semination (CAT/CEPID), Butantan Institute, and FAPESP
for the financial support.
C
1077
1078
1079
1080
1081
1082
1083
1084
1085
1086
1087
1088
1089
1090
1091
1092
1093
1094
1095
1096
1097
1098
1099
1100
1101
1102
1103
EReferences
Bingham, J., Jones, A., Lewis, R.J., Andrews, P.R., Alewood, P.F.,
1996. Conus venom peptides (conopeptides): inter-species,
intra-species and within individual variation revealed by
ionspray mass spectrometry, in: Lazarovici, P., Spira, M.,
Zlotkin, E. (Eds.), Biochemical Aspects of Marine Pharma-
cology. Alaken Inc., USA, pp. 13–27.
Conticello, S.G., Gilad, Y., Avidan, N., Ben-Asher, E., Levy, Z.,
Fainzilber, M., 2001. Mechanisms for evolving hypervaria-
bility: the case of conopeptides. Mol. Biol. Evol. 18 (2),
120–131.
Cruz, L.J., Corpuz, G., Olivera, B.M., 1976. A preliminary study of
Conus venom protein. The Veliger 18 (3), 302–308.
Endean, R., Rudkin, C., 1965. Further studies of the venoms of
Conidea. Toxicon 2, 225–249.
Eston, V.R., Migotto, A.E., Oliveira Filho, E.C., Rodrigues, S.A.,
Freitas, J.C., 1986. Vertical distribution of benthic marine
organisms on rocky coasts of the Fernando de Noronha
archipelago (Brazil). Bolm. Inst. Oceanogr. 34, 37–53.
Fainzilber, M., Zlotkin, E., 1992. A new bioassay reveals mollusk-
specific toxicity in molluscivorous Conus venoms. Toxicon 30
(4), 465–469.
Fan, C., Chen, X., Zhang, C., Wang, L., Duan, K., He, L.,
Cao, Y., Liu, S., Zhong, M., Ulens, C., Tytgat, J., Chen, J.,
Chi, C., Zhou, Z., 2003. A novel conotoxin from Conus
UNCORR
OXCON 2324—27/10/2004—15:54—ADMINISTRATOR—123241—XML MODE
TED PROOF
betulinus, k-BtX, unique in cysteine pattern and in function
as a specific BK channel modulator. J. Biol. Chem. 278 (15),
12624–12633.
Jimenez, E.C., Shetty, R.P., Lirazan, M., Rivier, J., Walker, C.,
Abogadie, F.C., Yoshikami, D., Cruz, L.J., Olivera, B.M., 2003.
Novel excitatory Conus peptides define a new conotoxin
superfamily. J. Neurochem. 85, 610–621.
Jones, R.M., Cartier, G.E., McIntosh, J.M., Bulaj, G., Farrar, V.E.,
Olivera, B.M., 2001. Composition and therapeutic utility of
conotoxins from genus Conus. Patent status 1996–2000. Exp.
Opin. Ther. Patents 11 (4), 603–623.
Kauferstein, S., Huys, I., Lamthanh, H., Stocklin, R., Sotto, F.,
Menez, A., Tytgat, J., Mebs, D., 2003. A novel conotoxin
inhibiting vertebrate voltage-sensitive potassium channels.
Toxicon 42, 43–52.
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.
Olivera, B.M., 2000. u-Conotoxin MVIIA: from marine snail to
analgesic drug, in: Fusetani, N. (Ed.), Drugs from the Sea.
Karger, Basel, pp. 74–85.
Olivera, B.M., Cruz, L.J., 2001. Conotoxins, in retrospect. Toxicon
39, 7–14.
Olivera, B.M., Rivier, J., Clark, C., Ramilo, C.A., Corpuz, G.P.,
Abogadie, F.C., Mena, E.E., Woodward, S.R., Hillyard, D.R.,
Cruz, L.J., 1990. Diversity of Conus neuropeptides. Science
249, 257–263.
Olivera, B.M., Hillyard, D.R., Marsh, M., Yoshikami, D.,
1995. Combinatorial peptide libraries in drug design:
lessons from venomous cone snails. TIBTECH 13
(10/141), 422–426.
Olivera, B.M., Walker, C., Cartier, G.E., Hooper, D., Santos, A.D.,
Schoenfeld, R., Shetty, R., Watkins, M., Bandyopadhyay, P.,
Hillyard, D.R., 1999. Speciation of cone snails and interespe-
cific hyperdivergence of their venom peptides—potencial
evolutionary significance of introns. Ann. NY Acad. Sci. 870,
223–237.
Rios, E.C., 1985. Seashells of Brazil. Fundacao Cidade do Rio
Grande, Rio Grande, pp. 129C plate 44.
Terlau, H., Olivera, B.M., 2004. Conus venoms: a rich source of
novel ion channel-targeted peptides. Physiol. Rev. 84, 41–68.
Westermeier, R., Naven, T., 2002. Proteomics in Practice. Wiley,
Germany.
L 4 – pp. 1–10
1104
1105
1106
1107
1108
1109
1110
1111
1112
1113
1114
1115
1116
1117
1118
1119
1120
Top Related