Mass spectrometric and high performance liquid chromatography profiling of the venom of the...

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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
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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

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http://www.elsevier.com/locate/toxicon

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M.C. Vianna Braga et al. / Toxicon xx (xxxx) 1–102


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RE
1. 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.
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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

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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


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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.
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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.




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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.

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the peak containing the novel conotoxin rg11a (named

according to Olivera and Cruz (2001)).

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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).
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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.


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

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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.



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REby iodoacetamide, the reaction product was digested with
trypsin. 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.
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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


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.




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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.



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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


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



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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.

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References

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intra-species and within individual variation revealed by

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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),

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Cruz, L.J., Corpuz, G., Olivera, B.M., 1976. A preliminary study of

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Chi, C., Zhou, Z., 2003. A novel conotoxin from Conus

UNCORR


TED PROOF

betulinus, k-BtX, unique in cysteine pattern and in function

as a specific BK channel modulator. J. Biol. Chem. 278 (15),

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Abogadie, F.C., Yoshikami, D., Cruz, L.J., Olivera, B.M., 2003.

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superfamily. J. Neurochem. 85, 610–621.

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Kauferstein, S., Huys, I., Lamthanh, H., Stocklin, R., Sotto, F.,

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channel targets, and drug design: 50 million years of

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