Isolation and Characterization of Conotoxins from the Venom

ISOLATION AND CHARACTERIZATION OF CONOTOXINS FROM THE VENOM

OF CONUS PLANORBIS AND CONUS FERRUGINEUS

Adriana Pak

A Thesis Submitted to the Faculty of

Charles E. Schmidt College of Science

in Partial Fulfillment of the Requirements for the degree of

Master of Science

Florida Atlantic University

Boca Raton, Florida

ISOLATION AND CHARACTERIZATION OF CONOTOXINS FROM THE VENOM OF C. PLANORBIS AND C. FERRUGINEUS by

AdrianaPak

This thesis was prepared under the direction of the candidate's thesis advisor, Dr. Frank Mari, Department of Chemistry and Biochemistry, and has been approved the members of her supelVisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science.

SUPERVISORY COMMITTEE:

Fran , --,....,...,·hesis Advisor ~vr/~-- L~ Deguo Du, Ph.D

Jer e E. Haky, Ph.D. Chair, Department of emistry and Biochemistry

us ell Ivy, Ph.D. Interim Dean, The Charles E. Schmidt College of Science

~~r2?r--~ , P~~.JDate

11 ACKNOWLEDGEMENTS

I would like to thank Dr. Frank Mari for all his guidance, time, and the opportunity to work with him.

I would also like to thank Dr. Lyndon West and Dr. Deguo Du for their guidance and for serving on my committee.

Furthermore, I would like to thank Dr. Katarzyna Pisarewicz for friendship and guiding me in the beginning. Thank you also Dr. Jose Riveira-Ortiz, Dr. Vered Marks,

Dr. Herminsul Cano and Alberto Padilla, for being great friends and lab mates. Thank you also to Pam Mongkhonsri, Tanya T. Kelley for friendship and helping me several challenge tasks. I also want to thank Dr. Carolina Moller for guiding me and giving me a good example of being mom and scientist at same time. Thank to all members of Dr.

Mari’s lab for a nice and warm environment in the lab.

Finally I would like to thank my husband Pedro, my daughters Alexis and

Michelle, my parents and my brother for all love and patience during this course of my life. And most important to God, my strength and my Savior.

iii

ABSTRACT

Author: Adriana Pak

Title: Isolation and Characterization of Conotoxins from the Venom of Conus planorbis and Conus ferrugineus

Institution: Florida Atlantic University

Thesis Advisor: Dr. Frank Mari

Degree: Master’s of Science

Year: 2014

The venom of marine gastropods belonging to the genus Conus has yielded numerous structurally and functionally diverse peptidic components. The increase variety of bioactive peptides identified in cone snail venoms is the product of the variety of molecular adaptations taken by Conus species in evolving neuroactive molecules to suit their diverse biological purposes. Toxins from cone snails are classified into two major groups. One group consists of disulfide-rich peptides commonly termed conotoxins; the second group comprises peptides with only one disulfide bond or none.

In this work, we present the discovery and characterization from the marine snails C. planorbis and C. ferrugineus. Both species are commonly found in the Indo-Pacific

region and are very similar and is not distinguishable by size and shape of the shell.

Novel P and T-Supefamiles were found in both species along with small linear peptides with have a high frequency of tyrosine residues. Each chapter contains a detailed look at the discovery process for the isolation and characterization of C. planorbis and C. ferrugineus. At discussion part, we also compared the peptides isolated in this work with other peptides from the literature.

ISOLATION AND CHARACTERIZATION OF CONOTOXINS FROM THE VENOM

OF CONUS PLANORBIS AND CONUS FERRUGINEUS

LIST OF FIGURES ...... viii

LIST OF TABLES...... xi

CHAPTER I - INTRODUCTION ...... 1

1.1 Conus planorbis and Conus ferrugineus ...... 5

1.2 Post Translational Modifications of Peptides ...... 6

1.3 Biology of Conus species...... 7

CHAPTER II - MATERIAL AND METHODS...... 10

2.1 Crude Venom Extraction ...... 10

2.2 Purification and Isolation of a Peptide...... 11

2.2.1 Size-Exclusion Superdex Peptide HR 10/30 High Performance Liquid

Chromatography (SE-HPLC)...... 11

2.2.2 Size-Exclusion High Liquid Chromatography (SE-HPLC)...... 11

2.2.3 Analytical Reverse Phase HPLC ...... 12

2.3 Mass Spectrometry...... 12

2.3.1 AB QSTAR XL MS/MS System ...... 12

2.3.2 Voyager-DE STR...... 12

2.4 Nuclear Magnetic Resonance ...... 13

2.5 Reduction and Alkylation of Cysteyl Residues ...... 14

2.6 Peptide Sequencing...... 14

2.7 Nomenclature...... 15

CHAPTER III - RESULTS...... 16

3.1 Isolation and characterization of the venom of C. ferrugineus...... 16

3.1.1 Purification of fraction Fer_B03g...... 16

3.1.2 Purification of fraction Fer_B05p...... 21

3.1.3 Purification of fraction Fer_D04ij ...... 24

3.1.4 Purification of fraction Fer_D05r ...... 34

3.1.5 Purification of fraction Fer_F06k ...... 37

3.1.6 Purification of fraction Fer_F07i ...... 42

3.2 Isolation and characterization of the venom of C. planorbis...... 45

3.2.1 Purification of Pla_A06j ...... 45

3.2.2 Purification of Pla_A06k ...... 46

3.2.3 Purification of Pla_A06l ...... 46

3.2.4 Purification of fraction Pla_A04j...... 55

3.2.5 Purification of fraction Pla_A04u...... 59

3.2.6 Purification of fraction Pla_B04f...... 62

3.2.7 Purification of fraction Pla_B05g ...... 66

CHAPTER IV - DISCUSSION AND CONCLUSION...... 72

REFERENCES ...... 77

vii

LIST OF FIGURES

Figure 1. 1: C. planorbis (left) and C. ferrugineus (right)...... 5

Figure 1. 2: Conus ferrugineus and Conus planorbis range map ...... 6

Figure 3. 1: Size Exclusion chromatogram of the venom from C. ferrugineus (Fer_B)

in Superdex-Peptide Column ...... 17

Figure 3. 2: Analytical RP-HPLC chromatogram of Fer_B 03 ...... 18

Figure 3. 4: MALDI-TOF MS of reduced and alkylated of Fer_B03g ...... 20

Figure 3. 5: Analytical RP-HPLC chromatogram of Fer_B 05p ...... 22

Figure 3. 6: TOF MS Spectrum of Fer_B05p...... 23

Figure 3. 7: Size Exclusion Chromatogram for C. ferrugineus (Fer_D) ...... 26

Figure 3. 8: Analytical RP-HPLC chromatogram of Fer_D 04...... 27

Figure 3. 9: Purification of peak i on Analytical RP-HPLC chromatogram of Fer_D04 . 28

Figure 3. 10: MALDI TOF MS Spectrum of Fer_D04ij before and after reduction and

alkylation...... 30

Figure 3. 11: 1D NMR Spectrum of Fer_D04ij...... 32

Figure 3. 12: 2D NMR TOCSY Spectrum of Fer_D04ij...... 33

Figure 3. 13: Analytical RP-HPLC chromatogram of Fer_D...... 34

Figure 3. 14: QSTAR TOF MS Spectrum of native Fer_D05...... 36

Figure 3. 15: QSTAR TOF MS Spectrum of Fer_D05r after reduction and alkylation ... 36

viii

Figure 3. 16: Size Exclusion Chromatogram for C. ferrugineus (Fer_F)...... 39

Figure 3. 18: MALDI-TOF MS Spectrum of Fer_F06k...... 41

Figure 3. 19: Analytical RP-HPLC chromatogram of Fer_F 07i...... 43

Figure 3. 20: MALDI TOF MS Spectrum of Fer_F07i ...... 44

Figure 3. 22: Analytical RP-HPLC chromatogram of Pla_A06 ...... 48

Figure 3. 23: MALDI-TOF MS Spectrum of Pla_A06j ...... 49

Figure 3. 24: 1D NMR Spectrum of Pla_A06j ...... 51

Figure 3. 25: 2D NMR TOCSY Spectrum of Pla_A06j ...... 52

Figure 3. 26: MALDI-TOF MS Spectrum of Pla_A06k...... 53

Figure 3. 27: MALDI-TOF MS Spectrum of Pla_A06l ...... 54

Figure 3. 28: Analytical RP-HPLC chromatogram of Pla_A04 ...... 55

Figure 3. 29: MALDI-TOF MS Spectrum of Pla_A04j before and after reduction

alkylation...... 57

Figure 3. 30: MALDI-TOF MS Spectrum of Pla_A04u before and after reduction and

alkylation...... 60

Figure 3. 32: Analytical RP-HPLC chromatogram of Pla_B04 ...... 64

Figure 3. 33: MALDI-TOF MS Spectrum of Pla_B04f ...... 65

Figure 3. 34: Analytical RP-HPLC chromatogram of Pla_B05 ...... 66

Figure 3. 35: MALDI-TOF MS Spectrum of Pla_B05g...... 67

Figure 3. 36: 1D NMR Spectrum of Pla_A(2)05g...... 69

Figure 3. 37: 2D NMR TOCSY Spectrum of Pla_A(2)05g...... 70

Figure 4. 1: Dendogram of P-superfamilies of conotoxins from C. planorbis and C.

ferrugineus compared to the literature...... 74 ix

Figure 4. 2: Dendogram of T-superfamilies of conotoxins from C. planorbis and C.

ferrugineus compared to the literature...... 75

LIST OF TABLES

Table 3. 1: Elution times of Fer_B crude venom fractions form Superdex 30...... 18

Table 3. 2: Elution times of fractions from Analytical RP of Fer_B peak 3 from Size

Exclusion...... 19

Table 3. 3: Sequence Analysis of Fer_B 03g...... 21

Table 3. 4: Elution times of Fer_B 05 crude venom fractions form Superdex 30...... 23

Table 3. 5: Sequence Analysis of Fer_B 05p...... 24

Table 3. 6: Elution times of Fer_D crude venom fractions form Superdex 30...... 27

Table 3. 7: Elution times of Fer_D 04 fractions in Analytical Reverse Phase ...... 28

Table 3. 8: Elution times of Fer_D04 i fractions from Analytical Reverse Phase...... 29

Table 3. 9: Sequence Analysis of Fer_D 04ij ...... 31

Table 3. 10: Elution times of Fer_D_05 (Analytical Reverse Phase)...... 35

Table 3. 11: Sequence Analysis of Fer_d 05r...... 37

Table 3. 12: Elution times of Fer_F (Superdex Peptide) ...... 39

Table 3. 13: Elution times of Fer_F 06 (Analytical Reverse Phase) ...... 40

Table 3. 14: Sequence Analysis of Fer_F06k ...... 41

Table 3. 15: Elution times of Fer_F 07i (Analytical Reverse Phase) ...... 43

Table 3. 16: Sequence Analysis of Fer_F07i...... 44

Table 3. 17: Elution times of Pla_A (Superdex Peptide)...... 48

Table 3. 18: Elution times of Pla_A06 (Analytical RP) ...... 49 xi

Table 3. 19: Sequence Analysis of Pla_A06j...... 50

Table 3. 20: Sequence Analysis of Pla_A06k...... 53

Table 3. 21: Sequence Analysis of Pla_A06l...... 54

Table 3. 22: Elution times of Pla_A04 (Analytical Reverse Phase)...... 56

Table 3. 23: Sequence Analysis of Pla_A04j...... 58

Table 3. 24: Sequence Analysis of Pla_A04u...... 61

Table 3. 25: Elution times of Pla_B SE Superdex Peptide column...... 63

Table 3. 26: Elution times of Pla_B04 on Analytical RP column ...... 64

Table 3. 27: Sequence Analysis of Pla_B04f...... 65

Table 3. 28: Elution times of Pla_B05 on Analytical RP column ...... 67

Table 3. 29: Sequence Analysis of Pla_B05g...... 68

Table 3.30: Amino acids assigned on 1D and 2D NMR of Pla_A(2)05g...... 68

Table 3.31: Table of isolated peptides from C. planorbis and C. ferrugineus ...... 71

xii

CHAPTER I - INTRODUCTION

The marine gastropods known as cone snails (Conus) constitute an unusually species-rich group of venomous predators, one of the largest single genera (~700 species) of living marine invertebrates.

The research on cone snails is the result of the long documented history of human interest of this group of molluscs. The most familiar molluscs are those that are eaten (e.g., oysters, escargots) although cone snails are harvested for food in some Pacific islands, they are not abundant enough to be a notable culinary resource. However, the strikingly beautiful patterns on their shells have attracted human interest from the earliest times, and in a wide variety of culture [1]. The other aspect of cone snails that has attracted human interest is that they can be deadly to humans like C. geographus [4, 5], which has caused more than thirty human fatalities [16].

The first Conus venom peptides were isolated and characterized over the last few decades, although the systematic investigation of cone snail toxins is continually growing with advances in biochemical, pharmacological, electrophysiological and the constant driver for developing new drugs as some marine organisms are proved to be the potent sources of new medicines [2], only a minuscule fraction (<2%) of peptides present in these venoms has been characterized pharmacology [53].

Cone snails use their venom as the primary weapon to capture prey and is also believed to be used defensively and competitively, and possibly for other biological purposes as well

[1].

They are classified by their prey as either vermivorous (worm-hunting), piscivorous (fish- hunting), or molluscivorous (mollusk- hunting). While the vermivorous cone snails make up the largest group of cone snails and the piscivorous snails make up the smallest group, peptides from piscivorous cone snails have been the most characterized since their venom rapidly immobilize fish [9, 10].

Most of venomous animals (e.g. snakes and arthropods) only produces one or a few poisons. The biological activity of Conus venom is due to a large complement (up to

5000) of unusually small, highly structured peptides, and each Conus (family Conidae) species has its own distinct repertoire of venom peptides [7]. Conopeptides are small peptides consisting of 6-40 amino acids, yet they have a well defined structure.

In the 1960’s, it had become clear that the venom of these beautiful creatures could be of pharmaceutical use [3], each conotoxins act as modulator to a special molecular target or receptor, which affects physiological functions in a biological environment.

Conotoxins usually targets ion channels, either voltage-gated or ligand-gated, and in few cases, G-protein-linked receptors. The bioactive peptides in Conus are classified into two broad groups: the non-disulfide-rich and the disulfide rich that are conventionally called conotoxins. The non-disulfide-rich class includes conopeptides with no cysteines

(contulakins [11] conantokins [12], and conorfamides [13], and conopeptides with two cysteines forming a single disulfide bond (conopressins [14] and contryphans [15]).

Conotoxins can be grouped into several superfamilies, (A, B, C, D, E, F, G, H, I, J, K, L,

M, N, O, P, S, T, V, Y) generally share a characteristic arrangement of Cys residues in the mature toxin region. Peptides within the same ‘superfamily’ share a characteristic cysteine arrangement and a conserved signal sequence in the precursors, and members within the same ‘family’ have in common the unique disulfide bonds and pharmacological activity [8]. Each superfamily may be subdivided into several families with distinct pharmacological activities: α-conotoxins behave pharmacologically as competitive antagonists of the nicotinic acetylcholine receptor (AChR) [18], the physiological targets of χ-conotoxins were identified to be a noradrenaline transporter

[21], and possess an alternate disulfide linkage pattern (C1-4, C2-3) compared to α-

conotoxins (C1-3, C2-4). κ-conotoxin known to interact with voltage-gated potassium channels by inhibiting Shaker-mediated currents [30-31], ρ-conotoxins possess α1- adrenoreceptor antagonist activity [38], δ-conotoxin, inhibits the inactivation of voltage dependent sodium channel [24], γ-conotoxins from has been shown to act as an agonist of neuronal pacemaker cation currents [28], it is suggested that ε-conotoxins may target presynaptic calcium channels (blocker) or act on G protein-coupled presynaptic receptors.

Most of venomous animals (e.g. snakes and arthropods) only produces one or a few poisons. The biological activity of Conus venom is due to a large complement (up to

A single mRNA encodes each Conus peptide and biosynthesis occurs through normal ribosomal translation mechanisms. The initial translation product is a prepropeptide precursor with a characteristic organization. The precursor, usually between 70 and 120 amino acids in length, has an N-terminal signal sequence (~20 amino acids), and intervening pro region and the mature toxin (usually 10-30 amino acids) region, always a single copy at the C-terminal end [19]. Signal sequences of peptides within the same superfamily are exceptionally conserved; in contrast to the mature toxin regions are hypermutated [20]. The biologically active toxin is produced by proteolytic cleavage from the precursor, an essential posttranslational step in conopeptide maturation [33].

The snails generate molecular diversity structures in venom peptides by hypermutating the mature toxin region. In effect, the snails are using a combinatorial library strategy to generate novel peptides sequences in their venoms, to survive and capture their prey and consequently, Conus has become the most species-rich of all marine invertebrate genera

[8].

1.1 Conus planorbis and Conus ferrugineus

Conus planorbis and C. ferrugineus (Figure 1.1) are worm-hunting cone snails and commonly found in the Indo-Pacific region (Figure 1.2). Both of these species belongs to distinct clade IX in the phylogenetic scheme for Conus described by Espiritu [23], equivalent to clade E5 of Duda [25].

Figure 1. 1: C. planorbis (left) and C. ferrugineus (right)

C. ferrugineus and Conus planorbis are vey similar, from which it is not distinguishable by size and shape of the shell. C. ferrugineus is usually called "C. planorbis Born". The only reliable difference is the uniformly white aperture of C. ferrugineus, in contrast to the violet to violet brown base of the aperture in C. planorbis. In addition, the white subshoulder band of C. planorbis rarely occurs in C. ferrugineus, and the granulose ribs on basal part of last whorl are often light coloured in C. planorbis but usually overlaid with a dark spiral line in C. ferrugineus, a character also stated in Born's description [26].

Figure 1. 2: Conus ferrugineus and Conus planorbis range map

1.2 Post Translational Modifications of Peptides

Venom peptides need to be sufficiently stable to survive chemical degradation in solution at ambient temperature and enzymatic degradation by processing proteases present in the venom itself [45], as well as those in the tissues of prey species. This stability is often achieved naturally through the use of post-translational modification

(PTMs), which can increase toxin potency [42, 43] and/or disulphide bonds that fold the peptide into a stabilized structure. Typical PTMs include amidation (C-terminal), sulphation (Tyr), bromination (Trp), glycosylation (Thr), γ-carboxylation (Glu), hydroxylation (Pro), pyroglutamation, and isomerization to D-amino acids. Specific enzymes are used during the production of venom peptides to introduce these modifications at specific locations, which can enhance peptide bioavailability and potency in addition to stability [34].

For any potential therapeutic use of conotoxins to progress to clinical trials, the issue of bioavailability is a concern because of their susceptibility to degradation within the body 6

[40]. Furthermore, they possess relatively large size and hydrophilic nature, which makes oral bioavailability of the venom petides (~10–40 amino acids) generally poor. Therefore, all candidates of conotoxins for therapeutic use would require invasive intrathecal delivery, intravenous, intraperitoneal, intramuscular, or subcutaneous injections.

1.3 Biology of Conus species

The shells of cone snails can grow up to 23 cm in length, and are widely distributed throughout all tropical and subtropical oceans. More than half of the species can be found in the Indo-Pacific region [26] and a single coral reef in the tropical Indo-Paciﬁc may have over 30 different species of Conus [41]. The species can be found from tidal waters to deeper areas (>150 m) living on sand or among rocks or coral reefs.

The conotoxin is synthesized in a long convoluted venom duct connected to a muscular bulb that ejects the venom through a hollow tooth held by the proboscis, much like a disposable hypodermic needle. The venom apparatus of a fish-hunting Conus species, is illustrated in Figure 1.3.

Figure 1. 3: (Top) Diagram of the venom apparatus of a cone snail. Adapted from Kohn

[44] Bottom panels. (A) The tip of the proboscis of the fish-hunting C. purpurascens with a harpoon-like radular tooth lodged at the end. (B) Scanning electron micrograph of the anterior tip of the hollow radular tooth, showing its barbed structure and the opening from which the venom is extruded. (C) Another view showing the harpoon-like outline of the radular tooth, which also serves as a disposable hypodermic needle.

The cone snail's harpoon is a modification of the radula, an organ in molluscs that acts as both tongue and teeth. When the snail detects a prey animal nearby, it turns its mouth - a 8

long flexible tube called a proboscis - towards the prey. The harpoon is loaded with venom and is ejected from the proboscis into the prey by a powerful muscular contraction. The prey is paralyzed quickly. The snail then back in the radula, bringing the prey directly the mouth. After the prey is digested, the cone snail will regurgitate any indigestible material such as spines and scales, along with the disposable harpoon.

CHAPTER II - MATERIAL AND METHODS

2.1 Crude Venom Extraction

Live specimens were placed in the refrigerator (0oC) for about 5 minutes in order to not disturb the body of the cone snail. The body was carefully removed by introducing a probe in the aperture of the cone snail and performing a concave motion of the cone snail.

The venom duct is located in the soft tissue of the body and a dissecting tool was used to tear the tissue and remove the venom duct. The venom duct is a highly coiled structure and off-white in color, which was ease for the identification of the duct. After dissection, the ducts were uncoiled and measured. Solutions of 0.1% trifluoroacetic acid (TFA) was prepared to store all the venom ducts and frozen at -80oC. After the lyophilization of the ducts, were then homogenized with 0.1% TFA and the extracts were centrifuged at 10,000 x g for 20 minutes, at 4oC. The pellets collected were washed with 0.1% TFA and re-centrifuged.

This process was repeated for three times to increase the extraction of the venom out of the ducts. All supernatants were pooled, lyophilized and stored at -80oC.

2.2 Purification and Isolation of a Peptide

2.2.1 Size-Exclusion Superdex Peptide HR 10/30 High Performance Liquid

Chromatography (SE-HPLC)

Some of the lyophilized crude venom were first submitted to purification using size exclusion Superdex Peptide HR 10/30 column (10 x 300 mm) purchased from Amersham

Biosciences. The mobile phase consisted of 0.1M NH4CO3 at flow rate 0.5 ml/min. The pump used was a LCD Analytical ConstaMetric 3500. Detector used was a Thermo

Separation Products Spectromonitor 5000 operating at wavelengths λ = 220, 250 and

280nm.

2.2.2 Size-Exclusion High Liquid Chromatography (SE-HPLC)

Highly concentrated crude venoms were submitted to size exclusion high performance liquid chromatography (HPLC) column Pharmacia Superdex-30 column, 2.5 x 100cm.

The pump used was from Thermo Separation ConstaMetric 3200MS. The mobile phase consisted of 0.1M NH4HCO3 at flow rate 1.5ml/min using isocratic gradient. Detector was from Thermo Separation Products and measured wavelengths at 220, 250 and

280nm.

2.2.3 Analytical Reverse Phase HPLC

The lyophilized samples from the SE-HPLC and Superdex Peptide were subsequently submitted to separation on reverse phase HPLC (5µm C18, 4.6 x 250mm Vydac) eluted at 1.00ml/min. The mobile phase was consisted of 0.1%TFA (buffer A) and 0.1% TFA in 60% Acetonitrile (ACN) (buffer B). Peptides were eluted with an incremental linear gradient of 1% B/min. Detector used from LDC Analytical Spectromonitor 5000 and measured wavelengths at 220, 250 and 280nm. The pump used from Perkin Elmer Series

200 Pump. All HPLC fractions were manually collected, lyophilized, and kept at -40 °C prior to further use.

2.3 Mass Spectrometry

2.3.1 AB QSTAR XL MS/MS System

Q-Star XL Q-TOF MS/MS (Applied Biosystems) spectrometer equipped with an oMALDI-2 source was used to determine the molecular mass of the fractions from

Analytical HPLC. All the samples was dissolved in 50% acetonitrile in 50% in water and

0.05% (TFA)

2.3.2 Voyager-DE STR

Molecular mass was determined by positive ion matrix laser desorption ionization time- of-flight (MALDI-TOF) mass spectrometry as detected by a Voyager-DE STR (Applied

Biosystems). Samples were dissolved in 50% acetonitrile (ACN) in water and applied between two layers of α-cyano-4-hydroxycinnamic acid matrix (Acros Organics) onto a magnetic plate. Spectra were acquired in either linear or reflector mode. Calmix 1 and

Calmix 2 were used as external calibration standards.

2.4 Nuclear Magnetic Resonance

Pure samples were submitted to one-dimensional NMR experiments to determine relative peptide concentration and to identify peptide signals. The NMR spectra were obtained by

Varian Inova 500 MHz instrument equipped with pulse field gradients, three radio frequency channels and waveform generators. Samples were dissolved in 37 µL of water

(HPLC grade), 1.5µL of deuterium oxide and 1.5µL of deuterium oxide with 2,2,3,3- tetradeutero-3-trimethylsilylpropionic acid (TSP) (Aldrich) as an internal reference. The total volume, 40µL was adjusted to pH around 3.6 using 0.01 M solutions of HCl and

NaOH and an Orion Micro pH electrode (Thermo Electron 9810BN) and placed in 1.7 mm NMR capillary tube (Wilmad), and the spectra were acquired at 25oC using a Varian gHCN (generation 5) 3mm probe (pw90 = 3 µs, at the upper limit of the linear range of the RF amplifier) Water suppression was carried out using either pre-saturation,

WATERGATE (wg), or double pulse field gradient spin echo (dpfgse) [46] pulse sequences. Samples with relatively high concentrations were submitted for further two

dimensional NMR experiments named NOESY and TOCSY, which confirm the presence of certain amino acids in the sequence.

2.5 Reduction and Alkylation of Cysteyl Residues

About 80% of each purified samples were submitted to reduction and alkylation to determine the number of cysteine residues in the peptide. Reduction and alkylations of cystine groups were carried out as previously described [47] with minor modifications.

The lyophilized samples were dissolved in 0.1M Tris-HCl (pH 6.2), 5mM ethylenediaminetetraacetic acid (EDTA) and 0.1 sodium azide and reduced with 20 mM dithiothreitol (DTT). The fraction was incubated at 60oC for 30 minutes. Peptides were alkylated with 50mM of iodoacetamide (IAM) and 2µl of NH4OH (pH 10.5), at room temperature for 1 hour, in the dark. The reduced and alkylated peptides were recovered and purified using a pre-equilibrated Zip Tip (C-18, SIZE P-10, Millipore) with 0.1%

TFA and 80% acetonitrile solution with 0.1% TFA. Alkylation of all cys residues was verified by MALDI-TOF mass spectrometry.

2.6 Peptide Sequencing

After the reduction and alkylation of the peptides, the lyophilized samples were dissolved in 16µl of water (HPLC grade) and were adsorbed onto Biobrene-treated glass fiber filters and amino acid sequences were carried out by Edman degradation using an

Applied Biosystems Procise model 491A Sequencer equipped with a micro gradient delivery system, model 610A, data analysis software model 140C and UV detector model

785A. Prior to running each of the new peptide sequence, a standard mixture of PTH- amino acids was used to obtain a reference elution profile of amino acids for accurate amino acid identification. The sequences are then established by confirming the expected molecular weight.

2.7 Nomenclature

The nomenclature assigned is as follows: we adopted a three letters to designate each of the Conus species, Pla and Fer for Conus planorbis and Conus ferrugineus respectively.

For the different batches submitted to purification, a capital letter next to the three letters as Fer_B, Fer_D and Fer_F. For each peak collected on the Superdex Peptide or

Superdex 30, fractions are labeled as numbers in numerical order for each peak such as pla_A01, pla_A02 and so on. The fractions from the semi-preparative or analytical columns take a non capital letter after the number from Superdex Peptide fractions such as pla_A01a, pla_A01b and so on.

CHAPTER III - RESULTS

3.1 Isolation and characterization of the venom of C. ferrugineus

The first step includes a prefractionation of the crude venom using Superdex-30 and/or

Superdex Peptide columns. The resulting fractions are then separated and refined using reverse phase C-18 semi-preparative and analytical columns. The crude venom collected from Conus Ferrugineus was divided in 6 batches, which this work only describe the purification of three batches, Fer_B, Fer_D and Fer_F.

3.1.1 Purification of fraction Fer_B03g

In order to begin the purification process, Fer_B (11.6mg) was dissolved in 0.1M

NH4HCO3 and submitted for purification on Superdex-Peptide column and ran for 100 minutes at wavelengths of 220, 250, 280 nm and a range 1, 0.5, 0.5 and 0.5 respectively

(Figure 3.1). This separation by size yielded 8 fractions, which fractions 3 and 5 were submitted for further separation (Table 3.1). The fraction 3 was submitted to purification on Reverse Phase analytical column and the run was monitored at wavelengths 220, 250 and 280nm for 80 minutes and ranges 2, 1.5, 1.5 respectively. Sixteen fractions were

collected (Figure 3.2, Table 3.2) and peak g, were analyzed by mass spectrometry as previously described in Materials and Methods. The sample was dissolved in 3µL of 50% of acetonitrile in water and 10% of solution was used for mass spectrometry. The sample was named Fer_B03g, which was then submitted to reduction with DTT and alkylation with iodoacetamide to determine number of cysteine residues and also to make the peptide linear to Edman degradation sequencing. Mass spectrometry of the reduced/carboxymethylate peptides (Figure 3.4) and of the native peptides (Figure 3.3) showed a mass difference consistent with the presence of four cysteine residues, which was determined by analyzing the molecular weight increase of the sample since the reduction and alkylation of each disulfide bond results in an increase of 58Da per cysteine residue (1666.57 – 1434.85 = 231.72 Da/58 = 4 Cys). This peptide has a C- terminal amidation, a post translational modification very common in T-1 subtype. Table

3.3 shows the sequence of the amino acids for Fer_B03g with six amino acids between the cysteines and belongs to T-Superfamily, which is the first report of this superfamily in C. ferrugineus.

1.22 1 3 5 250 r 0.5

-1.22 100 min

Figure 3. 1: Size Exclusion chromatogram of the venom from C. ferrugineus (Fer_B) in Superdex-Peptide column 17

Table 3. 1: Elution times of Fer_B crude venom fractions form Superdex 30 Fraction Elution time (minutes) 1 9.36 2 13.57 3 19.02 4 25.30 5 29.07

g 280 r 1.5

-0.03 0 80min

Figure 3. 2: Analytical RP-HPLC chromatogram of Fer_B 03

TABLE 3. 2: Elution times of fractions from Analytical RP of Fer_B peak 3 from Size Exclusion

Fraction Elution Time Fraction Elution Time (minutes) (minutes) a 3.00 i 41.02 b 4.32 j 43.90 c 5.21 k 46.43 d 9.80 l 52.32 e 10.20 m 54.02 f 12.05 n 59.43 g 32.89 o 63.03 h 34.34 p 64.01

1434.85 Da

Figure 3. 3: MALDI- TOF MS Spectrum of Fer_B03g, zoomed in to illustrate isotope

1666.57 Da

Figure 3. 4: MALDI-TOF MS of reduced and alkylated of Fer_B03g

Table 3. 3: Sequence Analysis of Fer_B 03g

Cycle # Assigned Residue 1 Gly 2 Ser 3 Cys 4 Cys 5 Ala 6 Ile 7 His 8 Leu 9 Asp 10 Ile 11 Cys 12 Cys 13 Asp 14 Ser Mass Spectrometer MW 1434.85 Da

Calculated MW 1434.55 Da

Sequence: G S C C A I H L D I C C D S* (T-Superfamily)

We attempted to acquire 1D and 2D NMR data on Fer_B 03g but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful.

3.1.2 Purification of fraction Fer_B05p

The peak 5 from Superdex-Peptide column (Figure 3.1) was submitted to reverse phase analytical column and the run was monitored at wavelengths 220, 250 and 280nm for 80 minutes and ranges 1, 0.5, 0.5 respectively. Nineteen fractions were collected for 21

Fer_B07 (Figure 3.5, Table 3.4), and the peak p was analyzed by mass spectrometry as previously described (Figure 3.6). The sample was dissolved in 3µL of 50% (ACN) in water and 10% of that solution was used for mass spectrometry. The sample was named

Fer_B05p, which was then submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry showed there was no difference between the molecular weight before and after the reduction and alkylation that indicate that

Fer_B05p is a linear peptide with absence of cysteine and subsequently disulfide bonds.

Table 3.5 shows the sequence of the amino acids.

6.25

220 r 1.0 p

-0.62 0 min 80 min

0.78

280 r 0.5

-0.078 80 min Figure 3. 5: Analytical RP-HPLC chromatogram of Fer_B 05p

Table 3. 4: Elution times of Fer_B 05 crude venom fractions form Superdex 30 Fraction Elution Time Fraction Elution time (minutes) (minutes) a 3.24 k 22.85 b 3.71 l 28.81 c 4.46 m 32.51 d 4.85 n 36.55 e 8.22 o 40.80 f 9.28 p 42.83 g 9.87 q 46.05 h 12.17 r 51.27 i 20.21 s 52.40 j 21.81

1124.43 Da

Figure 3. 6: TOF MS Spectrum of Fer_B05p

Table 3. 5: Sequence Analysis of Fer_B 05p

Cycle # Assigned Residue 1 Lys 2 His 3 Arg 4 Gln 5 Pro 6 Tyr 7 Tyr 8 Phe Mass Spectrometer MW 1124.43 Da

1123.57 Da Calculated MW

Sequence: L H R Q P Y Y F

We attempted to acquire 1D and 2D NMR data on Fer_B05p but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful.

3.1.3 Purification of fraction Fer_D04ij

The fourth batch of Fer_D (34mg) was injected into the Superdex 30 column and ran for

390 minutes at range 2.0 at wavelengths of 220, 250, 280 nm and a range 4.0 for the second 220 nm (Figure 3.7). This separation by size yielded 6 fractions (Table 3.6), which the fourth and fifth fractions (absorbance at 280nm) were chosen for further

separation. The fractions numbers 4 and 5 was submitted to reverse phase analytical column and the run was monitored at wavelengths 220, 250 and 280nm for 100 minutes and ranges 1, 0.5, 0.5 respectively. Twenty four fractions were collected for Fer_D04

(Figure 3.8, Table 3.7), the peak i, showed some impurifications and was resubmitted to reverse phase analytical column. The purification was monitored for 80 minutes at wavelengths 220, 280 and 250 at ranges 0.5, 0.2 and 0.2 respectively. Eighteen fractions were collected as shown (Figure 3.9, Table 3.8) and the peak j was analyzed by mass spectrometry as previously described. The sample was named Fer_D04ij, which was submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry of the reduced/carboxymethylated peptides and of the native peptides (Figure 3.10) showed a mass difference consistent with the presence of six amino acids (2930.59 –

2560.64 = 371.00 Da/58 = ~ 6 Cys). This peptide contains hydroxyproline, a distinctive post-translational modified amino acid of proline. Table 3.9 shows the sequence of the amino acids for Fer_D04ij. This peptide belongs to P-Superfamily of conotoxins and has a new cysteine framework, -CX3CX3CX3CXCX5C- where X can be any amino acid. This peptide resembles to the cysteine arrangement framework isolated from C. pulicarius,

Pu9.1, with following configuration of cysteines, CX3CX5CX3CXCX4C- [51]. The target for this framework still remains unknown, but the symptomatology elicited by C.

glorimaris, gm9a, with -CX3CX5CX3CXCX4C- framework and is reminiscent of spasmodic and spatic mouse mutants [17, 48]. It is interesting to note that because the P-

Superfamily peptides have no adjacent cysteines, these peptides may be able to exhibit a greater range of structural and functional diversity [22]. This is the first report of P- superfamily in C. ferrugineus. 25

5.835

4 5 220 r 2.0

0 -0.364 383

1.268

4 5 280 r 2.0

0.692

4 5 250 r 2.0

0 -0.058 383

Figure 3. 7: Size Exclusion Chromatogram for C. ferrugineus (Fer_D)

Table 3. 6: Elution times of Fer_D crude venom fractions form Superdex 30

Fractions Elution Time (minutes)

1 60.90 2 78.15 3 99.08 4 111.44 5 143.43 6 206.90

25.00

i 220 r 1.0

-2.50 100 min 6.25

i 280 r 0.5

0 min

-0.625 100 min Figure 3. 8: Analytical RP-HPLC chromatogram of Fer_D 04

Table 3. 7: Elution times of Fer_D 04 fractions in Analytical Reverse Phase

Fraction Elution Time Fraction Elution Time (Minutes) (Minutes) a 3.09 n 34.27 b 5.13 o 35.64 c 18.38 p 38.09 d 19.31 q 39.88 e 20.54 r 41.64 f 21.92 s 43.06 g 23.15 t 44.81 h 24.75 u 47.05 i 25.93 v 49.52 j 28.06 w 51.38 k 30.47 x 60.93 l 31.84 y 63.65

25.00

280 r 0.2

j 0 min 100 min

-2.50 Figure 3. 9: Purification of peak i on Analytical RP-HPLC chromatogram of Fer_D04

Table 3. 8: Elution times of Fer_D04 i fractions from Analytical Reverse Phase

Fraction Elution Time Faction Elution Time (minutes) (minutes) a 3.61 j 28.11 b 19.45 k 29.48 c 22.23 l 29.78 d 23.17 m 32.01 e 23.66 n 32.62 f 24.95 o 33.77 g 26.83 p 34.35 h 27.34 q 35.04 i 27.56 r 40.52

2560.64 Da

2930.59 Da

Figure 3. 10: MALDI TOF MS Spectrum of Fer_D04ij before and after reduction and alkylation

Table 3. 9: Sequence Analysis of Fer_D 04ij

Cycle # Assigned residue 1 Thr 2 Cys 3 Val 4 Gly 5 Ser 6 Cys 7 Val 8 Asn 9 Gly 10 Cys 11 Pro 12 Ser 13 Ile 14 Cys 15 Asp 16 Cys 17 Ile 18 Asn 19 Asn 20 Ser 21 Tyr 22 Cys 23 Ala 24 Ser 25 His Mass Spectrometer MW 2560.64 Da

Calculated MW 2549.96 – 6 Cys + 16= 2559.96 Da

Sequence: T C V G S C V N G C O S I C D C I N N S Y C A S H

(P-Superfamily)

Fer_D 04ij was lyophilized and prepared for NMR experiments as previously described.

Figure 3.11 and Figure 3.12 shows 1D NMR and 2D TOCSY spectrum of Fer_D 04ij. 31

According to the 1H NMR data the peptide seems folded because the δ methyl group of the isoleucine supposed to show around 0.89-1 ppm, but in the case of Fer_D 04ij shows around 0.63ppm, which reveals that this peptide can be folded as is indicate on high field of the spectrum.

Figure 3. 11: 1D NMR Spectrum of Fer_D04ij

Figure 3. 12: 2D NMR TOCSY Spectrum of Fer_D04ij

3.1.4 Purification of fraction Fer_D05r

The peak 5 on Superdex 30 (Figure 3.7) was submitted to analytical RP-HPLC. The run

was monitored at wavelength 220 and 280 at ranges 1 and 0.5 respectively and ran for

100 minutes. Twenty-three fractions were collected on the reverse phase analytical

column for Fer_D05 (Figure 3.13 and Table 3.10). The peak r from the analytical column

was analyzed by mass spectrometry as previously described. The sample was named

Fer_D05r, which was submitted to reduction with DTT and alkylation with

iodoacetamide. Mass spectrometry of the reduced/carboxymethylated peptides (Figure

3.15) and of the native peptides (Figure 3.14) showed a mass difference consistent with

the presence of four cysteine (1746.87 – 1514.06= 232.81 Da/58 = 4 Cys). Table 3.11

shows the sequence of the amino acids for Fer_D05r. This peptide has an amidated C-

terminus with no tail and elongated N-terminal tail (4 residues), two disulfide bonds and

belongs to the T-Superfamily of conotoxins with five amino acids between the cysteines.

5.271

220 r 1.0

adadaddadad r

-0.929 0 min 100 min Figure 3. 13: Analytical RP-HPLC chromatogram of Fer_D

Table 3. 10: Elution times of Fer_D_05 (Analytical Reverse Phase)

Fraction Elution Time (min) a 2.70 b 36.33 c 38.75 d 39.92 e 41.15 f 42.37 g 45.31 h + i 47.63 j 49.74 k 50.76 l 52.05 m 53.19 n +o 56.40 p 60.01 q 62.50 r 64.70 s 72.60 t 73.61 u 80.00 v 82.06 x 83.35

1514.22 Da

Figure 3. 14: QSTAR TOF MS Spectrum of native Fer_D05

1746.87 Da

Figure 3. 15: QSTAR TOF MS Spectrum of Fer_D05r after reduction and alkylation

Table 3. 11: Sequence Analysis of Fer_d 05r

Cycle # Assigned Residue 1 Gly 2 Lle 3 Phe 4 Ser 5 Thr 6 Leu 7 Cys 8 Cys 9 Gly 10 Val 11 Ile 12 Asn 13 Ser 14 Cys 15 Cys Mass Spectrometer MW 1514.22 Da

Calculated MW 1518.64 Da – 4 Cys = 1514.64 Da

Sequence: G I F S T L C C G V I N S C C* (T-Superfamily)

We attempted to acquire 1D and 2D NMR data on Fer_D 05r but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful.

3.1.5 Purification of fraction Fer_F06k

The sixth batch of crude venom from C. ferrugineus, named Fer_F (13 mg), was submitted to purification using Size Exclusion Superdex Peptide. The run was monitored at wavelengths 220, 250 and 280 at ranges 3, 0.5 and 0.5 respectively and ran for 100 minutes. Eleven fractions were collected on the SE Superdex peptide column (Figure 37

3.16, Table 3.12). The peaks 6 and 7 (Fer_F06 and Fer_07) were submitted to further purification by Reverse Phase Analytical column.

Fifteen fractions were collected from RP analytical column for Fer_F06. The run was monitored at wavelengths 220, 250 and 280 at ranges 1, 1 and 2 respectively and ran for

100 minutes (Figure 3.17, Table 3.13). Fifteen fractions were collected and peak k was analyzed by mass spectrometry as previously described. The sample was named

Fer_F06k, which was submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry (Figure 3.18) showed there was no difference between the molecular weight before and after the reduction and alkylation that indicate that

Fer_F06k is a linear peptide with absence of cysteine and subsequently disulfide bonds.

Table 3.14 shows the sequence of the amino acids. This sequence displays homology to a

14 amino acid long linear peptide from C. vitulinus from the same clade subtype of C. ferrugineus [25, 49].

11.17

7 220 r 3

-1.226 10 0 20.00 0

6 280 r 0.5 7

0 - 10 2.00 0 Figure 3. 16: Size Exclusion Chromatogram for C. ferrugineus (Fer_F)

Table 3. 12: Elution times of Fer_F (Superdex Peptide)

Fraction Elution Time Fraction Elution Time (minutes) (minutes) 1 11.97 7 36.42 2 18.41 8 41.34 3 21.71 9 46.44 4 25.63 10 49.48 5 28.55 11 51.55 6 30.29

5.587

220 r 1.0

0 -0.163 100

0.313

k 280 r 2.0

0 -0.031 100

Figure 3. 17: Analytical RP-HPLC chromatogram of Fer_F 06

Table 3. 13: Elution times of Fer_F 06 (Analytical Reverse Phase)

Fraction Elution Time Elution Time Fraction (minutes) (minutes) a 3.03 i 37.33 b 5.00 j 38.66 c 13.40 k 39.26 d 19.74 l 41.69 e 26.74 m 42.66 f 28.05 n 63.42 g 34.54 o 84.05 h 35.54

1469.62 Da

Figure 3. 18: MALDI-TOF MS Spectrum of Fer_F06k

Table 3. 14: Sequence Analysis of Fer_F06k

Cycle # Assigned Residue 1 Ala 2 Phe 3 Lys 4 Gln 5 Tyr 6 Asn 7 Trp 8 Gln 9 Arg 10 Met 11 Pro Mass Spectrometer MW 1469.62 Da

Calculated MW 1468.72 Da

Sequence: A F K Q Y N W Q R M P

We attempted to acquire 1D and 2D NMR data on Fer_F06k but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsucecessful.

3.1.6 Purification of fraction Fer_F07i

The peak 7 from the Size Exclusion Superdex Peptide column (Figure 3.16) was submitted for further purification by RP Analytical column. The run was monitored at wavelengths 220, 250 and 280 and ran for 100 minutes (Figure 3.19, Table 3.15).

Thirteen fractions were collected and peak i was analyzed by mass spectrometry as previously described. The sample was named Fer_F07i, which was submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry (Figure 3.20) showed there was no difference between the molecular weight before and after the reduction and alkylation that indicate that Fer_F07i is also a linear peptide with absence of cysteine and subsequently disulfide bonds. Table 3.16 shows the sequence of the amino acids. This peptide is similar with Fer_F06k, with the absence of the last two amino acids: methionine and proline.

1.39 7

i 220 r 1.0

0 -0.153 100 min

0.266

280 r 1.0

-0.078 100 min

Figure 3. 19: Analytical RP-HPLC chromatogram of Fer_F 07i

Table 3. 15: Elution times of Fer_F 07i (Analytical Reverse Phase)

Fraction Elution Time Fraction Elution Time (minutes) (minutes) a 2.93 h 30.36 b 3.33 i 32.63 c 5.60 j 35.97 d 23.73 k 38.41 e 24.43 l 51.31 f 27.04 m 63.80 g 28.05

Figure 3. 20: MALDI TOF MS Spectrum of Fer_F07i

TABLE 3. 16: Sequence Analysis of Fer_F07i

Cycle # Assigned Residue 1 Ala 2 Phe 3 Lys 4 Gln 5 Tyr 6 Asn 7 Trp 8 Gln 9 Arg Mass Spectrometer MW 1241.63 Da

Calculated MW 1241.40 Da

Sequence: A F K Q Y N W Q R

We attempted to acquire 1D and 2D NMR data on Fer_F07i but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful 44

3.2 Isolation and characterization of the venom of C. planorbis

Crude venoms of Conus planorbis were separated in two batches: Pla_A and Pla_B. Each batch of 9 mg was submitted separately on the SE Superdex Peptide.

3.2.1 Purification of Pla_A06j

Nine fractions were collected from SE Superdex peptide column for Pla_A. The run was monitored at wavelengths 220, 250 and 280 at ranges 3, 1.5 and 1.5 respectively and ran for 70 minutes (Figure 3.21, Table 3.17). Nine fractions were collected and peak 6 was submitted for further purification using Reverse Phase Analytical column. Eighteen fractions were collected on the reverse phase analytical column for Pla_A 06 (Figure

3.22). The peak j (Table 3.18) was analyzed by mass spectrometry as previously described. The sample was named Pla_A06j, which was submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry (Figure 3.23) showed there was no difference between the molecular weight before and after the reduction and alkylation that indicate that Pla_A06j is a linear peptide with absence of cysteine and subsequently disulfide bonds. This peptide 7 amino acid peptide has 2 tyrosine residues, an uncommon amino acid and is enriched with large hydrophobic residues such as leucine and isoleucine. This peptide is reminiscent of Conus planorbis described by Imperial [50].

Table 3.19 shows the sequence of the amino acids. Figure 3.24 and Figure 3.25 shows 1D

NMR and 2D TOCSY spectrum of Pla_A06j. 45

3.2.2 Purification of Pla_A06k

The peak k (Figure 3.21) collected from analytical RP named Pla_A06k was also analyzed by mass spectrometry and submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry (Figure 3.26) showed there was no difference between the molecular weight before and after the reduction and alkylation, which indicate that

Pla_A06k is a linear peptide with absence of cysteine and subsequently disulfide bonds.

Table 3.20 shows the sequence of the amino acids. This 9 amino acid long peptide is reminiscent of CPY-Pl1 from Conus planorbis described by Imperial [50].

3.2.3 Purification of Pla_A06l

The peak l (Figure 3.21, Table 3.17), from analytical RP column, named Pla_A06l was also analyzed by mass spectrometry (Figure 3.27) and submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry shows the same molecular weight before and after the reduction and alkylation indicating that Pla_A06l is also a linear peptide. Table 3.21 shows the sequence of the amino acids. This 11 amino acid long peptide is also reminiscent of CPY-pl1 from C. planorbis.

10.543

6 4 220 r 3.0

0 70.0 -1.857

11.509

4 280 r 1.5

-0.892 70.0

Figure 3. 21: Size Exclusion Chromatogram for Pla_A

Table 3. 17: Elution times of Pla_A (Superdex Peptide)

Fraction Elution Time Fraction Elution Time (minutes) (minutes) 1 12.16 6 31.16 2 17.35 7 38.75 3 21.31 8 47.31 4 23.01 9 51.14 5 28.09

2.793

j 220 r 2.0 k l

0 -0.306 100 0.698

j 280 r 1.0 k l

0 100 -0.077 Figure 3. 22: Analytical RP-HPLC chromatogram of Pla_A06

Table 3. 18: Elution times of Pla_A06 (Analytical Reverse Phase)

Fraction Elution Time Fraction Elution Time (minutes) (minutes) a 2.73 j 33.81 b 4.75 k 41.47 c 18.00 l 43.44 d 20.84 m 45.25 e 24.24 n 48.51 f 25.64 o 60.41 g 26.90 p 64.18 h 31.77 q 65.24 i 33.05 r 67.32

962.27 Da

Figure 3. 23: MALDI-TOF MS Spectrum of Pla_A06j

Table 3. 19: Sequence Analysis of Pla_A06j

Cycle # Assigned Residue 1 Leu 2 His 3 Arg 4 Tyr 5 Pro 6 Ile 7 Tyr Mass Spectrometer MW 962.27 Da

Calculated MW 962.14 Da

Sequence: L H R Y P I Y

Pla_A06j was lyophilized and prepared for NMR experiments as previously described.

Figure 3.4 and Figure 3.5 shows 1D NMR and 2D TOCSY spectrum of Pla_A06j. The

MALDI-TOF MS and Edman degradation sequencing show a clean peptide with 7 residues but according to the 1D 1H NMR we see a mixture of compounds. The aromatic peaks of tyrosine on 7.12ppm and 6.83ppm and the methyl groups of leucine and isoleucine clearly shows the presence of these amino acids on Pla_A06j.

Figure 3. 24: 1D NMR Spectrum of Pla_A06j

Figure 3. 25: 2D NMR TOCSY Spectrum of Pla_A06j

1179.45Da

Figure 3. 26: MALDI-TOF MS Spectrum of Pla_A06k

Table 3. 20: Sequence Analysis of Pla_A06k

Cycle # Assigned Residue 1 Ala 2 Arg 3 Phe 4 Leu 5 His 6 Pro 7 Phe 8 Gln 9 Tyr Mass Spectrometer MW 1179.39 Da

Calculated MW 1179.37 Da

Sequence: A R F L H P F Q Y

We attempted to acquire 1D and 2D NMR data on Pla_A06k but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful.

1443.64 Da

Figure 3. 27:MALDI-TOF MS Spectrum of Pla_A06l

Table 3. 21: Sequence Analysis of Pla_A06l

Cycle # Assigned Residue 1 Ala 2 Arg 3 Phe 4 Leu 5 His 6 Pro 7 Phe 8 Gln 9 Tyr 10 Tyr 11 Thr Mass Spectrometer MW 1443.84 Da

Calculated MW 1443.65 Da

Sequence: A R F L H P F Q Y Y T

We attempted to acquire 1D and 2D NMR data on Pla_A06l but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful.

3.2.4 Purification of fraction Pla_A04j

The peak 4 on SE Superdex Peptide column (Figure 3.21, Table 3.17) was submitted for further purification on RP Analytical column. The run was monitored at wavelengths

220, 250 and 280 at ranges 1, .5 and .5 respectively and ran for 100 minutes (Figure 3.28,

Table 3.22). Twenty one fractions were collected and peak j was analyzed by mass spectrometry and named Pla_A04j which was submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry of the reduced/carboxymethylated peptides and of the native peptides (Figure 3.29) showed a mass difference consistent with the presence of six cysteine (2853.51 Da – 2500.53 Da = 352.98 Da/58= ~6 cys).

Table 3.23 shows the sequence of the amino acids for Pla_A04j containing hydroxyproline, a posttranslational modification of the proline and belongs to P-

Superfamily.

11.174

j 220 r 1.0 u

100 0 -1.226 1.397

j 280 r 0.5 u

0 100 -0.513

Figure 3. 28: Analytical RP-HPLC chromatogram of Pla_A04

TABLE 3. 22: Elution times of Pla_A04 (Analytical Reverse Phase)

Fraction Elution time(min) Fraction Elution Time (min) a 3.28 l 33.85 b 19.65 m 34.68 c 22.40 n 35.90 d 23.74 o 38.71 e 27.30 p 41.67 f 28.06 q 42.90 g 28.91 r 52.74 h 30.39 s 63.37 i 31.30 t 71.40 j 32.15 u 75.10 k 33.03

2500.53 Da

2853.51 Da

Figure 3. 29: MALDI-TOF MS Spectrum of Pla_A04j before and after reduction alkylation

Table 3. 23: Sequence Analysis of Pla_A04j

Cycle # Assigned Residue 1 Ala 2 Cys 3 Val 4 Gly 5 Asn 6 Cys 7 Gly 8 Val 9 Ser 10 Ser 11 Gln 12 Cys 13 Hyp 14 Asp 15 Val 16 Gly 17 Cys 18 Gly 19 Cys 20 Ser 21 Asn 22 Leu 23 Gln 24 Cys 25 Trp Mass Spectrometer MW 2500.54 Da

Calculated MW 2499.94 Da

Sequence: A C V G N C G V S S Q C O D V G C G C S N L Q C W

P-Superfamily

We attempted to acquire 1D and 2D NMR data on Pla_A04j but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful. 58

3.2.5 Purification of fraction Pla_A04u

The peak u from the analytical column (Figure 3.28, Table 3.22) was also analyzed by mass spectrometry. The sample was named Pla_A04u, which was then submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry of the reduced/carboxymethylated peptides and of the native peptides (Figure 3.30) showed a mass difference consistent with the presence of four cysteine (2403.32 Da - 2639.13 Da=

235.81Da/58= ~4cys). Table 3.24 shows the sequence of the amino acids for Pla_A04u.

The peptide contains four cysteines and belongs to T-Superfamily with six amino acids between the cysteines. This peptide is similar to Fer_D05r from C. ferrugineus with three distinct differences: Pla_A04u has an elongated N-terminal tail with ten residues, while

Fer_D05r has six. The number of amino acids between the cysteines are also different, six residues for the peptide from C. planorbis and five from C. ferrugineus. Also

Pla_A04u has two amino acids at C-terminal tail, while Fer_d05r has no residues and has

C-terminal amidation. The comparison between the two peptides we can predict how diversified is this superfamily of conotoxins. The high variable toxin peptides may lead to the exhibition of different functions. This is the first report of the presence of T- superfamily in C. planorbis.

2403.32 Da

2639.13 Da

Figure 3. 30: MALDI-TOF MS Spectrum of Pla_A04u before and after reduction and alkylation

TABLE 3. 24: Sequence Analysis of Pla_A04u

Cycle # Assigned Residue 1 Gly 2 Ile 3 Leu 4 Ser 5 Gly 6 Val 7 Ile 8 Asn 9 Lys 10 Gly 11 Cys 12 Cys 13 Trp 14 Ala 15 Gln 16 Phe 17 Asp 18 Phe 19 Cys 20 Cys 21 Asn 22 Glu Mass Spectrometer MW 2403.32 Da Calculated MW 2407.03Da – 4 Cys= 2403.03 Da Sequence: G I L S G V I N K G C C W A Q F D F C C N E (T-Superfamily)

We attempted to acquire 1D and 2D NMR data on Pla_A04u but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful.

3.2.6 Purification of fraction Pla_B04f

The second batch of crude venom from Conus planorbis, Pla_B was submitted to purification on Size Exclusion Superdex Peptide column. The run was monitored at wavelengths 220, 250 and 280 at ranges 3, 1.5 and 1.5 respectively and ran for 55 minutes (Figure 3.31, Table 3.25). Seven fractions were collected and peak 4 was submitted for further purification using Reverse Phase Analytical column. Eight fractions were collected on the reverse phase analytical column for Pla_B04 (Figure 3.32, Table

3.26). The run was monitored at wavelengths 220, 250 and 280 at ranges 2, 1 and 1 respectively and ran for 100 minutes. The peak f was analyzed by mass spectrometry.

The sample was named Pla_B04f, which was submitted to reduction with DTT and alkylation with iodoacetamide. Mass spectrometry (Figure 3.33) showed there was no difference between the molecular weight before and after the reduction and alkylation, which is an indication that peptide is linear, and the sequence is shown on Table 3.27.

This peptide is 12 residues long and is reminiscent from CPYpl1 previously isolated from

C. planorbis.

11.174

220 r 3 4 5

0 -1.226 55 11.590

280 r 1.5 4 5

0 -0.810 55

Figure 3. 31: SE Superdex Peptide Column Chromatogram of crude venom C. planorbis Pla_B

Table 3. 25: Elution times of Pla_B SE Superdex Peptide column

Fraction Elution Time (minutes) 1 11.80 2 16.86 3 20.49 4 30.09 5 36.71 6 47.08 7 50.01

2.739

220 r 2 f

0 -0.306 100 0.349

280 r 1 f

0 0.349 100

Figure 3. 32: Analytical RP-HPLC chromatogram of Pla_B04

Table 3. 26: Elution times of Pla_B04 on Analytical RP column

Fraction Elution time (min) a 2.80 b 3.20 c 39.64 d 41.38 e 44.59 f 48.60 g 61.62 h 62.38

1556.85 Da

Figure 3. 33: MALDI-TOF MS Spectrum of Pla_B04f

Table 3. 27: Sequence Analysis of Pla_B04f

Cycle # Assigned Residue 1 Ala 2 Arg 3 Phe 4 Leu 5 His 6 Pro 7 Phe 8 Gln 9 Tyr 10 Tyr 11 Thr 12 Leu Mass Spectrometer MW 1556.85 Da

Calculated MW 1556.81 Da

Sequence: A R F L H P F Q Y Y T L

We attempted to acquire 1D and 2D NMR data on Pla_B04f but they were isolated in low picomolar quantities as we attempt to obtain 1D NMR data even after 4000 scans were unsuccessful.

3.2.7 Purification of fraction Pla_B05g

The peak 5 (Figure 3.31, Table 3.25) on SE Superdex Peptide column was submitted to further purifications on RP analytical column. The run was monitored at wavelengths

220, 250 and 280 at ranges 2, 1 and 1 respectively and ran for 100 minutes. Ten fractions were collected and peak g (Figure 3.34, Table 3.28) named Pla_B05g was analyzed by mass spectrometry (Figure 3.35) and submitted to reduction with DTT and alkylation with iodoacetamide. Mass Spectrometry showed no changes in the molecular weight before and after the reduction and alkylation, which indicate the presence of a linear peptide. Table 3.29 shows the sequence of Pla_B05g.

g 220 r 1

0 -0.613 100 1.397

280 r 2 g

0 100 -0.153

Figure 3. 34: Analytical RP-HPLC chromatogram of Pla_B05

Table 3. 28: Elution times of Pla_B05 on Analytical RP column

Fraction Elution Time Fraction Elution Time (minutes) (minutes) a 2.98 f 27.43 b 3.81 g 31.77 c 11.13 h 36.54 d 12.35 i 42.65 e 16.66 j 55.07

615.98 Da

Figure 3. 35: MALDI-TOF MS Spectrum of Pla_B05g

Table 3. 29: Sequence Analysis of Pla_B05g

Cycle # Assigned Residue 1 Tyr 2 Ile 3 Arg 4 Tryr Mass Spectrometer MW 615.98 Da

Calculated MW 614.72 Da

Sequence: Y I R Y

This 4 amino acid peptide is also reminiscent from CPYpl1 previously isolated from C. planorbis. Pla_B05g was lyophilized and prepared for NMR experiments as previously described. Figure 3.36 and 3.37 shows 1D NMR and 2D TOCSY spectrum of

Pla_A(2)05g. Table 3.30 shows the correlations between the amino acid sequence and the peaks assigned for each amino acid.

Table 3. 30: Amino acids assigned on 1D and 2D NMR of Pla_A(2)05g

N-H α ß Others Y 4.24 ppm 3.10 ppm Aromatic H’s ortho: 6.79 ppm (2H) Meta : 7.12ppm

I 8.32ppm 4.04 ppm 1.65 ppm γCH2: 1.41, 1.05ppm

γCH3: 0.65ppm, δCH3:0.83 ppm

R 8.24 ppm 4.29 ppm 1.78ppm γCH2=1.60ppm δCH2=3.17ppm 1.68ppm NH=7.15ppm Y 7.91 4.42 ppm 3.09 ppm Aromatic H’s ortho: 6.83ppm 2.85 ppm Meta: 7.09 ppm

Figure 3. 36: 1D NMR Spectrum of Pla_A(2)05g

Figure 3. 37: 2D NMR TOCSY Spectrum of Pla_A(2)05g

Table 3. 31 summarizes all the peptides isolated from C. planorbis and C. ferrugineus in this work. Signs in red indicate the post translation modification of the peptides.

Table 3.31: Table of isolated peptides from C. planorbis and C. ferrugineus

C. ferrugineus Sequence Gene Superfamily Fer_B03g GSCCAIHLDICCDS* T Fer_B05p LHRQPYYF Fer_D04ij TCVGSCVNGCPSICDCINNSYCASH P Fer_D 05r GIFSTLCCGVINSCC* T Fer_F06k AFKQYNWQRMP Fer_F07i AFKQYNWQR C. planorbis Pla_A06j LHRYPIY Pla_A06k ARFLHPFQY Pla_A06l ARFLHPFQYYT Pla_A04j ACVGNCGVSSQCPDVGCGCSNLQCW P Pla_A04u GILSGVINKGCCWAQFDFCCNE T Pla_B04f ARFLHPFQYYTL Pla_B05g YIRY

CHAPTER IV - DISCUSSION AND CONCLUSION

The linear peptides isolated from C. planorbis, were aligned to CPY-Pl1, a 30 amino acid long linear peptide isolated from C. planorbis, described by Imperial et al. that affect voltage gated K+ channels. [50]. CPY-Pl1 has sequence homology with the vertebrate neuropeptide

Y. Our peptide seems to be fragments related to CPY-Pl1, possibly they are products of degradation from CPY-Pl1.

Pla_A06l ARFLHPFQYYT------11 Pla_B04f ARFLHPFQYYTL------12 Pla_A06k ARFLHPFQY------9 CPYpl1 ARFLHPFQYYTLYRYLTRFLHRYPIYYIRY 30 *********

Fer_B05p and Pla_A06j showed some sequence similarities and they were aligned with

Vt0C01, from C. planorbis vitulinus, a subtype of clade E5 equivalent of clade IX of C. planorbis and C. ferrugineus described by Espiritu [23]. The linear peptides isolated in our work, could be a product of degradation of the mature toxin Vt0C01 or to a similar peptide, since no complete alignment of sequence was shown as we can see below.

Pla_A06j ------LHRYPIY---- 7 Fer_B05p ------LHRQPYYF--- 8 Vt0C01 ARYLSPFQYYKLYRYLARFLHRFPFYYIRY 30 *** * *

Identical signal sequence of the conotoxins precursors was found on both CPY-Pl1 and

Vt0C01, indicating that our linear peptides mentioned above, belong to the M-superfamily of conotoxins, since the similarities between the ER signal sequence of the conotoxin precursors classifies the gene superfamilies.

Fer_F06k and Fer_F07i showed no homology to previously discovered conopeptides from the literature, but some similarities was found to conp-vt, isolated from the vermivorous C. planorbis vitulinus, the same clade subtype of C. planorbis and C. ferrugineus.

Fer_F06k AFKQYNWQRMP--- 11 Fer_F07i AFKQYNWQR----- 9 conp-vt AFVKGSAQRVAHGY 14 ** : ** where * (asterisk) indicates fully conserved residue and : (colon) indicates conservation between groups of strongly similar properties

Conp-vt is an excitatory peptide that has significant sequence homology to the peptides of the myoactive tetradecapeptide (MATP) family as described by Dutertre [54]. MATPs are important neuromodulators in molluscs, annelids and insects. From the comparisons of our linear peptides to the literature, we can conclude that these conopeptides are homologous to neuropeptides families as neuropeptide Y and MATP that can function either directly or indirectly to modulate synaptic activity.

A dendrogram was made to compare the P-superfamilies of conotoxins isolated from C. planorbis and C. ferrugineus to other conotoxins in the literature within the same superfamily

(Figure 4.1). Fer_D04ij and Pla_A04j have different number of amino acids between the cysteines, -CX3CX3CX3CXCX5C- and -CX3CX5CX4CXCX4C- respectively but in the dendrogram below, they belong to the same clade, which was expected since both species belong to the clade IX in the phylogenetic scheme. These two species are also related to 73

pu9.1 from vermivorous C. pulicarius, which belongs to clade XIV. Even if a correlation is found on the dendrogram between these species, the sequences are very different from each other, showing the diversity of amino acid sequences of the conotoxins.

TCVGSC--VNGCP-SICDCINNSYCASH

ACVGNCGVSSQCPDVGCGCSN-LQCW--

SCTGSCSSSSFCP-PGCDCFH-AECT-- :* * * ** * * : *

where * (asterisk) indicates fully conserved residue and : (colon) indicates conservation between groups of strongly similar properties

Figure 4. 1: Dendogram of P-superfamilies of conotoxins from C. planorbis and C. ferrugineus compared to the literature.

A dendrogram was made to compare the T-superfamilies isolated from C. planorbis and C. ferrugineus to other conotoxins in the literature within the same superfamily (Figure 4.2). As we can see from the dendrogram below, the T-superfamily of conotoxins from C. planorbis and C. ferrugineus are not directly related to each other, since Conus species within the same or closely related clade could present some similarities. Fer_B03g, showed close relation in sequence to txd021 from the molluscivorous C. textile, both with C-terminal amidated peptides. C. textile belongs to clade V on the phylogenetic scheme described by Espiritu [23].

Fer_B03g and txd021 showed some relation to ar5.2 from the C. arenatus, which belongs to clade XIV. In this “molecular clade” relationship, we have three different species of cone snails from different clades.

** : ** ------SGCC-VIDSN-CC—

------GSCC-AIHLDICCDS LLGLVTGACCAVLKFSFCCGKK

** ** :***** GILSGVINKGCCWAQF-DFCCNE ------TCCKFQFLNFCCNE

GIFSTLCCGVINS-CC---

------CCIKFHP-CCHNG ** :: ** where * (asterisk) indicates fully conserved residue and : (colon) indicates conservation between groups of strongly similar properties

Figure 4. 2: Dendogram of T-superfamilies of conotoxins from C. planorbis and C. ferrugineus compared to the literature.

Pla_A04u showed close relation in sequence to ca5.1, from the vermivorous C. caracteristicus that belongs to clade XIV in the phylogenetic scheme, especially at C- terminal tail.

Fer_D05r is similar to im5.5, from the vermivorous C. imperialis that belongs to clade XVII on the phylogenetic scheme. The Conus species in this clade, apparently devour

amphinomids (“ﬁreworms”), a distinctive group of errant polychaete annelids defended by sharp spicules that would seem to make them singularly unattractive prey [52].

To summarize, C. planorbis and C. ferrugineus are different species of cone snails and belong to the same phylogenetic clade IX. Both P- and T-superfamilies conotoxins were found for the first time in the venom from both species. The linear peptides described in this work, are products of degradation of M-superfamily conopeptides and they are homologous to neuropeptide Y and myoactive tetradecapeptide families of neuropeptides.

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