Variability in the Venom of regius

By

Alejandro Uribe-Benninghoff

A Thesis Submitted to the Faculty of

the Charles E Schmidt College ofScience in Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, FL

December 1999 Variability in the Venom of Conus regius

By

Alejandro Uribe-Benninghoff

This thesis was prepared under the direction of the candidate's thesis advisors, Dr. Jim Hartman, Department of Biological Sciences, and Dr. Frank Mari, Department of Chemistry & Biochemistry, and has been approved by the members of his supervisory 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.

SUP ERVISORY COMMITTEE:

Dr. Fran . Mari Thes i<:.. Co Advisor Dr~' a v idBi n n i ~~

------Ch~it;~partm~ e nt of Biological ~cienccs

Vice Provost

ii ACKNOWLEDGEMENTS

Special gratitude to both Dr. Frank Mari and Dr. Jim Hartmann, thesis advisors and friends, for giving me the opportunity to come to Florida Atlantic University and make one of my lifelong dreams come true.

I would also like to thank my friend and soul mate, Teresa Koledin, for giving me her help during endless hours in the lab during every step of the project, both above and below the water. Herminsul Cano for his invaluable guidance in HPLC procedures in cone snail venom analysis and give special thanks to all the members of the U.S.S.

Conotoxin diving team. Their precious help took this project to "soaring" new depths.

Acknowledgements for Cognetix Inc., Salt Lake City, for its help with the cDNAIPCR experiments.

Finally and most importantly, I would like to thank my parents, Alejandro Uribe and

Olga Maria Benninghoff, my aunt, Diana Benninghoff, my brother and my sisters, for their never-ending love and support throughout all of these years.

Ill ABSTRACT

Author: Alejandro Uribe-Benninghoff

Title: Variability in the Venom of Conus regius

Institution: Florida Atlantic University

Thesis Co-Advisors: Dr. James X. Hartmann & Dr. Frank Mari

Degree: Master of Science

Year: 1999

The venom of two different geographical populations of Conus regius has been isolated and characterized. Comparisons between the chromatographic profiles of the venom of these two populations exhibited similarities and differences among the venom's constituents. MALO I-TOF and PCR analysis techniques ratified the differences present in the venom of both populations. It is postulated that these differences could reflect the rapid adaptive nature of cone snails in an actual stage of speciation. Molecular weights of the _venom's constituents were compared with those of patented conopeptides in the

Swiss Protein Database. Results of this comparison indicated that a number of the peptides isolated for both of the populations of C. regius had the same molecular weight as other patented conopeptides. In combination with the PCR analysis of these conopeptides, it has been proposed that some of the venom constituents of the venom of

C. regius could have pharmacological applications for vertebrate systems.

IV TABLE OF CONTENTS

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

1. INTRODUCTION ...... 1

1.1 General Background ...... 1

1.2 Gross Anatomy of the Venom Apparatus ...... 4

1.3 Cone Snail Venom ...... 8

1.4 Conopeptide Action ...... 13

1.5 Conopeptide Evolution ...... 15

1.6 Conus Peptides and Phylogenetic specificity ...... 16

1. 7 Purpose and Scope of the Research ...... 19

1.8 Objectives ...... 20

2. MATERIALS AND METHODS ...... 22

2.1 Venom Collection ...... 22

2.2 Venom Extraction ...... 22

2.3 Peptide Isolation ...... 24

2.4 Mass Spectrometry ...... 25

2.5 eDNA Analysis ...... 26

3. RESULTS AND DISCUSSION ...... 27

3.1 Venom Characterization ...... 27

3.2 eDNA Analysis ...... 40

v 3.3 Possible Venom Constituents ...... 42

- 4. CONCLUSIONS ...... 46

5. REFERENCES ...... 49

VI LIST OF TABLES

Table 3.1a: MALDI-TOF results for the chromatographic profiles of the venom of Conus regius specimens belonging to the Florida Keys ...... 30

Table 3.1b: MALDI-TOF results for the chromatographic profiles of the venom of Conus regius specimens belonging to Curacao ...... 31

Table 3.1c: Molecular weight similarities and retention volumes of peaks shared by the chromatographic profiles of C. regius for both geographical areas ...... 36

Table 3.1d: Molecular weight differences and retention volumes of peaks in the chromatographic profiles of C. regius for both geographical areas ...... 3 8

Table 3.3a: Molecular weights of venom components of C. regius compared to molecular weights ofthe Swiss Protein Database of patented conopeptides ...... 45

VII LIST OF FIGURES -.

Figure 1.7a: Conus regius ...... 21

Figure 1.7b: Conus regius citrinus ...... 21

Figure 1. 7c: Conus regius shells showing "transitional stage" ...... 21

Figure 2.1a: Collection sites for C. regius and C. regius citrinus ...... 23

Figure 3.1a: Reversed-Phase HPLC chromatogram for the venom

of a Conus regius specimen collected in the Florida Keys

(Pickles Reef, Plantation Key) ...... 28

Figure 3.1b: Reversed-Phase HPLC chromatogram for the venom

of a Conus regius specimen collected in the island ofCuracao ...... 29

Figure 3.1c: Chromatographic profiles for two Conus regius

specimens (Florida Keys) showing presence of four "signature peaks" ...... 33

Figure 3.1d: Chromatographic profiles for two Conus regius

specimens (Curacao) showing presence of four "signature peaks" ...... 34

Figure 3.1e: Chromatographic profiles of C. regius from the

Florida Keys (top) and C. regius from Curacao (bottom)

showing conserved areas (enclosed by boxes) ...... 37

VIII 1. INTRODUCTION

1.1 General Background

The class contains the largest number of mollusk species. Within this class, the families , Turridae, and Terebridae constitute the suborder Toxoglossa.

Members of the Toxoglossa suborder are characterized by the possession of a venom apparatus, of which the most venomous are the marine snails belonging to the genus

Conus. A number of human fatalities and intoxications have resulted from their stings.

1 There are around 500 species in the family Conidae , all of them members of the single genus Conus, and with few exceptions these are confined to tropical and subtropical waters. 2 Only a few number of species extend outside the tropics and those that do usually show a high level of endemicity in such cool areas as South Africa. southern Australia, and southern Japan. There are approximately 150 species present in the Western Atlantic out of which 60 are endemic to the Florida coastline. Despite great variation among these snails in shell size and shape, they all have a rough conical shape. 3

The shells are distinguished for rich colors and varied patterns. The whorls are rolled upon themselves below a small, sharp apex; the narrow aperture is as long as the body whorl and usually notched near the suture.4 Color patterns in cones are very important but

1 variable features in their identification. Colors are usually the product of depositing

metabolic waste products, although they are not fully understood. 5 -.

Cone snails are predominantly nocturnal in habit, burrowing in sand, under coral

or beneath rocks during the daytime. They become active at night, when feeding usually

occurs. Generally, each cone snail species is a highly specialized venomous predator.

Certain Conus will feed on only a single prey species. Various Conus species feed on fish

(piscivorous), others feed on gastropod mollusks (molluscivorous), or polychaete worms

as well as two smaller phyla (hemichordates and echiuroid worms). This last, and most

numerous group is known as vermivorous cone snails.3

Cone snails are mainly sublittoral inhabitants, ranging from tidal reef areas to

depths of several hundred meters. They can be found in a variety of microhabitats that

include coral reefs, sandy or coral rubble substrates and muddy bottoms. In such type of

habitats, vermivores may be very numerous and common, with a good diversity of

species. Molluscivores are generally second in abundance, with piscivores being actually

rare.

Sexes in cones are apparently always separate, presenting in some cases sexual

dimorphism in size or shape of shell and perhaps in color and habitat preference. In at

least some species, mating occurs only during a few short weeks and in an environment

not inhabited by the species the rest of the year. Other species apparently breed all year in

the same habitat.5

2 Females usually lay several large flask-shaped or pouch-shaped egg capsules in

clusters under coral slabs and rocks. The total number of eggs can vary between a few

dozen to tens of thousands. In species with large numbers of small eggs, the larvae hatch

into a veliger that is pelagic for several days or weeks, before settling and

metamorphosing into a more adult-looking shell. Such species are usually widely

distributed and show little variation form area to area as the veligers serve for distributing

the species over the range. 5

Species with egg capsules containing fewer and larger eggs usually hatch directly

into what is called a veliconcha, which looks like a metamorphosed shell of the typical

species. Such species have a very limited ability to expand their range, as there is no free­

swimming larval stage. This type of development results in the development of small

colonies of the species and a higher speciation or at least variation from colony to

colony.5

Cone snails are recently evolved when compared to other taxa of venomous

(i.e., snakes, spiders, and scorpions); however, the genus exhibits the greatest diversity of prey of any generic group of venomous predators. The first cone snails

6 appeared after the Cretaceous-Tertiary Pleistocene epoch , with Conus fossils becoming common only in the Cenozoic period. Paleontological evidence indicates that Conus evolved from a more ancient group of venomous toxo-glossid snails, the turrids or slit shells, which were already well established by the late Mesozoic. Since turrids prey on

3 worms, it seems likely that the primitive Conus were also wonn-hunters.7 An expansion of the genus is observed through the upper Miocene, which is then counteracted by a drop in the number of species while entering the Pliocene. This reduction is characteristic of this epoch, as seen in gastropod fossil records as well as in other invertebrate genera. 8

Then very rapid speciation occurs in the Pleistocene leading to the recent count of 500.

1.2 Gross Anatomy of the Venom Apparatus

Members of the family Conidae have a radular system consisting of three basic parts: a large transverse poison bulb, a long and highly intricate poison duct, and the radular sac containing harpoon-like radular teeth (Figure 1.2.a). The venom apparatus lies in a cavity posterior to the rostrum, on the dorsal part of the . The poison bulb is not responsible for the production of the venom. It apparently serves some type of function in helping the ejection of the poison through the venom duct. The bulb is usually the most conspicuous part of the system. It is generally bean-shaped, with the venom duct projecting from the slimmer right end.

The venom duct does not only function as a channel (conducting liquid from one point to the other), but is also responsible for the actual secretion of the venom. The duct originates at the right end of the venom bulb. It then passes sharply under the ventral face of the bulb and then over the esophagus to the left side of the cavity where the venom apparatus is contained.

4 A radular sac is found associated with the venom duct, containing harpoon-like

barbs that serve as hypodermic needles for the venom (radular teeth) (Figure 1.2.b). The

- sac varies in shape and size from species to species, but generally consists of two distinct

arms. The "reserve arm" apparently generates the teeth, while the "ready arm" contains

only a few well-developed barbs. The sac is attached to the gut near the base of the

proboscis sheath and opposite to the genital opening. The method through which the

venom moves from the venom duct into the radular teeth is not yet understood.

The pharynx and the proboscis, part of the digestive system of the cone snail, play

an important role as accessory organs. When the cone is hunting its prey, one radular

tooth is passed from the radular sac into the pharynx and is finally transported to the tip

of the proboscis. Once food is detected, the cone becomes active, extending its proboscis

about. When prey come within range, the proboscis lunges forward stabbing the victim,

perforating its body wall with the barbed tooth. Venom is released into the wound upon

penetration. The effects of the venom are immediate and the victim is soon paralyzed.

5 Venom Bulb ______

Radular teeth

Radular sac

Proboscis

Venom Duct Esophagus

Nerve Ring

Salivary gland

Figure 1.2a - Diagram of the venom apparatus of a cone snail

6 SHAFT

CUSP ------

SHEATH------,

Figure 1.2b- Parts of a vermivorous radular tooth

7 1.3 Cone Snail Venom

The biologically active agents in Conus venoms are exceptionally small peptides,

10-30 amino acids in length, a dominant characteristic of the venoms of all species

examined. Polypeptide toxins directly encoded by genes from other animal venoms are

significantly larger, usually 40-100 amino acids in length (snakes, spiders, scorpions, and

sea anemones).9

Cone snail venom is composed of conopeptides (major paralytic components), conopressins (the smallest of all peptides), conantokins (which contain large numbers of y-carboxyglutamate residues) and other peptides (King Kong, scratcher, etc). 10 There is a large array of different peptides in every venom, and each appears to be specifically targeted to a particular receptor, interfering with its normal function. 11 Physiological targets have been identified for several peptides found in Conus venoms. However, for most Conus venoms that have already been biochemically characterized (over 130 peptides so far, from 12 venoms), the receptor targets remain unknown. 12

For those peptides that have had their targets identified, it has been found that some target nicotinic acetylcholine receptors, voltage sensitive calcium channels, sodium channels, and N-methyl-D-aspartate (NMDA) receptors. Peptides targeting the same receptors have been divided into the following pharmacological families (which contain their probable pharmaceutical applications):

8 1. a-conopeptides: these conopeptides block neuromuscular or neuronal subtypes of

nicotinic acetylcholine receptors.

Potential applications: Treatment of schizophrenia, certain neuromuscular disorders,

certain types of cancer and urinary dysfunction.

2. ro-conopeptides: inhibit pre-synaptic neuronal subtypes of voltage-gated calcium

channels that mediate synaptic release of neurotransmitters

Potential Applications: Regulation of Ca influx to nerve cells limiting brain damage

from 0 2 deprivation caused by stroke or injury; alleviation for chronic pain.

3. g-Conopeptides: block currents of voltage-gated sodium channels in muscle.

Appear to inhibit neuronal sodium channels.

Potential Applications: Muscle relaxants, anti-seizure compounds or pam

therapeutics.

4. b-Conopeptides: activate neuronal sodium channels.

Potential Application: Highly toxic to non-mammalian systems it may be used as

pesticides.

5. -K-Conopeptides: target voltage sensitive K+ channels.

Potential Applications: nervous system, cardiovascular and gastrointestinal diseases.

6. \ji-Conopeptides: possible nicotinic receptor channel blocker.

In addition to the paralytic conopeptides, a number of biologically active peptides, which do not by themselves cause paralysis of prey, have also been identified. Some of these have been biochemically characterized.

9 The first non-paralytic peptide described from Conus venom was conantokin-G, extracted from the venom of Conus geographus. It has recently been shown that this peptide targets to glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype.

This is a novel biological activity for a peptide, since no previous examples have been described which exhibit activity against the NMDA receptor. However, the precise specificity for this peptide is still under research. 13

A second non-paralytic peptide, also extracted from the venom of Conus geographus, is conopressin G, a vasopressin analog. Conopressins are the smallest of the family of constrained peptides. Its central nervous system effects are similar to those induced by vasopressin.

The high affinity and specificity of conopeptides is dependent on their highly constrained and somewhat rigid conformations. 20 to 50% of all amino acids constituting conopeptides are Cys residues, and it is these residues which are involved in the formation of multiple disulfide bonds. In most conopeptides, the covalent cross-linking through multiple disulfide bonds presumably stabilizes the biologically active conformation. Conopeptides actually have some of the highest known densities of disulfide bonding found in biological systems.

Along with pharmacological families of Conus peptides, there are structural classes that can be defined by both the characteristic patterns of arrangement of Cys

10 residues or the post-translationally modified amino acid, y-carboxyglutamate (Gla).9 Gla

2 residues have the potential for stabilizing a-helical regions in the presence of Ca +. A

structural class comprises multiple pharmacological families of peptides. More than 30% .... of all sequenced Conus peptides belong to a single structural class which is characterized

by the arrangement of cysteine residues in ro-conopeptides (i.e., C-C-CC-C-C, the "4-

loop Cys framework). 11

a-conopeptides belong to the 2-loop structure classification. These conopeptides

were the first to be isolated from Conus venom and received their alpha designation due

to the similarity in their action when compared to alpha neurotoxins from snake venom

(i.e., alpha bungarotoxin). The 2-loop structure is shown hereinafter:

-ccr---c-----fI I

J..t-conopeptides have three disulfide bridges and belong to the three-loop structure

classification. The disulfide bonds are located between the first and fourth cysteine, the

second and fifth cysteines and the third and sixth cysteines. This produces the

characteristic three-loop moiety, which is also shared by \jl-conopeptides and is shown

below:

I I I I --cc-----c----c----cc-

11 w-conopeptides have a typical distribution of 25 to 28 residues in length and three disulfide bonds. These type of conopeptides belong to the four loop structural class, in which the disulfide bridges occur between cysteines one and four, two and five , and three and six. Although ro-conopeptides have the same number of disulfide bonds as ~­ conopeptides, they belong to the four-loop moiety instead of the three loop structural class. This is due to the fact that there is only one pair of cysteines located one after the other, while the rest are spread over the length of the peptide, giving rise to an extra loop.

~-conopeptides contain two pairs of cysteines.

c------~------cc---t=------c

Two distinctive subclasses have been characterized for the four loop structural class. They are designated as Group I (the w-conopeptide subclass) and Group II (the

King Kong peptide class). These classes are distinguishable by the overall biochemical characteristics of their peptides. In the case of w-conopeptides, the amino acids are strongly hydrophilic, and the peptides have a high net positive charge. In the case of the

KK peptides, the inter-cysteine amino acids are considerably more hydrophobic, with a net negative charge.

Conus peptides are currently being used in hundreds of research laboratories, for a wide variety of physiological and pharmacological investigations in both vertebrate and invertebrate nervous systems. Some Conus peptides have become well-established

12 neurobiological tools and powerful painkillers (e.g., conopeptide MVIIA from the venom of Conus magus, which produced the new drug Ziconotide).

1.4 Conopeptide action

In the rich marine environments where cone snails are present, speed of capture and paralysis of prey may be critical. Cone snails must swiftly immobilize their prey, and at the same time prevent being preyed upon while being exposed. In addition to their primary use of venom in paralyzing prey, it seems likely that some cones may use their venoms defensively. The evidence, although circumstantial, is presented by recent observations of cone snail attacks on humans. These suggest stinging as a defensive behavior of the snail.

The paralytic components of the venom that have been the focus of recent investigation are the alpha, omega and mu-conopeptides. Each of these conopeptides act by preventing neuronal communication, but each targets a different aspect of the process to achieve this. The alpha-conopeptides target nicotinic ligand gated channels, the mu­ conopeptides target the voltage-gated sodium channels and the omega conopeptides target the voltage-gated calcium channels.

Alpha and Psi-conopeptides block neuromuscular or neuronal subtypes of nicotinic acetylcholine receptors. The mechanism by which paralysis is brought about is thought to involve the alpha and psi-conopeptides binding to the alpha subunit of the nicotinic

13 ligand-gated ion channel. This causes blockage of the binding of acetylcholine and of

agonists such as nicotine. By preventing the agonist-induced conformational change in

- the receptor ion channel, which is required for the influx of sodium (essential for

membrane depolarization), the alpha conopeptides inhibit neurotransmitter action and

induce paralysis.

The best defined omega-conopeptides are GVIA, from Conus geographus, and omega

conopeptides MVIIA, MVIIIV and MVIID, from Conus magus venom. They are also

known as 'shaker peptides', as they induce persistent tremors in mice when injected intra­

cerebrally.9 GVIA, GVIB and GVIC all block the neuromuscular junction of skeletal

muscle and act by blocking calcium channels without interfering with cellular action

potentials. Differential effects are observed upon the application of GIV A - it appears that

of the three types of calcium channel, the toxin will block the L and N type in neurons,

but only has effect on the N type in muscle. This channel subtype specific response may

be useful in the identification and manipulation of calcium channels.

Mu-conopeptides act upon sodium channels in muscles. They also act on a very

limited extent in neurons, where they bind to a site designated "binding site 1" at the

mouth of the sodium channel. This inhibits the influx of sodium into the cell, rendering

the organism paralyzed. The 22-residue mu-conopeptide (from Conus geographus -

known as geographutoxin) is a competitive inhibitor of the binding of saxitoxin (STX)

and derivatives to the sodium channels in muscle but not in nerve. In contrast to

tetrodotoxin (TTX) and STX, the binding of mu-conopeptide is voltage-dependent. The

14 toxin discriminates between nerve and muscle sodium channels which otherwise show very little difference in their kinetics and in their responses to TTX and STX. Cardiac

"TTX-resistant" sodium channels are also refractory to mu-conopeptides, which indicates that the mu-conopeptide and the structurally unrelated STXffTX toxins all bind to common or overlapping sites on the sodium channel.9

1.5 Conopeptide Evolution

The rate of conopeptide evolution is higher than that of most other known proteins.

Gene duplication and diversifying selection result in the formation of functionally variable conopeptides that are linked to ecological diversification and the evolutionary success of the genus. 14 Nucleic acid templates directly encode Conopeptides, a characteristic that provides opportunities to study toxin genomes of great complexity. 15

An "antibody-like evolutionary strategy" probably generated the array of small Conus peptides with diverse ligand specificity, but relatively conserved folding pathways and stru_ctural frameworks. The net result is that every species has its own characteristic mixture of peptides, and this conopeptide cocktail is highly potent in the particular prey type on which the species specializes. Consequently, it not only has a rather unusual biochemical strategy been used for paralyzing prey (i.e., small, highly constrained peptides), but also generates the wide diversity of peptides in cone snail venom, a novel genetic and evolutionary strategy is employed by the genus as well. 16

15 Other results show that conopeptide diversity is associated with an ongoing process of locus duplication and rapid divergence. Because conopeptides are intricately related to a species' ability to paralyze its prey, the rapid adaptive evolution of these loci suggests that conopeptides are under strong selection in response to changes in the availability of or accessibility to a particular prey species over time. It can also represent a type of "arms race" between conopeptides and the cell channels and receptors of prey. Co-evolution of predator and prey may generate evolutionary forces similar to those seen in host­ pathogen evolution and provide means by which ecologically relevant genetic loci may rapidly diversify. 13

Reports also exist that peptides present a high rate of nucleotide changes in the toxin exon; even synonymous changes were increased 10-fold over other exons.17

1.6 Conus Peptides and Phylogenetic specificity

Endean and Rudkin (1963 and 1965), carried out pharmacological experiments with crude venom of the genus Conus, revealing that venom samples from piscivore species had no effect on mollusks or worms, and vice versa. These early studies suggested that the venom samples from Conus species were presumably strongly selected to act upon protein targets in the prey, and that the more unrelated the taxa to the prey, the more unlikely that it would have targets for the biologically active components in the venom.

16 Studies of individually isolated conopeptides have sustained the vtew of phylogenetic specificity of Conus venoms. The conopeptides of the piscivore Conus venoms have been particularly well studied. In every specific case studied, the peptides have been inactive in invertebrate systems. a-conopeptides GI and MI from Conus geographus and magus, respectively, are broadly active in vertebrate systems, and act at nicotinic acetylcholine receptors, not only in fish but also in all mammalian systems tested. In contrast a-conopeptide SI seems much more phylogenetically narrow in its biological activity, inhibiting nicotinic acetylcholine receptors in fish and elasmobranchs, but its activity in mammalian systems is orders of magnitude less.

Studies of individually isolated conopeptides have supported this general idea of phylogenetic specificity. A conopeptide from the venom of a particular species is generally specific for targets in the phylum to which the prey belongs. The conopeptides from fish-hunting cone snails have been the most studied. In all cases specifically tested, the peptides have been inactive in invertebrate systems, varying in how broadly they act.

Even though the sequence changes are not very great, apparently there are striking different conformations assumed by these two peptides. The same situation has been exposed for both w-conopeptides and 1-1-conopeptides, which target voltage-sensitive calcium channels and sodium channels, respectively, and are also inactive in invertebrate systems. 15

Consequently, the general view of Conus venoms and conopeptides from studies on the fish-hunting species is that the agents to be found are likely to be vertebrate-

17 specific. Studies on the crude venom suggest that vermivore species are likely to have conopeptides specific to annelids and molluscivore cones are likely to have conopeptides that are mollusk-specific. 18 However, peptides with broad phylogenetic specificity are advantageous as tools for investigating the role of their receptor targets over evolutionary time. Most receptors and ion channels evolved early in eukaryotic evolution, and are widely used by all of the higher eukaryotic taxa.

1. 7 Purpose and Scope of the Research

The vast majority of research being performed in the present has dealt with piscivorous cone snails located mainly in the Indo-Pacific and Eastern Atlantic Oceans.

This is probably due to the fact that almost three fourths of the species that have been identified inhabit these areas. At the same time, research has focused mainly on fish­ hunting cone snails due to the pharmacological and toxicological properties of the venom in bioassays conducted with vertebrates. Few data is available describing the ecology and venom of western Atlantic cones; just one tropical Atlantic cone snail has had its venom analyzed (Conus ermineus). The importance of this research relies on the fact that, even within the same genus Conus, no two venoms are alike. Each is a unique brew of chemical constituents, including toxic enzymes and other proteins and peptides that act on envenomed animals in various ways. This project provides information on the ecology and characteristics of the venom of a western Atlantic cone snail known as the crown cone.

18 The vermivorous crown cone, Conus regius, is an inhabitant of the coral reefs and

sandy bottoms in shallow waters along the coast of North Carolina, Florida, the

Caribbean and the western coast of Brazil. The pattern of their shells is quite variable but

usually consists of two spiral rows of large blotches that may be variably fused. The body

whorl is white in color, heavily covered with brown to olive, sometimes yellowish or

reddish, blotches arranged above and below a clearer midbody area (Figure l.7a).

There is a variant that has received the classification of subspecies due to its

distinctive shell coloring. Conus regius citrinus has a totally yellow or orange shell, a

white spire sometimes with some pattern (Figure l.7b). Shells are occasionally found having partially normal and partially citrinus patterns, a somewhat interesting stage

(Figure 1.7c). It is considered to be only a locality variation of Conus regius with its distribution restricted to Curacao and Cuba.19The fact that the species in itself is so variable has caused a great deal of confusion in regard to nomenclature in the past.

Although the sting of a Conus regius has not been proved to be deadly for a human its effects have been published by one author. 20 The effect of the sting of the crown cone was compared to that of the Antillean yellow scorpion - no immediate pain, but a gradual building up of discomfort and a hard swelling which numbed the fingers and palm of the hand for some hours. The sting was produced after the animal was disturbed and removed from its habitat.

19 1.8 Objectives

The main objectives of this project were the standardization of both extraction and characterization methods for the venom of the cone snail specimens of both reg10ns

(Conus regius from the Florida Keys and Conus regius var. citrinus from Curacao).

Due to the fact that the specimens had to remain in controlled conditions while they were being analyzed, special care was given to the environment of the salt-water aquarium they were kept in, trying to keep their environment as similar to the one they were collected from. The specimen's diet was another factor of importance during this project; Conus specimens mainly being fed Hermodice carunculata and Chloeia viridis

(both different species of fireworms).

Results from the analysis of the characterization of the specrmens from both geographical regions are presented. Comparisons between the chromatograms of specimens from both areas will provide insight on the importance of taking special care when analyzing the venom of the species Conus regius.

20 Figure 1.7a Conus regius

Figure l.7b Conus regius citrinus

Figure 1.7c Conus regius shells showing "transitional stage"

21 2. MATERIALS AND METHODS

2.1 Venom Collection

Diverse sized specimens of Conus regius (3 to 7 em in length) were collected along the South Florida Coast, specifically in the Florida Keys by scientifically certified divers using SCUBA equipment (Figure 2.1a). Conus regius citrinus was collected near the island of Curacao in the Caribbean Sea using the same technique. Half of the animals collected were dissected immediately in order to collect their venom ducts. The rest of the specimens were kept in a saltwater aquarium for dissection at a future time.

Ocean salt water was used to fill the 50-gallon aquarium. The water was directly pumped from the beach, run through filters and put into containers at Gumbo-Limbo

Natural Park. Specific gravity of the water was maintained between 1.026 and 1.032, a pH of 8.2, temperature oscillating between 68° F and 72° F. Water in the tank was filtered by a Marineland Emperor 2080 system. 40% water changes were preformed evety two weeks. Water was tested weekly for the presence of Ammonia, Nitrites,

Nitrates and pH.

2.2 Venom Extraction

Specimens that were analyzed were weighed in a Digital OHAUS Portable plus scale, measured and had their shells cracked before dissection. Each specimen

22 Figure 2.la Collection sites for C. regius and C. regius citrinus

23 was dissected and analyzed individually. The venom duct was dissected from each specimen and placed within a plastic centrifuge tube which had one of its faces cut out.

The tube was positioned at a 45° angle on a Styrofoam base, allowing the collected venom to drip down to the bottom of the tube.

To maintain the venom duct moist during the procedure, the duct was washed with drops of 0.1% trifluoroacetic acid. Using a small spatula, the venom is pushed down into the tube in small sections. Once the venom had been extracted from the duct, it was poured into a rnicrocentrifuge tube and placed within an Eppendorf Centrifuge (5414) during 5 minutes at 15,000 rpm. The duct was then saved and frozen at -70°C for further eDNA analysis.

The supernatant of the tube was collected and then filtered using a 0.45 Jlm

PVDF Whatman microfilter. The filtered portion was then lyophilized overnight in a

Savant Speed Vac Concentrator used in combination with a Labconco freeze dry/shell freeze system and a Savant Refrigerated Condensation trap.

2.3 Peptide Isolation

The standardization of the procedure for the venom analysis of Conus reg ius and

Conus regius citrinus was based on procedures previously published for other cone snail venoms 21 · 22 · 23 · 24 ' 2s · 26 . Th e l yop h.l.1 1ze d venom was t h en we1g· he d m· a D enver Instrument

Company Analytical scale. 1 mg of lyophilized venom was then separated and 0.4 Ill of

24 0.1% trifluoroacetic acid was added. 0.20 J.ll of venom solution was injected for reverse phase High Performance Liquid Chromatography (HPLC). The HPLC used consisted of a

Constametric 4100 multiple solvent delivery system and a Spectromonitor 4100 programmable, variable wavelength detector. For isolation of the peptides from the venom, buffer A consisted of 0.1% trifluoroacetic acid and buffer B was 0.1% trifluoroacetic acid in 60% acetonitrile. Acetonitrile (UV grade) and trifluoroacetic acid

(sequencing grade) was from Fisher. Milli-que water was used for buffer dilutions.

The peptides were separated on C 18 Vydac and Phenomenex columns (4.6 mm x

25 em, 5 J.lm particle size) with a flow rate of 1.0 ml/min. For the analytical runs, the venom was eluted with a linear gradient of 100% buffer A to 100% buffer B during 30,

60 and 100 minutes at a 220 nm wavelength and a 0.5 detection range. Among these, the

HPLC run that presented the most complete chromatogram was elected as the standardized procedure for peptide isolation. Sample collection was done manually for each peak representing a peptide for further analysis. Reverse phase HPLC chromatograms from specimens of both Conus regius and Conus regius citrinus were used to determine if there were visible differences between the peptide components of the two types of venoms.

2.4 Mass Spectrometry

The standardization of the procedure for the individual peptide analysis through mass spectrometry was based on procedures previously published for other cone snail

25 venoms.27 Purified peptide samples for mass spectrometric analysis were dissolved in acetonitrile/water (1: 1), and an aliquot was loaded onto the probe tip followed by addition of 1 Jll of sinapinic acid matrix 100 mM (ACN:MeOH:H20). A Hewlett-Packard

G2025A Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometer was used to obtain the molecular weight of each of the purified peptide samples. The molecular weights obtained from the analysis of each specimen were compared to those of other conopeptides that have been already patented and studied to determine if there has been a discovery of a new conopeptide. The molecular weights obtained from the specimens of Conus regius were compared to those of Conus regius citrinus to try to identify a difference in the components of their venoms.

2.5 eDNA Analysis

Cognetix Incorporated, a pharmaceutical company located in Salt Lake

City, Utah, will perform eDNA analysis of cone snail specimens from both regions. mRNA will be extracted from a portion of the venom ducts of both types of specimens

28 29 and_eDNA will be synthesized using variations on methods already published. · PCR primers will be used to amplify genes encoding for the a-conopeptides, J..L-conopeptides, ro-conopeptides, \j/-conopeptides, conantokins and contulakins.

Data obtained by eDNA analysis will be used to confirm if there is a genetic differentiation between specimens of the two different localities, which is then represented by the production of slightly different types of venom.

26 3. RESULTS AND DISCUSSION

3.1 Venom Characterization

Conus regius is the first Tropical-Atlantic cone snail species to have had its venom analyzed, and the third within its dietary group, the vermivores (C. abbreviatus

14 and C. lividus) • It is also the third cone snail species to have been studied pertaining to the Atlantic Ocean. The first one was C. ermineus, also known as the Atlantic Fish­ hunting snail, and the second was the alphabet cone, C. spurius at/anticus of which little

30 31 is known. ,

Chromatographic profiles of crude venom obtained from dissected venom ducts for specimens of both geographical regions (Florida Keys and Curacao) are shown in

Figures 3.la and 3.lb, respectively. Between 50 to 70 peptides have been separated by

20 25 the procedure previously described. - Molecular weights for the majority of the peaks are shown on Tables 3.la and 3.1 b. Peaks with retention volumes lower than 22.0 mL did not produce molecular weights within the range of conopeptide structures (between 1000

- 4000 Da). These highly polar constituents could have molecular weights under 800 Da and hence not produce a reading within the spectrum analyzed.

A closer analysis of the chromatographic profiles reveals differences between the venom of the specimens that inhabit these two areas. Similar peaks are also evident.

Although the differences could be explained as a natural variability present in individual

27 1674.2 Da 3141.5 Da

800 mV ' '

2001.5 Da

1439.3 Da ' '

Retention Time (min)

Figure 3.1a- Reversed-Phase HPLC chromatogram for the venom of a Conus regius

specimen collected in the Florida Keys (Pickles Reef, Plantation Key)

28 1680.3 Da 3162.0 Da

800 mV ' '

2018.1 Da 1436.0 Da '

Retention Time (min)

Figure 3.lb- Reversed-Phase HPLC chromatogram for the venom of a Conus regius

specimen collected in the island of Curacao

29 Table 3.la- MALDI-TOF results for the chromatographic profiles of the venom of Conus reg ius

specimens belonging to the Florida Keys

Peak Number Retention Molecular Weight (SP- signature peak) Volume (ml) Conus regius -Florida (Da) II 22.75 I489.0 12 27.IO I930.2 I3 27.85 I871.3 I4 (SP) 28.20 1439.3 15 29.40 3742.5 17 32.96 2731.3 18 35.00 1670.3 19 36.14 1834.1 20 37.96 3387.4 21 (SP) 38.95 1674.2 23 41 .50 2999.7 24 42.10 3185.8 25 42.79 1589.1 28 46.90 2556.1 29 47.80 1702.4 30 48.90 2078.1 31 (SP) 49.70 3141.5 32 50.70 2522.1 34 52.52 2494.5 35 54.60 1849.3 36 56.60 1520.3 37 57 .75 3165.1 38 58.47 2959.8 39 59.20 2780.6 40 60.01 2557 .2 41 61.50 3154.1 42 (SP) 63.40 2002.7 43 65.35 1976.2 44 67.70 2001.5 45 70.01 3645.1 47 72.65 1739.2 48 73 .50 3342.0 49 74.80 2145.9 50 75.60 2646.7 51 77.02 2260.1 52 79.29 3615.0 53 80.10 3638.1 55 84.64 3111.0 57 87.30 3187.5 58 88.65 2977.4 59 91.15 3319.7

30 Table 3. lb- MALDI-TOF results fo r the chromatographic profi les of the ve nom of

Conus regius specimens belongi ng to Curacao

Peak Number Retention Molecular Weight (SP- Signature peak) Volume (ml) Conus regius -Curacao (Da) 11 23 .71 146 1. 1 12 (SP) 25.46 1436.0 13 27. 21 11 69.9 14 27.75 3617.9 15 29.20 18 17.0 16 30.28 27 12.7 18 3 1.36 1753.9 19 32.26 1691.3 23 36 .1 3 1837.2 25 (SP) 37.2 1 1680.3 26 38.05 3042.7 27 38.60 1823.7 28 39.86 32 12.0 29 4 1.06 1578 .3 31 43 .1 6 2032.4 32 44. 13 1748.6 33 44.86 2 11 7.9 34 45.46 3448. 6 35 (S P) 46.96 3 162 .0 37 48.90 2526.1 38 49.13 213 1.1 4 1 5 1.95 19 11.6 42 52.75 1829.1 43 53 .81 2548.8 45 57.4 1 1443.0 47 59.90 2037 .2 49 62.28 1458.7 50 (SP) 64.33 20 18.1 5 1 65.43 3679.5 52 67.8 1 1786.0 56 7 1.56 1829.8 57 72.4 1 2 179.6 59 84.26 3367.5 60 86.65 3225.7

31 snails within a given species (due to diet, age or season) results indicated otherwise.

Similarities were found when comparing chromatographic profiles of different specimens

inhabiting the same geographical area (Figure 3.1a and 3.1c, Figure 3.1 b and 3.ld

respectively). These chromatographic profiles contained the same number of peaks, the

same retention volumes and the same molecular weight for each one of its peaks.

Although differences exist amidst the relative abundance of the peaks in each individual

profile, a parallelism can be made within a profile group of a region in general. This

parallelism is not as evident when comparing chromatographic profile groups of the two

regwns.

Among the similarities, four distinct peaks shared by the specimens of the two

geographical regions can be observed. The first of these peaks belongs to that which

produces the highest abundance reading after the 22 minute mark. Peaks before this time

limit were not chosen due to a lack of MALO I-TOF data. The second and third peaks

were chosen due to their constant presence in every chromatographic profile and their

high relative abundance. The fourth peak was elected because it presented the starting

point of a common area of peaks, shared by the two groups of chromatographic profiles.

These "signature peaks" have been labeled with arrows and although they do not

represent all the similarities within both types of profiles, they do represent the most

visible ones. Molecular weights of the signature peaks are also displayed in each of the

figures (3.la and 3.lb, respectively) being extremely similar. Variations between the

molecular weights of the signature peaks by 40 Da or less can be explained by the calibration of the MALOI- TOF system ( + 1-3 Da), the interaction of the matrix with

32 800 mV

'

Retention Time (min)

800 mV

Retention Time (min)

Figure 3.lc- Chromatographic profiles for two Conus regius specimens (Florida Keys)

showing presence of four "signature peaks"

33 800 mV

0.0 ' 103' Retention Time (min)

800 mv

' 0.0 103' Retention Time (min)

Figure 3.ld- Chromatographic profiles for two Conus regius specimens (Curacao)

showing presence of four "signature peaks"

34 the sample ( + 1-5 Da) or loose sodium ( +22 Da) or potassium ( +34 Da) ions. This would imply the possibility that signature peaks found for specimens of both regions would actually be representing similar or identical conopeptides present in the species in general. Figures 3.lc and 3.ld show chromatographic profiles for different specimens belonging to the same areas, all showing the same signature peaks.

Peaks preceding or following signature peaks also exhibit very similar molecular weights when compared in both sets of chromatographic profiles. Table 3.lc presents peaks that have similar retention volumes and molecular weights for the profile groups of both areas. These peaks, in conjunction with signature peaks, generate "conserved areas" where the venom constituents of the specimens for both regions are almost identical. Two to five peaks form the conserved areas for both geographical groups. Figure 3.le depicts these proposed conserved areas, which are enclosed by boxes.

Although the chromatographic profiles for the specimens inhabiting two distinct geographical areas contain similarities, over 50% of the peaks in each profile group

(pertaining to each geographical area) have different retention volumes and molecular weights (Table 3.ld). As it has been mentioned before, these differences are only present when comparing specimens from the Florida Keys with those belonging to the island of

Curacao. These differences are non-existent in specimens that inhabit the same geographical areas.

35 Table 3.1c- Molecular weight similarities and retention volumes of peaks shared by the

chromatographic profiles of C. regius for both geographical areas

Retention Molecular Weight Retention Molecular Weight Volume (ml) Conus regius - Volume (ml) Conus regius - Florida Keys Curacao (Da) (Da) EJ22.75 1489.0 23 .71 1461.1 28.20 1439.3 25.46 1436.0

35.00 1670.3 32.26 1691.3 u36.14 1834.1 36.13 1837.2 38 .95 1674.2 37.21 1680.3

41 .50 2999.7 38.05 3042.7

42.79 1589.1 41.06 1578.3 047.80 1702.4 44.13 1748.6 49.70 3141.5 46.96 3162.0

50.70 2522.1 48.90 2526.1

63.40 2002.7 59.90 2037.2

67.70 2001.5 64.33 2018.1 070.01 3645.1 65.43 3679.5 72.65 1739.2 67.81 1786.0

74.80 2145.9 72.41 2179.6

36 800 mV

Retention Time (min) 2 3

800 mV

4

1

0.0 - 103' Retention Time (min) Figure 3.1e- Chromatographic profiles of C. regius from the Florida Keys (top) and

C. regius from Curacao (bottom) showing conserved areas (enclosed by boxes)

37 Table 3.ld- Molecular weight differences and retention volumes of peaks in

the chromatographic profiles of C. regius for both geographical areas

Retention Molecular Weight Retention Molecular Weight Volume (rnl) Conus regius - Volume (rnl) Conus regius - Florida Keys Curacao (Da) (Da) 27.10 1930.2 27.21 1169.9 27.85 1871.3 27.75 3617.9 29.40 3742.5 29.20 1817.0 32.96 2731.3 30.28 2712.7 37.96 3387.4 31.36 1753.9 42.10 3185.8 38.60 1823.7 46.90 2556.1 39.86 3212.0 48.90 2078.1 43.16 2032.4 52.52 2494.5 44.86 2117.9 54.60 1849.3 45.46 3448.6 56.60 1520.3 49.13 2131.1 57.75 3165.1 51.95 1911.6 58.47 2959.8 52.75 1829.1 59.20 2780.6 53.81 2548.8 60.01 2557.2 57.41 1443.0 61.50 3154.2 59.90 2037.2 63.40 2002.7 62.28 1458.7 65.35 1976.2 71.56 1829.8 73.50 3342.0 84.26 3367.5 75.60 2646.7 86.65 3225.7 77.02 2260.1 79.29 3615.0 80.10 3638.1 84.64 3111.0 87.30 3187.5 88.65 2977.4 91.15 3319.7

38 These results could represent a probable divergence in the evolutionary path of the Conus regius species. Ecological diversification and evolutionary success has been widely demonstrated for the genus Conus6 and has numerous examples in the ever- changing world of taxonomical classification. 5 It has been established that conopeptides are associated with trophic diversification, intricately related to a species' ability to paralyze its prey. Conopeptide diversity is also associated with an ongoing process of locus duplication and rapid divergence, suggesting that conopeptides are under a strong selection response to changes in the availability of or accessibility to a particular prey species. over time.. 14

By inhabiting two distinct geographical locations, which are approximately 1800 kilometers apart, these two different populations of Conus regius would encounter different types of prey and predators, modifying their venom composition to suit the conditions of the environment. A difference in the diets of both C. regius populations would also explain the variance in their shell coloration patterns. As was mentioned before, it has been hypothesized that shell coloration is produced by the deposition of metabolic waste products.5 Dietary differences in these two populations would cause a modification of their metabolic waste products and hence shell coloration.

Although these two populations may maintain a number of similarities (signature peaks and conserved areas in terms of HPLC retention times and mass), the variation in the components of the venom for this species would indicate the ability to adapt to new

39 conditions in a very swift manner. This exhibition of geographic variation could prove to be the expression of an underlying genetic differentiation.

Although specimens from both geographical regions were kept alive under controlled conditions for almost a year, during which time live fireworms were placed in the aquarium as their unique source of food, differences between the chromatographic profiles of C. regius and C. regius var. citrinus were maintained.

This was not the case for their shell coloration. Two specimens belonging to the area of Curacao, C. regius var. citrinus, started to show a change in the coloration of their shell. This color alteration was very apparent since it went from their typical orange to the characteristic white body mottled with red blotches of the C. regius of the Florida

Keys. When the venoms of these two specimens were analyzed, their chromatographic profiles remained similar to those observed for the region they inhabited, a somewhat interesting phenomenon with unknown consequences.

3.2 eDNA analysis

PCR primers were used to amplify genes encoding for the a-conopeptides, ).l­ conopeptides, w-conopeptides, \jl-conopeptides, conantokins and contulakins. There were strong PCR products for the w-family and a-family primers. The ro-family primers gave four distinct products for the specimen belonging to the Florida Keys and 3 for the one belonging to Curacao. The ).l-family primer pair gave a weak product in C. regius var.

40 citrinus (Curacao) and no visible product for C. regius (Florida Keys). The conantokin primer pair gave a moderate product for C. regius and a very weak product in C. regius var. citrinus.

It appears that each of the cone snails is expressing different conopeptide genes.

Cloning and sequencing of the PCR products would be the next step to know how different the expressions are and if there really are any genetic differences.

It can be hypothesized, however, that these two populations do contain differences within their genetic material due to the two distinct bio-geographical areas they inhabit. Changes in the gene pool of both populations will be more than likely to occur due to the rapid adaptation characteristics of the conopeptide loci, leading to a prompt speciation process. This does not necessarily imply, however, that these specimens which belong to two different geographical areas are two completely different spectes.

A factor that could spark this genetic differentiation could be a difference in the prey they feed on. Although both of the populations hunted and consumed two different types of fireworms while they were in captivity (Hermodice carunculata and Chloeia viridis), this does not mean that the population from Curacao had a preference for another type of prey that was not present in the tank. If the specimens belonging to Curacao hunt for different species of worms than those present in the Florida Keys, they may have had

41 to change their venom to target that specific type of prey, a change that would be made at the genetic level.

Up to date, this is the first study that has taken into consideration the possible variability in the venom of two populations of the same species of cone snail. It is unknown if prevwus investigations obtained their specimens from one or various geographic areas. If the variability observed m the venom constituents of these two populations is considered a common occurrence (due to the rapid adaptive nature of these organisms), then it could be possible that the same is true for other cone snail populations. The discovery of a larger number of biologically active conopeptides could be done by analyzing populations which inhabit different geographical areas of species that have already had there venom analyzed (C. magus, C. aulicus, C. purpurascens, C. striatus, C. textile, C. episcopatus, C. ermineus, C. Imperialis among others).

3.3 Possible venom constituents

MALDI-TOF results from the venoms of both regions were compared with the

Swiss Protein Database for similarities with known conopeptides. This procedure has been previously employed to obtain sequence information on novel peptides.24 Table

3.3a presents results of this analogy. Peaks exhibiting similar or identical molecular weights when compared to patented conopeptides are shown.

42 It is apparent from Table 3.3a that masses of several previously known peptides from species belonging to the Pacific Ocean (C. magus, C. goegraphus, C. striatus, C. aulicus, and C. episcopatus) are very similar to those found for C. regius in both regions.

Although this information cannot be used directly to establish the identity of the peptides, it sheds information on possible sequences found in the fractions. By combining this information with that obtained through the PCR analysis, it is fairly evident that the venom of C. reg ius contains conopeptides of the families a, ~ and ffi.

This approach could be of great use due to the fact that it is impractical to sequence every peptide, given the complexity of the venoms. By using this comparison method, the selection process of peptides for future pharmaceutical use could be made much easier. Although no peptide sequencing or synthesis was done in this project, it is highly recommended to follow on with sequencing procedures for those peaks that show a molecular weight similarity with conopeptides already known.

If what is proposed here is true, some of the constituents of the venom found in both populations of C. regius would be able to block neuromuscular or neuronal subtypes of nicotinic acetylcholine receptors and be used as drugs for neuromuscular disorders.

These peaks would be: 11, 14, 18, 21 , 25, 30 and 47 from the Florida Keys and 11 , 12,

25 , 29, 49 and 52 from Curacao, all of them possible a-conopeptides. Other peaks would be able to inhibit pre-synaptic neuronal subtypes of voltage-gated calcium channels that mediate synaptic release of neurotransmitters and be used for treating brain damage

(peaks 17, 50 and 55 from the Florida Keys, possible ffi-conotoxins) . Finally, those peaks

43 that block currents of voltage-gated sodium channels in muscle and inhibit neuronal sodium channels could be used as another pain-killing drug (28 and 40 from the Florida

Keys and 43 from Curacao, possible J.l-conotoxins).

Although the sting of a C. regius has not proven to be deadly, its effects in humans have been compared to those produced by the sting of a scorpion, as it has been mentioned before. The characteristics of the symptoms presented after the sting imply the action of the venom in a localized area and an obvious activity in mammalian systems.

These observations, combined with the MALDI-TOF data, and PCR analysis would tend to confirm the action of a number of venom constituents in vertebrate systems.

Consequently, the current view of vermivore cone snails containing a venom that acts

18 32 weakly in vertebrate systems ' , would change dramatically.

Another aspect that must be taken into consideration is the high percentage of vermivorous cone snails present in the world today. Almost 80% of the 500 species of cone snails identified are vermivorous. The possibilities for pharmaceutical research in this area would be easily quadrupled. The number of possible biologically active peptides would increase even more, once the variations among the venom constituents of different populations of the same species were taken into account.

44 Table 3.3a- Molecular weights of venom components of C. regius compared to

molecular weights of the Swiss Protein Database of patented conopeptides

Peak Number Molecular Weight Molecular Patented Weight (SP- signature Conus reg ius- Patented Conopeptide Nameffype/sp. peak) Florida Conopeptide (Da) (Da) 11 1489.0 1499.0 a-conopeptide MI (C. magus) 14 (SP) 1439.3 1422.0 a-conopeptide Gil (C. goegraphus) 17 2731.3 2746.0 w-conopeptide SVIB (C. striatus) 18 1670.3 1673.0 a-conopeptide AUIC (C. aulicus) 21 (SP) 1674.2 1673.0 a-conopeptide AUIC (C. aulicus) 25 1589.1 1578.0 a-conopeptide AUIB (C. aulicus) 28 2556.1 2553.0 11-conopeptide GIIIC (C. geographus) 30 2078.1 2082.0 a-conopeptide EI (C. ermineus) 40 2557.2 2553.0 11-conopeptide GIIIC (C. geographus) 47 1739.2 1731.0 a-conopeptide AUlA (C. aulicus) 50 2646.7 2646.0 w-conopeptide MVIIA (C. magus) 55 3111.0 3104.0 w-conopeptide MVIIA (C. magus) Peak Number Molecular Weight Molecular Patented Weight (SP- signature Conus regius- Patented Conopeptide Nameffype peak) Curacao Conopeptide (Da) (Da) 11 1461.1 1461.0 a-conopeptide SIA (C. magus) 12 (SP) 1436.0 1422.0 a-conopeptide Gil (C. goegraphus) 25(SP) 1680.3 1673.0 a-conopeptide A UIC (C. aulicus) 29 1578.3 1578.0 a-conopeptide A UIB (C. aulicus) 43 2548.8 2553.0 11-conopeptide GIIIC (C. geographus) 49 1458.7 1461.0 a-conopeptide SIA (C. magus) 52 1786.0 1792.0 a-conopeptide EPI (C. episcopatus)

45 4. CONCLUSIONS

The characterization of the venom of two populations of Conus reg ius (one belonging

to the Florida Keys and the other to the island of Curacao) produced novel results. The

chromatographic profiles of both populations presented a complex venom, constituted by

almost 70 different peptides.

Similarities and differences were observed between the chromatographic profiles of

these two populations. Among the similarities found, four peaks having the same retention volume and identical molecular weights were present in each one of the chromatographic profiles for specimens belonging to both geographical areas. These

peaks were labeled as "signature peaks".

Peaks preceding or following signature peaks also exhibited very similar molecular weights when compared in both sets of chromatographic profiles. These peaks, in conjunction with signature peaks, generate "conserved areas" where the venom constituents of the specimens for both regions are almost identical. Two to five peaks form the conserved areas for both geographical groups.

On the other hand, over 50% of the peaks in each profile group (pertaining to each geographical area) had different retention volumes and molecular weights. These differences were only present when comparing specimens from the Florida Keys with those belonging to the island of Curacao. These differences were non-existent in

46 specimens that inhabited the same geographical areas. This would indicate differences in

the constituents of the venom of two populations belonging to the same species, a finding

that has never been reported before. The implications of this discovery may be

widespread. If the variability observed in the venom constituents of these two populations

is considered a common occurrence (due to the rapid adaptive nature of these organisms),

then it could be possible that the same is true for other cone snail populations. The

discovery of a larger number of biologically active conopeptides could be done by

analyzing populations which inhabit different geographical areas of species that have

already had there venom analyzed. A comparison of the chromatographic profiles of

venoms belonging to different populations of already analyzed cone snails is strongly

encouraged.

Other results seem to prove that some of the constituents of the venom found in

both populations of C. regius would be able to block neuromuscular or neuronal subtypes

of nicotinic acetylcholine receptors and be used as drugs for neuromuscular disorders.

Other peaks would be able to inhibit pre-synaptic neuronal subtypes of voltage-gated calcium channels that mediate synaptic release of neurotransmitters and be used for treating brain damage. Another group of peaks possibly block currents of voltage-gated sodium channels in muscle and inhibit neuronal sodium channels and could be used as a pain-killing drug. All of these characteristics were attributable only to piscivore cone snails. The current view of vermivore cone snails is that they contain a venom that acts weakly in vertebrate systems and, therefore, is of a low priority in pharmaceutical and neuro-physiological research. These results will definitely change this conception.

47 With over 80% of the cone snails identified up to date being verrnivores, a whole new gamma of possibilities has opened for the conopeptide community and pharmaceutical industry in general.

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