Isolation and Characterization of Novel Conopeptides from
Conus nux
By
Rani Elizabeth Ramlakhan
A Thesis Submitted to the Faculty of
The Charles E. Schmidt College of Science
In Partial Fulfillment of the Requirements for the Degree of
Master of Science
Florida Atlantic University
Boca Raton, FL
December 2002 Isolation and Characterization of Novel Conopeptides from Conus nux
By
Rani Elizabeth Ramlakhan
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 by the members of her 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.
SUPERVISORY COMM TTEE: / ~~(}
partment of Chemistry and Biochemistry
hmidt College of Science ~w~ Vice Provost Date
11 AKNOWLEDGEMENTS
Special thanks to Dr. Frank Mari for the opportunity ofjoining and becoming a member
of his research team. I would also like to thank Florida Sea Grant College Fund for
providing funding for this research.
I would like to thank Dr. Gregg Fields and Dr. Craig Byrdwell for all their help and
support over the past few years.
It was my pleasure working with Fred Pfleuger, Herminsul Cano, Aldo Franco, David
Mora, Husam Abbasi, Carolina Moller and all the rest of the members of this research
team. I thank and wish each of them success in finding new scientific breakthroughs in
this field of research.
Finally, I would like to thank my parents, Harry and Surnintra Ramlakhan, and my
brothers, Enrick, Michael, and David, for all their support in all my educational
endeavors over the past few years.
ill ABSTRACT
Author: Rani Elizabeth Ramlakhan
Title: Isolation and Characterization ofNovel Conopeptides from
Conus nux
Institution: Florida Atlantic University
Thesis Advisor: Dr. Frank Mari
Degree: Master of Science
Year: 2002
Cone snails are marme gastropods belonging to the genus Conus that inhabit in tropical habitats throughout the world. They are predators that paralyze their prey by injection of venom, containing a complex mixture of conopeptides. The venom of cone snails has been found to be a valuable source of specific drugs for disorders ranging from stroke to chronic pain. For this work, the venom of Conus nux, a Panarnic cone snail species, was extracted and analyzed. Components of the venom were isolated using Size Exclusion Chromatography (SEC) and Reversed Phase High Performance
Liquid Chromatography techniques. A novel conopeptide sequence, determined by
Edman Degradation is reported. The arrangement of the cysteines residues within this sequence suggest that it member of the M-superfarnily of conotoxins.
lV TABLE OF CONTENTS
LIST OF TABLES ...... vii
LIST OF FIGURES ...... ix
1. INTRODUCTION ...... 1
1.1 General Background ...... 1
1.2 Cone Snail Venom Apparatus ...... 2
1.3 Venom Composition ...... 3
1.4 Conotoxins ...... 4
1.5 Conus Peptides as Drugs ...... 7
1.6 Objectives ...... 10
2. MATERIALS AND METHODS ...... 11
2.1 Cone Snail Description and Collection ...... 1 I
2.2 Crude Venom Extraction ...... 13
2.3 Peptide Isolation ...... 16
2.4 Reversed Phase HPLC ...... 18
2.5 Size Exclusion Chromatography ...... 20
2.6 Analytical Reversed Phase HPLC ...... 22
2. 7 Reduction/ Alkylation ...... 23
3. RESULTS AND DISCUSSION ...... 24 v 3.1 Chromatographic Analysis ofVenom ...... 24
3.2 Peptide Sequencing ...... 51
3.3 M-superfamily ...... 53
4. CONCLUSIONS ...... 62
5. REFERENCES ...... 64
Vl LIST OF TABLES
Table 1.4a Classification of Co no toxins (II) ...... 6
Table I.4b Structural Classification of Conotoxins (23) ...... 7
Table I.5 Potential Therapeutics of Conopeptides ...... 9
Table 2.2 List of Crude Venom Batches of Conus nux ...... I6
Table 2.4 Fluorescent Indicator Dyes ...... 20
Table 3.Ia Retention times, MW, and Bioassays results for Conus nux (NUX_A)
fractions I-I8 ...... 26
Table 3.Ib Retention times, MW, and Bioassays results for Conus nux (NUX_A)
fractions I9-33 ...... 29
Table 3.Ic Retention times, MW, and Bioassays results for Conus nux (NUX_A)
fractions 34-47 ...... 30
Table 3.Id Retention times, MW, and Bioassays results for Conus nux (NUX_A)
fractions 48-63 ...... 3I
Table 3.Ie Retention times, MW, and Bioassays results for Conus nux (NUX_A)
fractions 64-81 ...... 32
Table 3.I f Retention times, MW, and Bioassays results for Conus nux (NUX_A)
fractions 82-103 ...... 33
Table 3.1g Molecular Weight (Da) vs. Retention Time (min) for Conus nux
(NUX_A) ...... 34
Table 3.Ih Molecular Weight (Da) vs. Retention Time (min) for Conus nux (NUX_A)
and Conus jaspideus, an Atlantic cone snail species ...... 34 Vll Table 3.li Retention times of the fractions collected from NUX Ea ...... 37
Table 3.1j Retention times of the fractions collected from NUX Eb ...... 37
Table 3.1k Retention times and Intensity of fractions collected from NUX_E01 ...... 40
Table 3.11 Retention times and Intensity of fractions collected from NUX_E02 ...... 40
Table 3.1m Retention times and Intensity offractions collected from NUX_E03 ...... 41
Table 3.1n Retention times and Intensity offractions collected from NUX_E04 ...... 42
Table 3.1o Retention times, Intensity, and MW offractions collected from
NUX E05 ...... 43
Table 3.lp Retention times, Intensity, and MW offractions collected from
NUX E06 ...... 44
Table 3.1q Retention times, Intensity, and MW offractions collected from
NUX E07 ...... 45
Table 3.1 r Retention times, Intensity, and MW of fractions collected from
NUX E08 ...... 46
Table 3.1 s Retention times and Intensity of fractions collected from NUX_E09 ...... 47
Table 3.1t Retention times and Intensity of fractions collected from NUX_El 0 ...... 48
Table 3.1u Retention times and Intensity of fractions collected from NUX_E11 ...... 49
Table 3.1 v Retention times, Intensity and MW of fractions collected from
NUX E12 ...... 50
Table 3.2 Peptide Sequence of fraction NUX_E0905 (nux-1) ...... 52
Table 3.3a Comparison of the sequence ofnux-1 and published J..~.-conotoxins ...... 54
Table 3.3b Table of J.!-conotoxins (Patent W0021 07678) ...... 55
viii LIST OF FIGURES
Figure 1.2a Diagram ofthe Venom Apparatus of Cone Snails ...... 2
Figure 1.2b Photograph of the Venom Apparatus of a Cone Snail...... 3
Figure 2.1 a Photograph of marine cone snail, Conus nux, collected by F AU ...... II
Figure 2.1 b Map of Collection Sites for Conus nux ...... 12
Figure 2.2 Schematic Drawing of the dissection procedure of a cone snail ...... 14
Figure 2.3 Protocol for the extraction and characterization of Conus nux ...... 17
Figure 2.4 Semi-preparative Reversed Phase HPLC Chromatogram of Conus nux
(NUX_A) ...... l9
Figure 2.5a Size Exclusion HPLC Chromatogram of NUX_D ...... 21
Figure 2.5b Size Exclusion HPLC Chromatogram ofNUX_E ...... 21
Figure 2.5c Size Exclusion HPLC Chromatogram ofNUX_F ...... 22
Figure 3.la Biologically Active Fractions ofSample NUX_A ...... 25
Figure 3.1 b Semi-preparative RP-HPLC of Conus nux (NUX_A) fractions 1-18 ...... 26
Figure 3.1c MALDI-TOF mass spectrometry ofNUX_A fraction 17 ...... 27
Figure 3.1d MALDI-TOF mass spectrometry ofNUX_A fraction 18 ...... 28
Figure 3.1e Semi-preparative RP-HPLC ofConus nux (NUX_A) fractions 19-33 ...... 29
Figure 3.1f Semi-preparative RP-HPLC ofConus nux (NUX_A) fractions 34-47 ...... 30
IX Figure 3.1g Semi-preparative RP-HPLC of Conus nux (NUX_A) fractions 48-63 ...... 31
Figure 3.1h Semi-preparative RP-HPLC of Conus nux (NUX_A) fractions 64-81 ...... 32
Figure 3.1i Semi-preparative RP-HPLC of Conus nux (NUX_A) fractions 82-103 ...... 33
Figure 3.1j Size Exclusion chromatograms ofNUX_Ea at the wavelengths 220run,
280run, and 250nrn ...... 36
Figure 3.1 k Size Exclusion chromatograms of NUX_ Eb at the wavelengths 220run,
280nrn and 250nrn ...... 38
Figure 3.11 Semi-preparative RP-HPLC of Conus nux sample NUX_E01 ...... 39
Figure 3.1m Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E02 ...... 40
Figure 3.1n Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E03 ...... 41
Figure 3.lo Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E04 ...... 42
Figure 3.lp Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E05 ...... 43
Figure 3.lq Semi-preparative RP-HPLC ofConus nux SEC fraction NUX_E06 ...... 44
Figure 3.1 r Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E07 ...... 45
Figure 3.ls Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E08 ...... 46
Figure 3.lt Semi-preparative RP-HPLC of Conus nux SEC fractionNUX_E09 ...... 47
Figure 3.lu Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_EIO ...... 48
Figure 3.1 v Semi-preparative RP-HPLC of Conus nux SEC fractionNUX_E 11 ...... 49
Figure 3.lw Semi-preparative RP-HPLC ofConus nux sample NUX_El2 ...... 50
Figure 3.lx Analytical reversed phase separation of Conus nux, NUX_E0905 ...... 51
Figure 3.2 MALDI-TOF mass spectrometry of fraction NUX_E0905 ...... 53
X 1. INTRODUCTION
1.1 General Background
Marine snails of the genus Conus (cone snails) are venomous predatory gastropods found on or near coral reefs in tropical waters throughout the world. The families Canidae, Turridae, and Terebridae constitute the superfamily Conoidea, members of which are characterized by the possession of a venom apparatus. The most deadly are the Canidae family that includes approximately 1000 species of the genus
Conus (11). Approximately 250 species have been found in the Atlantic Ocean, including the West African species and approx. 50 species have been found in the Panarnic Region
(tropical west coast of the Americas) (24). Cone snails are of interest because they have an elaborate biochemical strategy for evenomating their prey (15). The natural prey of
Conus consists of polychaete worms, other gastropods and small fish. Cone snails are generally classified by their feeding habits: verrnivorous (worm eaters), molluscivorous
(mollusk eaters), and piscivorous (fish eaters). Cone snails feed by envenomating their prey by using a disposable harpoon-like radula tooth. Injection of the venom rapidly immobilizes their prey, which is subsequently engulfed by the snail (11). 1.2 Cone Snail Venom Apparatus
The venom apparatus in cone snails consists of following major parts: the venom bulb, the venom (poison) duct, a proboscis, the pharynx and a radular sac containing harpoon-like radular teeth (shown in Figure 1.2a). The venom bulb aids the ejection of the venom through the venom duct; the duct originates at the right end of the venom bulb and the other end is associated with radular sac (Figure 1.2b). The radular teeth contained in the radular sac serve as disposable hypodermic needles for venom injection
(16). When the cone snail is hunting its prey, one radular tooth is transported from the radular sac to the tip ofthe proboscis. As the cone snail extends the proboscis, the victim is stabbed suddenly and the tooth penetrates its body releasing the venom, thereby paralyzing their prey.
Figure 1.2a Diagram of the Venom Apparatus of Cone Snails. 2 b- bulb, p-proboscis, d-venom duct, ph- pharynx
Figure 1.2b Photograph ofthe Venom Apparatus of a Cone Snail.
1.3 Venom Composition
Cone snail venom is a cocktail of neuropeptides, comprising of 20-200 different components. The primary function of the venom of the predatory marine snails is to paralyze their prey (15). However, over the years, many divers and shell collectors drawn to their colorful shells have inadvertently been stung while handling cone snails. Some cone snails are even capable of inflicting human fatalities (~ 100 fatalities reported). The species most frequently associated with human envenomations are Conus textile and
Conus geographus (lethal) (11 ). Human symptoms from cone snail stings vary from severe pain to a spreading numbness leading to muscular paralysis, depending on the species involved. Each species of cone snail has unique venom that is highly specific to
3 its species. The venom consists of a large number of different peptides that, when separated, exhibit a wide range of biological activities. The venom is a highly complex mixture of conopeptides, which includes conotoxins, conopressins, contryphans and other peptides. The most studied conopeptides are the conotoxins, primarily because of their high selectivity and potency towards neuronal targets.
I. 4 Conotoxins
The biologically active components present in cone venoms consist primarily of proteins and peptides. The most intensely studied group are the peptides between 10-40 amino acid residues in length. These conopeptides can be divided into two major groups:
(1) those with multiple disulfide bonds (2 or more), referred to as conotoxins, and (2) non-conotoxins, those with a single disulfide linkage or none at all. Although there are approximately 1000 species of cone snails, the functions of only a minority of these peptides have been determined. For peptides where the function has been determined, three classes oftargets have been elucidated: voltage gated ion channels; ligand-gated ion channels and G- protein-like receptors.
Conus peptides that target voltage-gated ion channels include those that delay the inactivation of sodium channels, as well as blockers specific for sodium channels, calcium channels, and potassium channels. Peptides that target ligand-gated ion channels include antagonists of NMDA and seratonin receptors, as well as competitive and noncompetitive nicotinic receptor antagonists. Peptides that act on G-protein coupled receptors include neurotensin and vasopressin receptor agonists (12). 4 These peptides are generally grouped into 'superfamilies.' The three most extensively studied superfamilies are the A, M and 0 superfamilies. Members of each superfamily have a similar highly conserved protein sequence in their precursors, and family members within each superfamily have a characteristic arrangement of cysteine residues in the mature peptides. Peptide families found within the A-superfamily include the a-conotoxins and aA-conotoxins, both of which are competitive antagonists of the nicotinic acetylcholine receptor. In addition to these members, KA-conotoxins act by blocking voltage-gated potassium channels. In theM-superfamily, most notable are the Jl conotoxins that block voltage-gated sodium channels. There are also \j/-conotoxins, which are noncompetitive antagonists of the nicotinic-acetylcholine receptor. At the present time, the 0-superfamily appears most diverse in terms of pharmacological function.
Family members include ro-conotoxins, which blocks voltage-sensitive calcium channels,
8-conotoxins, which delays the inactivation of voltage-sensitive sodium channels, J.!O conotoxins, which block voltage-gated sodium channels and K-conotoxins, which block voltage-gated potassium channels. For most worm, fish and mollusk-hunting Conus species, these three superfamilies (A, M and 0) account for the major proportion of venom peptides. There is, however, evidence of other additional superfamilies in Conus venoms (Table 1.4a) (II).
5 Superfamily Cysteine Family Molecular target Prototypical Example Arrangement A CC-C-C a nAChR a-GI
CC-C-C p a 1-antagonist p-TIA CC-C-C-C-C a A nAChR aA-EIVA CC-C-C-C-C KA Possible K+ channel KA-SVIA (antagonists) Na+ channel M CC-C-C-CC ~ ~-PillA CC-C-C-CC \If nAChR '1'-PIIIE
0 C-C-CC-C-C 8 Na+ channel 8-TxVIA
C-C-CC-C-C ~0 Na+ channel ~0-MrVIB 2 C-C-CC-C-C (J) Ca+ channel w-MVIIA C-C-CC-C-C K K+ channel K-PVIIA C-C-CC-C-C y Pacemaker channel y-PnVIIA
p C-C-C-C-C-C Spastics Unknown Tx9a
p . +2 T CC-CC 't re-synaptlc c a 1::-TxiX (tx5a) channel (blocker) X Noradrenaline x-Mri transporter inhibitor
s C-C-C-C-C-C- cr 5-HT3 (antagonist) cr-GVIIIA C-C
Table 1.4a Classification of Co no toxins (11 ).
The unprecedented pharmacological selectivity of conotoxins can be defined in part by their specific disulfide bond frameworks combined with the hypervariable amino acid within the disulfide loop (12). Therefore, conotoxins are also structurally classified by their disulfide-bridging pattern (shown in the Table 1.4b).
6 Structural class of Disulfide Bridging Co no peptide Examples Pattern
2-loop structural class cc .. c ... c a-Conotoxins: GI, GIA, Gil, & MI I I I I
cc.~ ... c.c2 3-loop structural class I I I I ~-Conotoxins: GIIIA, GIIIB, & GIIIC
ro-Conotoxins: GVIA, GVIB, & 4-loop structural class c. .. c.J:c.c .. 2: I I I I GVIC
Table 1.4b Structural Classification of Co no toxins (23).
1.5 Conus Peptides as Drugs
The potential pharmaceutical uses of various conotoxins have inspired a new wave of studies. In recent years, conopeptides have mainly been used as tools for neuroscience research, but the ability of individual conotoxins to bind selectively to some types of receptors and ion channels, but not to others, makes them attractive drug
7 candidates (7). Due to the high potency and exquisite selectivity of conopeptides, several of them are in various stages of clinical development for the treatment of human disorders. For example, two Conus peptides are being developed for the treatment of pain. The most advanced is co-conotoxin MVIIA (Ziconotide), anN-type calcium channel blocker (US patent 5,859,186). co-conotoxin MVIIA, isolated from Conus magus, is approximately I 0-100 times more potent than morphine, yet it does not produce the tolerance or addictive properties of opiates. co-conotoxin MVIIA has completed Phase III
(fmal stages) of human clinical trials and has reached approvable status by the FDA as a therapeutic agent. This conotoxin is introduced into human patients by means of an implantable programmable pump with a catheter threaded into the intrathecal space.
Preclinical testing for use to reduce post-surgical pain is being carried out on another
Conus peptide, contulakin-G, isolated from Conus geographus. Contulakin-G is a sixteen amino acid 0-linked glycopeptide with a C-terrninus that resembles neurotensin. It is an agonist of neurotensin receptors, but appears significantly more potent than neurotensin in inhibiting pain.
Since there is a large number ofbiologically active substances in Conus species, it
IS highly desirable to further identify and characterize peptides capable of treating disorders involving voltage gated ion channels, rangmg from stroke to chronic pain.
Shown below is a list of different conopeptides and there potential treatments (Table 1.5)
8 Peptide class Mechanism of action Potential Therapeutic Indication a-Conopeptides Antagonists of neuronal and Anxiety, Parkinson's disease, pain,
\jl- Conopeptides Skeletal muscle nAChR muscle relaxants, anti hypertensive agents, cancer
~- Conopeptides Skeletal muscle sodium channels Neuromuscular block co- Conopeptides Calcium channels Stroke, pain
K- Conopeptides Potassium channels Hypertension, arrhythmia, asthma
Conopressin Vasopressin receptors Blood pressure regulation Contulakin-G Neurotensin receptors Pain and CNS disorders Conantokins NMDA receptors Epilepsy, pain, stroke, Parkinson's disease
Table 1.5 Potential Therapeutics of Conopeptides ( 19).
~-conopeptides can be used for the treatment of disorders associated with voltage gated sodium channels. ~-conopeptides can be useful as neuromuscular blocking agents, local anesthetic agents, analgesic agents, neuroprotective agents and as a treatment for neuromuscular disorders. Novel conotoxins, especially those that can be used for the treatment of disorders involving voltage gated ion channels could address the need for a safer and more effective treatment.
9 1. 6 Objectives
The general goal of the project described in this thesis is to develop a methodology to isolate and characterize the Conus venom by usmg extensive chromatographic techniques (Size Exclusion Chromatography and Reversed Phase
HPLC) with the aim of discovering novel conopeptide sequences from Conus nux, an unstudied Panamic cone snail species.
10 2. MATERIALS AND METHODS
2. I Cone Snail Description and Collection
Conus nux, (also known as the Nut Cone) is the smallest of the Panamic cone snails species. The shell of this cone snail is reddish-brown with white markings and spots arranged in indistinct bands (Figure 2.1 a). There is also a very distinct purple blotch on the anterior tip, which makes the cone commonly show two purple bands on a white surface (1 0). The proboscis is thin and reddish-brown and the foot of the animal is pink.
Conus nux generally measures up to 25 mm in length and up to 14 mm in diameter.
Figure 2.1 a Photograph of marine cone snail, Conus nux, collected by F AU.
11 This vermivorous marine mollusk is distributed commonly in intertidal areas. Conus nux
is a favorable Conus to for venom isolation and characterization because it lives in highly
populated colonies spanning over most of the Pacific Coast of the Americas (from
Mexico to Peru) assuring an abundant supply of cone snails. The specimens used in this
research were collected off the coast of Costa Rica (Figure 2.1 b).
Figure 2.1 b Map of Collection Sites for Conus nux.
The specimens were transported alive to our laboratory at Florida Atlantic University
located in Boca Raton. Upon arrival the water was changed immediately with saltwater
provide by Gumbo-Limbo Natural Park (Boca Raton). This water was pumped directly 12 from the beach allowing us to mimic to the greatest extent possible, the same natural
conditions of temperature and chemicals concentrations as the habitat of the cone snails.
The majority ofthe cone snails collected was kept alive and then dissected the next day
when the venom was extracted. Any cone snails that didn't survive the journey were
frozen at -70 OC and studied separately.
2. 2 Crude Venom Extraction
The first part of the venom extraction consisted of taking the animal out of the
shell. At this stage, one can choose to break or not to break the shell; in either case, the snail will perish. Removal of the snail from each specimen without cracking the shell was the selected method. Since live specimens were used, each cone snail was placed on ice for a period of five minutes, to insure that the body would come out intact. A long thin dissecting probe, or in some cases a fme needle, was inserted into the shell. The body was then extracted, by applying pressure against the interior walls of the shell and then pulling the body out in one fluid motion. Finding the venom duct within this small cone was the most difficult part of the entire extraction procedure. For the smallest cones collected, a dissecting microscope was used. The body was held down with a needle, while a thinner needle was used to dissect and tear through the tissue to remove the venom duct, without destroying its shape. In most cases, the beige color venom duct of
Conus nux could be found tightly clustered together and was usually visible under a thin and clear layer oftissue. Once the duct was isolated, it was placed on a flat or cylindrical surface so that the duct could be elongated. While holding the upper part of the duct 13 (usually the venom bulb) with a needle, a small spatula was used to squeeze the venom out, pushing the duct with the venom against the walls gently. By doing so, the venom spilled out into the tube. In some cases, if the duct was long, a small portion ofthe venom duct was cut off. Typically, the venom duct of Conus nux is around 30mm long or shorter; therefore, the duct is cut only once. All the pieces of the venom ducts were placed on dry ice and later stored at -80°C for further analysis. During the extraction of venom, the venom duct was rinsed with minimal amounts of 0.1% TF A in water to avoid drying of the venom and the duct, which could complicate the entire extraction process
(Figure 2.2).
- --~ '1 -- -,..------:-, ------
l t
11
radula tooth
Figure 2.2 Schematic Drawing ofthe Dissection Procedure of a Cone Snail. 14 Once the venom was extracted, it was then rinsed again with enough 0.1% TF A solution (usually I 0- 20ml depending on the number of specimens dissected at the same time). This was done in order to dissolve as much crude venom as possible. The venom solution was placed in a centrifuge for about 15 minutes at approximately 4000 revolutions per minute to separate impurities from pure venom. The supernatant was separated from the solid and the solid pellet is stored at --40°C. The venom was filtered using 0.45 J..tm luer-lock membrane filters attached to a plastic syringe and then transferred to 5 ml eppendorf tubes and placed at --40°C until it became completely frozen. Finally, the frozen venom was lyophilized to obtain a fluffy white/beige colored powder. The venom at this stage was regarded as crude venom. The crude venom was weighed, labeled and placed at - 70 o C.
Several batches of Conus nux crude venom from were prepared as the specimens were collected on different trips to Costa Rica; each batch of crude venom was given a different code. The amount of snails sacrificed and the amount of venom extracted varied in each batch preparation.
15 Conus nux Number of Cone Snails Total Crude Venom Sample Preparations Specimens (extracted) NUX A 12 (larger cones) 13.6mg
NUX B 15 6.0mg
NUX C 58 31.0 mg
NUX - D ---- 14.5 mg (Pooled from A, B, C)
NUXE 90 (80 medium size, 10 small) 40.0 mg
NUX F 52 (dead snails) 14.0 mg
Table 2.2 List of Crude Venom Batches of Conus nux.
2.3 Peptide Isolation
After obtaining the crude venom from the cone snails, two different methods, involving combined chromatographic techniques, were used to isolate the peptide (as shown in Figure 2.3). The frrst separation technique involved direct separation of the crude venom using Semi-preparative RP-HPLC. At frrst, this technique was useful to obtain a chromatographic profile that outlined the complexity of the venom from Conus nux. However, this chromatographic technique by itself did not meet the primary goal, which is to isolate "pure" peptides with a low molecular weight in as few steps as possible, because of severe chromatographic overlap of the peptide components.
Therefore, isolation was improved by using the second method: Size Exclusion HPLC
16 and then Semi-preparative RP-HPLC and fmally Analytical RP-HPLC for further purification of selected fractions (when needed).
I Collection of specimens I I I Venom Extraction I I ICentrifuge I Filter/ Lyophilize I I I CRUDE VENOM I I I I IReversed Phase HPLC I I SE-HPLC I 1 I I I RP·HPLC I Bioassays II MALDI·TOF MS I I I Purified Fractions Active fractions I I I Reduction/Alkylation I Sequencing Edman Degradation I IMass Spectrometry j
Figure 2.3 Protocol for the extraction and characterization of Conus nux.
17 2.4 Reversed Phase HPLC
A 7.2 mg sample of Conus nux crude venom (NUX_A) dissolved in 0.1 %
TFAIH20 was injected into the Semi-preparative Reversed Phase HPLC (Figure 2.4).
For this separation, a Vydac 5J.lm C-18 protein/peptide semi-preparative column (250mm x 10mm) and UV detector (Thermo Separation Products SM-4100) measuring the absorbance at A. = 220 nm and 280 nm were used. The solvent gradient (Thermo
Separation Products CM-41 00) established for the separation of conopeptides is a 100 min. linear gradient starting at 100% of Buffer A and ending with I 00% of Buffer B.
The mobile phase used for this separation is the following:
Buffer A: 0.1% TF A I H20
Buffer B: O.I%TFA I 60% ACN I H20
The flow rate used for all the Semi-preparative RP-HPLC separations in this research was
3.5rnl/min.
18 0 Time (min) 100
Figure 2.4 Semi-preparative Reversed Phase HPLC Chromatogram of Conus nux (NUX_A).
The chromatogram obtained for NUX_A provided a general profile of the venom of
Conus nux. A total of 108 fractions were collected from this separation. A portion of each fraction was used to determine the purity of the components isolated and the molecular weight by Matrix-Assisted Laser Desorption Ionization -Time of Flight (MALDI-TOF) mass spectrometer (Hewlett Packard Model G2025A) using a DHB ( dihydroxybenzoic acid) matrix and laser energy of2.3-3.1 ~-
The remainder of each fraction was subjected to fluorescence-based in vitro assays for screening the venom from Conus nux. These assays allow the identifications of venom fractions that possess biological activity at both ion channels and neurotransmitter receptors. These fluorescence-based functional Bioassays where performed in murine 19 cortical cell cultures, usmg Di-8-ANEPPS (4-[2-[6-(dioctylamino)-2-naphthalenyl] ethenyl]- I -(3-sulfopropyl)-pyridinium) to determine the membrane potential dye. The following dyes used for each ion channel are shown below (Table 2.4).
Fluorescent Indicator Dyes Voltage-gated Ion Channel
SBFI dye (4,4'-[ I ,4, 10-trioxa-7 , 13- diazacyclopentadecane- Na+ channel 7, 13-diylbis(5- methoxy-6,2-benzofurandiyl))bis- I ,3-Benzenedicarboxylic acid) PBFI dye I ,3-Benzenedicarboxylic acid, 4,4'-[ I ,4, I 0, 13- K+ channel tetraoxa-7, 16- diazacyclooctadecane-7, 16- diylbis(5- methoxy-6,2-benzofurandiyl)]bis- Fluo3 dye (N-[2-[[[[2-[bis( carboxymethyl)am ino]-5- (2, 7- Ca+ channel dichloro-6-hydroxy-3-oxo-3H- xanthen-9- yl)phenoxy]methyl]methyl]oxy]- 4- methylphenyl]-N-(carboxymethyl) glycine)
Table 2.4 Fluorescent Indicator Dyes.
In the case of Sodium, an increase in fluorescence indicated a depolarization, meaning that it is a possible Na channel opener. A decrease in fluorescence indicated a hyperpolarization and indicated the opposite, establishing a possible Na+ channel blocker.
A total of 12 fractions from Conus nux showed some bioactivity.
2.5 Size Exclusion Chromatography
Three batches of crude venom (NUX_D, NUX_E, and NUX_F) were separated usmg the second method, involving Size-Exclusion Chromatography. A Chromoflex
Pharmacia Superdex-30 column (560mm x 25.4mm), using an isocratic system was
20 utilized for these separations. Each run was approximately 300-350 min long. The mobile
phase used for these separations was 0.1 M Ammonium Bicarbonate (at a flow rate of 1.5
ml/min). A UV Detector (TSP SM-51 00 PDA Detector) measuring the wavelengths at
220nm, 280nm, and 250nm. Examples of the SE-HPLC chromatograms can be seen
below.
A-=220 nm
9
6 7 5 11 12 13
0 Time (min) 300 Figure 2.5a Size Exclusion HPLC Chromatogram ofNUX_D.
A-=220 nm 9
2 10 11
I I I I I I I 0 Time (min) 300
Figure 2.5b Size Exclusion HPLC Chromatogram of NUX_E.
21 Both Chromatograms of NUX_D and NUX_ E show some similarity to the SEC profile
of Conus nux. Even the sample of dead snails (NUX_F) shows some similar aspects.
However, in this chromatograph, an extra peak not shown in the other samples denoted
some possible sample decomposition.
A-=220 nm
3
8 7 14 6 5 10 11 13 12 15
0 Time (min) 300
Figure 2.5c Size Exclusion HPLC Chromatograph ofNUX_F.
2.6 Analytical Reversed Phase HPLC
Selected fractions collected from the Semi-preparative RP-HPLC of each of the
SEC fraction runs were subjected to further purification. For this separation, an analytical Phenomenex Jupiter 5)lm C18 column (250mm x 4.60mm) and UV detector
(TSP SM-4100) (measuring the absorbance at A.= 220nm and 280nm) were used. The quaternary pump (TSP CM-41 00) produced a linear gradient starting at 100% of Buffer
A and ending with 100% of Buffer B. The mobile phase used for these separations was
22 the same as used for the Semi-Preparative HPLC runs (Buffer A: 0.1% TFA I H20, Buffer
B: 0.1%TFA I 60% ACN I H20). The flow rate used for all the Analytical RP-HPLC
separations in this research was 1.0 rnllmin.
2.7 Reduction/Alkylation
Once the co no peptide was isolated and purified, the sample had to be reduced and alkylated before sequencing because of the possibility of disulfide bridges being present.
The sample was reduced and alkylated prior to Edman Sequencing at the Biotechnology
Research Laboratory at Yale University using the following protocol:
1. The sample was dried from 0.1% TF A I H20 in 2 ml screw capped Eppendorf
tubes.
2. The sample was reconstituted with 60J.!l d-H20.
3. The sample was taken to dryness.
4. The dried peptide was dissolved in 30 Jll 10%ACN/10%N~HC0 3 (made
fresh before use).
5. The pH was checked to ensure that it was in the range 7.5-8.5.
6. 5J.!l of 45mM DTT (dithiothreitol) was added to the sample, and incubated at
3 7 degrees for 20 min.
7. 5J.!l 100mM (iodoacetamide) was added to the sample, and incubated at room
temperature for 20 min.
Approximately 75% of the sample was used for sequencing. The rest was used for mass spectrometry. 23 3. RESULTS AND DISCUSSION
3.1 Chromatographic Analysis of Venom
When using the initial method of separation (direct Semi-preparative Reversed
Phase HPLC of the crude venom) a total of 103 fractions were collected from the
NUX_A. From the chromatogram shown earlier, it is clear that the venom composition of
Conus nux is quite complex. Therefore, it was necessary to adopt another form of chromatography to enhance the separation ofthe components ofthe venom. The fractions collected from sample NUX_A were separated into two parts. The first part was used for fluorescence-based functional bioassays. The information from the results of the bioassays confirmed that Conus nux did indeed have biologically active components. A total of twelve fractions showed activity on the sodium, potassium, and calcium channels
(Figure 3.1 a). These fractions appeared to show the most activity on the voltage-gated sodium channel, rather than the other two channels.
24 0 Time 100
Figure 3.la Biologically Active Fractions ofSample NUX_A.
The other portion of the sample was analyzed usmg MALDI-TOF mass spectrometry to determine how many components were in each fraction. From the data, more than one fraction had several components with different molecular weights. The data ofNUX_A has been compiled and it is shown in great detail below.
25 1-2 14
16
13
15
4 12 3 17 5 10
Figure 3.1b Semi-preparative RP-HPLC of Conus nux (NUX_A) fractions 1-18.
Peak# Time M.W. (Da) M.W. (Da) M.W. (Da) #Cpds Na-Di8 Na-SBFI K-PBFI Ca-Fiuo3 1 3.94 2 4.17 3 5.40 4 5.61 3473.7 6512.1 2 5 14.40 6 15.54 7 16.36 8 17.27 719.3 914.0 1067.4 4 1358.0 9 18.47 731 .1 914.5 1086.2 4 1355.3 10 20.31 727.1 900.8 1876.3 7 2987.8 3277 3397 3939.6 11 20.81 728.7 1285.2 1716.3 5 3267.5 3383.9 12 21.29 1715.4 1884.7 1980.8 3 13 21.53 1695.7 1962.1 2033.2 5 2363.9 2688.9 14 22 .11 1927 2042.7 2925.6 4 3270.4 15 22 .84 1285.8 1460.6 1887.5 5 2039.7 2942.6 16 23.42 1283.2 2022.7 2257.6 5 2919.3 31.59.3 17 23.95 1292 2272.8 3182.9 2 18 24.62 1926.8 2711 .6 3176.5 4 6.5 6.7 -0.6 0.3 3375.8 Table 3.la Retention times, MW, and Bioassays results for Conus nux (NUX_A) fractions 1-18.
26 C :\NlWDEEP\HYP70."l'OF• [l\.03 .00 , #1979} Sample Name Conu5 Nux Peak #17 P reparc.t t ion ACN/Wat<'r 50 : 50 Matrix DHB User Name Rl\NI Department Name Chemistry and BlochP. m.i.3try Application rnaldi Co l lected Snt Mar 01 12;13;19 1980 Pr oc .. s, ..d Snt Mar 01 12;17:28 1 980 P rinted Sat Mar 01 12:17 : 41 1980 Sequence 11et.hod DATA METHOD !'OR AYALA58. TOF* Co llec-:: ion Mode Single Shots ( l33 of 223 ) H
0
I I
- _ ____ jI 2 ---·-----T- I ----,------,------, .1 r----___:1 0 7 00 1400 2100 2800 3500 4200 4900 5600 6300 7000 m/z
Figure 3.1c MALDI-TOF mass spectrometry ofNUX_A fraction 17.
27 C:\NAVDEEP\ HYP7l.TOF• [A.03.00 ' #1980) Sample Name Con us Nux Peak # 1& Prepara tion ACN/Wate< 5 0:50 Ma t rix DH B User Na me RAN I De part ment Name Chemistry and Biochemiatry Application maldi Col lected Sat Mar 01 12 : 18 : 26 1980 Processed Sat Mar 01 1 2:21:07 1 960 Printed Sat Mar 01 12 :21 :1 9 1980 Sequence Method DATA METHOD FOR AYALA58.TOF* Co llection Mode Singl e Shots (71 o f 142 ) Mesa 8 [ 1-1 20] Laset: Energy 2 .62 {0 .11) uJ Vacuum B.60e-007 torr Mass Range 7000 Da I on Opti cs 28 . 0/7 . 0 kV Mas s Filte L 600 Da Detector - 4.75 kV Data Interval 10.0 nsec Digitizer 1 0 00 mVFS Pol arity Positive Filter None A2 5, 2:16399 0 Al - 0 .4271150 AO 0.0087430 res 0.5489027 Ca libration - Program Calculated {2-Param@ter ) Cal ibrati on Date Thu Feb 02 13 :02: 01 1995 Calibrator System Operator Ca lib Da ta File C:\HPTOF\ DATA\ WSR\ ISTD\PEPPOSl.TO< [ #276]
2900
2500
21 00 Cl'" «! ,._ -N :::!< Ul Cl"' . <0 ' Cl N Lll'" a ~ .,;,.._ IX:!'" "' Lll,.._ ::::;; M :::;; "" Ul "' (/) ::::;; . Ul ' "' ...
1400 2J.OO 2800 3500 42 0 m/z
Figure 3.ld MALDI-TOF mass spectrometry ofNUX_A fraction 18.
28 33
111
24
23 29 25 11
Figure 3.le Semi-preparative RP-HPLC of Conus nux (NUX_A) fractions 19-33.
Peak# Time M.W. (Da) M.W. {Oa) M.W. (Da) #Cpds Na-Di8 Na-SBFI K-PBFI Ca-Fiuo3 19 24.96 1139.3 2710.8 3114.3 5 3226.2 3357.3 20 25.88 1138.8 2183.6 3104.2 5 6.5 10.1 2.5 0.6 3217.7 3368.3 21 26.68 1086.8 2252.3 2385.8 6 3166.9 3367.8 4836.2 22 27.15 1051 .7 1194 1911 9 2363.8 3042.1 3360.9 3830.5 4115.6 4812.2 23 27.55 1197.1 4835.7 2 24 28.34 1084.5 1565.7 1861 .1 4 4814.4 25 29.29 1083.4 1352.1 1568.7 6 2862.1 3277.9 4822.9 26 29.95 1402.9 1512.7 2563.7 7 2856.6 3121 .5 3720.2 4807.9 27 30.55 3137.3 1 28 31 .30 3011.5 3104.6 2 29 31.72 1573.3 3089.1 3481 .8 3 30 32.42 1086.6 1212.4 1573.4 7 2379.8 3090.5 3367 3480.6 31 33.15 1557.2 1933.2 2526.6 5 3097.1 3485 32 33.83 2536.4 3107.3 3782.6 3 33 34.69 1971 .1 3117.4 3643.6 5 4476.7 5100.4
Table 3.1b Retention times, MW, and Bioassays results for Conus nux (NUX_A) fractions 19-33. 29 1R
40 4'i
37 .u
Figure 3.1fSemi-preparative RP-HPLC of Conus nux (NUX_A) fractions 34-47.
Peak# Time M.W. (Da) M.W. (Da) M.W. (Da) #Cpds Na-Di8 Na-SBFI K-PBFI Ca-Fiuo3 34 36 .09 1326.9 3169.4 3423.7 5 4460.6 5084.8 35 36 .30 1704.7 2991 .5 3177.3 6 3425.9 4492.1 5096 36 36 .51 3614.8 4403.15 4972.8 7 5692.7 37 37.10 3155.9 3700.9 3816.6 4 4606.4 38 37.50 1993.5 2774.4 3172.6 5 3804.1 4865 39 38 .80 2785.9 3187.6 3814 3 40 39.10 1564.6 1934.4 2531.1 6 3091 .6 3496.3 3782.4 41 40.45 1447.6 2448.3 3171 .6 6 3799.1 4206.8 4856.4 42 40 .85 1446 3166.8 3289 5 3797.3 4199.7 43 41 .87 3177.3 3292.7 3801 4 4203.2 44 42.20 1327.1 1443.8 3160.1 7 3280.3 3499.8 3794.7 5244.4 45 42 .90 1446.9 2413.3 3305.2 6 3501 3805.3 5268 46 43.44 1278 2422.1 2810.7 8 6 8 -0.6 1.7 3166.4 3289.1 3486 3709 5230 47 43 .94 1291 .9 2204.8 3169.2 4 3698.8
Table 3.1c Retention times, MW, and Bioassays results for Conus nux (NUX_A) fractions 34-4 7.
30 fiO
58
Figure 3.lg Semi-preparative RP-HPLC of Conus nux (NUX_A) fractions 48-63 .
Peak# Time MW. (Da) MW. (Da) MW. (Da) #Cpds Na-Di8 Na-SBFI K-PBFI Ca-Fiuo3 48 44.71 2653.7 3164.4 3379.3 5 3504.4 4077.6 49 45.90 2659.6 3175.8 3388 5 3496 4070.4 50 46.27 1388.1 2645.3 3188 5 3491 .1 5870 51 46.70 1363 1386.5 4642.4 4 8.4 6.6 0.2 0.9 5867.5 52 47.15 1614.1 4658.6 2 53 47.59 1614.1 3122.1 3681.7 3 54 48.30 2230.9 2408.6 2535.4 3 55 48.59 1195.4 1220.7 1512.7 5 6.3 12 -1 .1 0.1 2016.3 2244 56 49.35 1220.8 1527.8 1582.3 7 1651 .9 2446.9 2597.9 3572.9 57 49.60 1615.4 2433.6 2 6.5 8.9 3.1 0.4 58 50.29 1614.1 2430.2 2 59 51 .13 1497.6 1683.1 2774.1 3 60 51 .59 1498.2 1 61 52.64 987.5 1330.2 1500.6 7 6.3 9.9 3.4 0.6 2870.3 3419.8 3569.4 4610.3 62 53.60 2882.6 3442.8 2 63 54.33 1566.9 1870 2070.8 8 2434.9 2866.4 3967.8 Table 3.1d Retention times, MW, and Bioassays results for Conus nux (NUX_A) fractions 48-63.
31 69
Figure 3.1h Semi-preparative RP-HPLC ofConus nux (NUX_A) fractions 64-81.
Peak# Time M.W. (Da) M.W. (Da) M.W. (Da) # Cpds Na-Di8 Na-SBFI K-PBFI Ca-Fiuo3 64 54.73 1251 1503.8 2778.7 7 2845.4 3639.6 3948.4 4490.8 65 55.35 3765.1 3943.1 4503.6 5 5045.8 6687.8 66 55.93 2478.3 3161 .7 3935.8 7 4496.8 5202 5578.1 6658.2 67 56.42 0 68 56.96 1185.7 1272.1 2024.2 6 2156.6 3889.8 6139.2 69 57.61 1336.9 1940.9 3381 .1 3 70 58.74 1338.4 3383.4 2 -5.9 6.3 4 -0.5 71 59.55 1341.6 1 72 59.95 1101 .1 1339.7 3994.7 4 4191 .1 73 60.34 1100.6 1940.7 2827.1 6 4015 4517.8 6549.5 74 61 .02 1705.1 1755.3 1942.9 3 75 61.30 1756.2 1 76 61 .89 4495.1 1 -6 7.4 3 0.3 77 62.50 727.3 910.2 1080.5 5 1896.8 3205.9 78 36.16 1899.5 2596 .2 3205.3 3 79 64.33 1884.9 2282.4 2585.8 3 80 64.87 1881 .1 1995.6 2502.7 5 3134.7 3755.6 81 65.65 1995.9 2239.2 2943.5 4 3244.2
Table 3.1e Retention times, MW, and Bioassays results for Conus nux (NUX_A) fractions 64-81 .
32 98
Figure 3.li Semi-preparative RP-HPLC of Conus nux (NUX_A) fractions 82-103.
Peak# Tirre MW. (ll:l) MW. (ll:l) MW. (ll:l) #Cpds Na-[)8 Na-SEFI K-PBFI Ca-RUO: 82 66.00 1972.6 22.37.7 2610.5 6 2955.1 3123.2 3244.8 83 66.67 2282 2493.9 ~.1 3 ~.7 7.5 3.7 -{).6 84 67.50 1669.2 2284.3 2004.7 6 ~ 10.3 -0.4 0.1 3127.4 3820.5 4594.4 85 68.57 2235.9 2984.4 3004.6 3 00 69.15 3048.8 4835.4 2 87 70.97 1833.4 2303.3 3152 3 88 72.03 3128.7 3395.2 2 89 73.00 2256.8 3153.9 3 00 74.73 1473.9 1994 2232.8 4 3240.1 91 75.50 3168.2 3402.4 2 92 76.08 2263.8 3158.6 3370.1 3 93 76.00 2270.2 3141 .9 3370.7 3 94 77.34 3135.1 3351.6 2 95 78.40 3119.5 3414.8 2 -9.3 8.7 2.8 -1 00 79.49 3008.6 3403.4 3759.3 3 97 80.51 3111.5 3405.4 3763.8 3 98 81 .56 3120.1 3300.2 3700 3 99 82.61 2007 3003.7 3362 4 5032.7 100 85.27 3051 .2 4946.2 2 101 88.22 2983.4 3120.8 2 102 93.77 3002 3121.6 2 103 95.95 3146.8 4018.4 6540.3 3
Table 3.1fRetention times, MW, and Bioassays results for Conus nux (NUX_A) fractions 82-1 03.
33 MY vs_ Retention Time
8000
7000 • • .. 6000 e. ~000 ..,.. ,. • • .!'.. • > 4000 • • • :; •• J. • • • ••• ~ • • • . "-...... _:...... ::.. I • .. 3000 ...... 0 • .. .•• • ... • . = :f ...... - ,;. . - .• •= 2000 .. · .~ ... ••••• : .. ·-· • • •· • ·..r:.... -• • -. ., .. ~ • •• • I .. .. t-:, • 1000 ••~roC"-• • ..••• . ··- •
0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 Retention Time (min)
Table 3.lg Molecular Weight (Da) vs. Retention Time (min) for Conus nux (NUX_A).
3000 ------
1000 • ... • • f>OOO • • • • .. ... • Q 5000 •• • • • • ~ • • • .!' ••• • •• • - j+ C.nux :) 4000 ·-;...: . . -.. • I• C.j::a~pidau ~ .. ••••• t•""'1t • • .. • - .... • :.- ~ ~ -~ :.· _... i . . - . _ :1 3 000 ...... ·~,.l~• . . . *:!:t-'--* . : ...... ,...... + .., • .• ... •• • ...... •• . *'··.,...... 2000 ~ ' .. , .:._:: • : ~ -....r • • . ... .,. ·~· .. .,. . ' .. ·.·- • -:-...• • :. ~. ~*'!. • 1000 :::.-- ~- : . .. : o+-----~------~----~------~----~------~----~----~------~----~ 0.00 10.00 20.00 30.00 40.00 50.00 60.00 TO.OO 30.00 ~0.00 100.00
Table 3.lh Molecular Weight (Da) vs. Retention Time (min) for Conus nux (NUX_A) and Conus jaspideus, an Atlantic cone snail species.
34 After completing the Semi-preparative RP-HPLC analysis of the venom of Conus
nux, the data obtained made it clear that further separation techniques must be used. From
the MW vs. Retention plots (Table 3.1g and Table 3.lh) showed that the venom of Conus
nux is very complex and has almost twice the amount of components as an Atlantic cone
snail, Conus jaspideus. Many of the components present in the venom of Conus nux have
a very high molecular weight, which is not desirable for this project. Components with
molecular weights below 3000 Da are more suitable for this project since easier to
characterize and synthesize. Therefore, Size Exclusion chromatography was adopted,
because it separated components of the venom on the basis of the molecular weight and
size. Several batches of venom were separated by this method, to evaluate its reproducibility; chromatograms of NUX_D, NUX_E, and NUX_F samples gave essentially identical separations. The NUX_E sample was chosen to carry out a complete separation of the venom of Conus nux, because it was the batch with largest amount of sample (obtained from the venom of ninety cone-snails). As mentioned earlier, NUX_E was a 40 mg sample. The sample was separated into two equal 20 mg samples (NUX_Ea and NUX_Eb) and then run on the Size Exclusion Chromatography Column. The sample was run at the following wavelengths: 220nm (for presence of amino acids), 280nm (for the presence of aromatic amino acids present in most conopeptides), and 250nm (for cysteines bridges present). The data for both samples ofNUX_E are shown below.
35 A-=220 run 9
3 5
2 10 11
A-=280 run
!...... r...... r ... , .. r.. w .... r...... r...... r...... r.. ""'"'''""l!l' .... ,.r ... "' ... r..... 111 .t ..... ,1 ...... ,1 .. " ••• r"'""''''"'"'""'""r ...... r...... r...... r...... r...... w!.,, ... !. .... " .. r...... r...... "'rw, .. "rw .... 1. ,,,,,
A-=250 run
r, .... ".J .. r ..t ...... r, ... ..,r,, .. ,r ...... ,.r .. 11 ..... r...... r"""""''"lll"'""'"''' '"''"'""'''""""'r ...... 111 r...... ,r, ...... r""'""'' ,,,,r...... r..... ,r...... r.. ,,,,,,,,, .... t. 1111 .... r, .. , .. r...... ,r .. , .. ,r 0 Time (min) 300
Figure 3.lj Size Exclusion chromatograms of NUX Ea at the wavelengths 220run,
280nm, and 250run.
36 Peak Elution Time Retention Time NUX E01 57.80-74.74 66.49 NUX E02 74. 74-81. 30 78.55 NUX E03 81 . 30-91. 04 88.64 NUX E04 91.04-97.52 94.14 NUX E05 . 97.52-113.28 102.66 NUX E06 113.28-119.25 115.49 NUX EO? 119.25-127.90 124.20 NUX E08 127.90-135.29 130.03 NUX E09 135.29-160.26 152.60.. NUX E10 160.26-169.02 164.24 NUX E11 169.02-190.71 174.69 NUX E12 209.90-220.80 213.89
Table 3.1 i Retention times of the fractions collected from NUX Ea.
Peak Elution Time Retention Time NUX Eb01 57.80-7 4. 66 66.50 - NUX Eb02 74.66-81.80 78.73 . NUX Eb03 81.80-91.28 87.91 NUX Eb04 91.28-98.06 94.40 NUX Eb05 98.06-110.74 102.73 NUX Eb06 11 0. 74-119. 34 115.68 NUX Eb07 119.34-126.99 123.24 NUX Eb08 126.99-135.69 130.43 NUX Eb09 135. 69-160. 60 152.12 NUX Eb10 160. 60-168. 59 164.28 NUX Eb11 168. 59-192. 34 174.78 NUX Eb12 207.46-230.58 213.93
Table 3.lj Retention times of the fractions collected from NUX_Eb.
37 1 A-=220 run
9 3 5 7 2 4 6 8
12
I I A-=280 run
,, • I
A-=250 run
0 Time (min) 300
Figure 3.1k Size Exclusion chromatograms of NUX Eb at the wavelengths 220run, 280run and 250run.
38 From the Size Exclusion data, it is quite clear that both the NUX_ Ea and NUX_ Eb
separations are almost identical. Therefore, the fractions collected from each sample were
pooled together. Each Size Exclusion Chromatography fraction from NUX_ E was
separated usmg Semi-preparative Reversed Phase HPLC. MALDI-TOF mass
spectrometry analysis was done on each of the pooled fractions. The first two fractions
showed components with molecular weights greater than 6000 Da. Therefore, when SEC
fractions were run on the Semi-preparative RP-HPLC column, using the linear gradient described earlier, a poorly defined chromatogram was obtained, possibly because the higher molecular weight components were extremely hydrophobic and may require a different solvent system for their proper separation. As shown below, fewer components were found in each of the Semi-preparative RP-HPLC chromatograms of each fraction of
NUX E.
3
0 Time (min) 100
Figure 3.11 Semi-preparative RP-HPLC ofConus nux sample NUX_EOI. 39 Retention Peak Time Intensity NUX E0101 53.41 w NUX E0102 54.90 w NUX E0103 58.18 M NUX E0104 62.93 w NUX E0105 64.71 w NUX E0106 67.05 w NUX E0107 68.45 w NUX E0108 70.08 w NUX E0109 74.40 w NUX E0110 95.38 w
Table 3.1k Retention times and Intensity of fractions collected from NUX_EOl.
3
0 Time (min) 100
Figure 3.lm Semi-preparative RP-HPLC ofConus nux SEC fraction NUX_E02.
Retention Peak Time Intensity NUX E0201 63.23 w NUX E0202 74.85 w NUX E0203 95.05 w
Table 3.11 Retention times and Intensity of fractions collected from NUX_E02.
40 9
0 Time (min) 100
Figure 3.In Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E03.
Peak Retention Time Intensity NUX E0301 38.38 w NUX E0302 38.86 M NUX E0303 51.63 w NUX E0304 52.97 w NUX E0305 53.97 w NUX E0306 54.60 w NUX E0307 56.02 w NUX E0308 58.80 w NUX E0309 60.45 s NUX E0310 61.36 w NUX E0311 62.48 w NUX E0312 68.98 w
Table 3.lm Retention times and Intensity of fractions collected from NUX_E03.
41 9-10
12 II
0 Time(min) 100
Figure 3.lo Semi-preparative RP-HPLC ofConus nux SEC fraction NUX_E04.
Peak Retention Time Intensity NUX E0401 19.26 w NUX E0402 35.26 w NUX E0403 38.08 w NUX E0404 39.28 w NUX E0405 40.18 w NUX E0406 40.91 w NUX E0407 49.16 w NUX E0408 54.91 w NUX E0409 58 .30 s NUX E0410 59.76 w NUX E0411 82.90 w NUX E0412 95.28 w
Table 3.ln Retention times and Intensity of fractions collected from NUX_E04.
42 9
4
15
0 Time (min) 100
Figure 3.1p Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E05.
MALDI-TOF Peak Retention Time Intensity MW (Da) NUX E0501 20.43 w NUX E0502 24.75 w NUX E0503 26.05 s NUX E0504 28.11 w NUX E0505 29.15 w NUX E0506 30.65 w NUX E0507 31.66 w NUX E0508 32.45 w NUX E0509 34.51 s 1274.4 NUX E0510 36.68 w NUX E0511 39.86 w NUX E0512 42.91 w NUX E0513 47.46 w NUX E0514 53.58 w NUX E0515 95.84 w
Table 3.1o Retention times, Intensity, and MW offractions collected from NUX_E05.
43 5
12 8
0 Time(min) 100
Figure 3.1q Semi-preparative RP-HPLC ofConus nux SEC fraction NUX_E06.
Peak Retention Time Intensity MW (Da) NUX E0601 17.58 w NUX E0602 22.78 w NUX E0603 24.75 w NUX E0604 28.01 w NUX E0605 31.68 M 1276.8 NUX E0606 33.91 w NUX E0607 37.83 w NUX E0608 42.41 w NUX E0609 56.30 w NUX E0610 64.03 w NUX E0611 75 .81 w NUX E0612 95.45 w
Table 3.1 pRetention times, Intensity, and MW of fractions collected from NUX_E06.
44 7
15
14
0 Time (min) 100
Figure 3.1r Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E07.
Peak Retention Time Intensity MW (Da) NUX E0701 9.43 w NUX E0702 16.83 w NUX E0703 17.96 w NUX E0704 21.25 w NUX E0705 22.06 w NUX E0706 23.30 w NUX E0707 25.15 s 1280.7 NUX E0708 26.78 w NUX E0709 28.63 M 2842.3 NUX E0710 30.38 w NUX E0711 32.23 w NUX E0712 34.90 w NUX E0713 35.93 w NUX E0714 37.06 w NUX E0715 41.78 M 3290.4 NUX E0716 55.13 w NUX E0717 65.30 w
Table 3.lq Retention times, Intensity, and MW offractions collected from NUX_E07.
45 18
20
ll 9
6 10 15 16 17
0 Time (min) 100
Figure 3.1 s Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E08. MALDi-TOF Peak Retention Time Intensity MW (Da) NUX E0801 9.51 w NUX E0802 14.13 w NUX E0803 22.77 w NUX E0804 25.66 w NUX E0805 26.28 w NUX E0806 31.75 w NUX E0807 35.86 w NUX E0808 37.90 w NUX E0809 41.75 w NUX E0810 50.00 w NUX E0811 51.86 w NUX E0812 54.01 w NUX E0813 55.00 w NUX E0814 59.25 w NUX E0815 64.61 w NUX E0816 69.36 w NUX E0817 75.00 w NUX E0818 94.33 s 3058.3 NUX E0819 95.68 M NUX E0820 96.41 M
Table 3.lr Retention times, Intensity, and MW offractions collected from NUX_08.
46 23
2
8 5 7 17. 2 2 6 15 3 4
0 Time (min) 100
Figure 3.1t Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E09.
Peak Retention Time Intensity NUX E0901 6.53 w NUX E0902 9.81 w NUX E0903 10.36 w NUX E0904 13.23 w NUX E0905 23.91 M NUX E0906 28.94 w NUX E0907 29.76 w NUX E0908 31.61 w NUX E0909 35.83 M NUX E0910 37.26 w NUX E0911 37.98 w NUX E0912 38.90 w NUX E0913 44.15 w NUX E0914 49.76 w NUX E0915 54.41 w NUX E0916 56.98 w NUX E0917 58.70 w NUX E0918 59.33 M NUX E0919 71.90 w NUX E0920 72.68 w NUX E0921 89.03 M NUX E0922 91 .76 s NUX E0923 94.16 s NUX E0924 98.03 w Table 3.1s Retention times and Intensity offractions collected from NUX_E09. 47 7
4 9
3 1 2 5 6
0 Time (min) 100
Figure 3.1u Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_EIO.
MALDI-TOF Peak Retention Time Intensity MW (Da) NUX E1001 6.98 w NUX E1002 9.66 w NUX E1003 11.75 w NUX E1004 15.76 M NUX E1005 34.66 w NUX E1006 43.75 w NUX E1007 49.25 s 1504.5 NUX E1008 53.86 w NUX E1009 95.53 w
Table 3.1t Retention times and Intensity of fractions collected from NUX_El 0.
48 3 5
7
0 Time (min) 100
Figure 3.1 v Semi-preparative RP-HPLC of Conus nux SEC fraction NUX_E 11.
Retention Peak Time Intensity NUX E1101 4.45 w NUX E1102 5.16 w NUX E1103 5.61 s NUX E1104 7.70 w NUX E1105 9.50 s NUX E1106 41.95 w NUX E1107 49.10 w NUX E1108 51.78 w NUX E1109 55.35 w NUX E1110 56.68 w
Table 3.1 u Retention times and Intensity of fractions collected from NUX_E 11.
49 3
5 II 6
0 Time (min) 100
Figure 3.lw Semi-preparative RP-HPLC of Conus nux sample NUX_El2.
MALDI-TOF Peak Retention Time Intensity MW (Da) NUX E1201 5.35 w NUX E1202 9.81 w NUX E1203 23.50 s 852 .9 NUX E1204 40.60 w NUX E1205 42.25 M 1320.3 NUX E1206 43.60 M 1271.3 NUX E1207 45.68 w NUX E1208 52.96 w NUX E1209 57.28 w NUX E1210 59.51 w NUX E1211 95.83 M 852.5
Table 3.1 v Retention times, Intensity and MW of :fractions collected from NUX E. 50 After separating all SEC-HPLC fractions on the Semi-Preparative Reversed Phase
HPLC, fraction NUX_E0905 was selected for further purification using Analytical
Reversed Phase HPLC. The chromatogram ofNUX_E0905 is shown below.
20 Time (min) 30
Figure 3.lx Analytical Reversed Phase HPLC Separation of Conus nux, NUX_E0905.
3.2 Peptide Sequencing
Fraction NUX_E0905 was reduced and alkylated since disulfide bridges may be present and then was sequenced by Edman degradation at the Biotechnology Research
Laboratory at Yale University. The fraction was sequenced using an ABI Procise cLC
51 amino acid sequencer, capable of sequencing samples at pico molar levels. The results
are summarized in Table 3.2.
Peptide Sequence Analysis
Cycle Assigned Residues 1 ARG 2 CYS* 3 CYS* 4 HYP 5 HYP 6 GLN 7 ARG 8 CYS* 9 SER 10 THR 11 HIS 12 CYS* 13 ARG 14 SER ,•', 15 CYS* •' 16 CYS* 17 GLY
MALDI MASS (M+H da)=2270.09 MASS Predicted (M+H da)=2272.44 ..
Sequence: RCCOOQRCSTHCRSCCG * carboxyam mdomethyl-cysteme (CAM-Cys)
Table 3.2 Peptide Sequence effraction NUX_E0905 (nux-I).
52 : tiDU~. Ramlakllan - -· TofS.pec-5~ ;t124p79 2 (1 .788) Cn (Cen,3, 50.00, Ht); Sb (1 ,20.00 ); Sm (Mn, 2x2.00); Cm (1 :2) TOF LD+ 100 2270.09 8.41e3
%
2213.80 2254.28 2154.41 2280.52 2323.69 2382.21 2562 O 2026.18 2062.81 2124.49-; 1, : 2542 .92 .4° 2590.67 1 Ill II I !I II t124p79 2 (1.788) Sb (1 ,20.00 ); Sm {Mn, 2x2.00); Cm {1:2) TOF LD+ 100 2270.02 8.43e3
%
,, ' 2213.93 1 I 2253.72 2381 .98 2180.03 2324.74
O r-~-=~~~~~~~~~--,------~---- 20bO 2050 2100 21 '50 2200 ' 2250 2300 2350 2400 2450 2500 2S50 z6oo mlz I
Figure 3.2 MALDI-TOF mass spectrometry offraction NUX_E0905.
The protein sequence obtained from fraction NUX_E0905 is now referred to as nux-1. As shown above, nux-1 is a 17-residue conopeptide that has six cysteines
(characteristic of conotoxins) and two modified amino acids (hydroxyprolines) with a
MALDI-TOF mass of 2270.09 Da (Figure 3.2). The distribution of cysteine residues indicates that nux- I belongs to theM-superfamily.
53 3. 3 M-Superfamily
The isolated conopeptide, nux-1 has the characteristic six cysteines arrangement
(-CC-C-C-CC) of the M-superfamily. As mentioned earlier the M-superfamily Is comprised of two families of conotoxins, the J.l-conotoxins and \j/-conotoxins.
J.l-conotoxins are paralytic toxins, which target the voltage-gated Na channels.
Some of the published the J.l-conotoxins are around 22 amino acids in length with three disulfide bonds (Table 3.3a). These peptides contain the modified amino acid hydroxyproline and they are the first polypeptide toxins known to compete for binding with the guanidinium toxins, tetrodotoxin and saxitoxin. However, they differ in being much more selective for the skeletal muscle subtype than for axonal Na channels
(inhibited by the guanidinium toxins). Some of the most studied published J.l-conotoxins are shown below along with the sequence ofnux-1 (Table3.3a).
nux-1 RCC- OOQRC-STHCR-S-CCG Conus nux J.!GIIIA RDCCT OOKKCKDRQCKPQRCCA Conus geographus J.!GIIIB RDCCT OO RKCKDRRCKPMKCCA Conus geographus J.!GIIIC RDCCT OOKKCKDRRCKPLKCCA Conus geographus J.lPIIIA RLCCGFOKSCRSRQCKOHRCC* Conus purpurascens
Table 3.3a Comparison ofthe sequence ofnux-1 and published J.l-conotoxins.
54 From the table, nux-1 shows much similarity to the other four Jl-conotoxins (f..lGIIIA, flGliiB, flGliiC, JlPIIIA), despite the fact that it is only a 17 residues sequence.
Currently, there are many Jl-conotoxins that have been discovered in various species of cone snails, but none of them have been published in the regular scientific literature. For this reason, an extensive search was required to determine if the sequence of nux-1 was found in any other species of cone snails. Many conotoxins sequences can be found in the patent literature, most of then have been deduced indirectly from the sequence ofthe isolated rnRNA ofthe corresponding precursor proteins. The following is a library ofJ..l-conotoxins found in patent WO 02107678.
Alignment of J.1-Conopeptides
A3.4 ---CCKVQ-CES--C---TPCC!A Ak3. ---CCELP-CGPGFC---VPCCA Ar3.1 ---CCERP-CNIG-C---VPCCA Bn3.1 ---CCNWP-CSMG-C---IPCCYYA Bt3.1 ---CCELP-CH-G-C---VPCCWPA Bt3.2 ---CCGLP-CN-G-C---VPCCWPS" Bt3.3 ---CCSRN-CAV--C---IPCCPNWPA" Bt3a ---CCKQS-CTT--C---MPCCWA Bt3b --ACCXQS-CTT--C---MPCCA Bt3c ---CCEQS-CTT--C---MPCCW? Ca3.3 R--CCRYP-CPDS-C--HGSCCYKA Ca3.4 ---CCPPVACNMG-C---KPCC# Ca3.5 ---CCDDSECDYS-C---WPCCMF# Ca3.6 ---CCRR--CYMG-C---IPCCF"
55 Circling ---CCPPVACNMG-C---KPCCGA Comatose/Death SKQCCHLAACRFG-C---TOCCNA Cp3.1 S--CCR--DCGED-C---VGCCRA Ct3.1 ---CCDWP-CIPG-C---TPCCLPA Da3.1 ---CCDDSECDYS-C---WPCCILSA Da3.2 --QCCPPVACNMG-C---EPCC# Da3.3 ---CCNAGFCRFG-C---TPCCWA Di3.1 Z--CCVHP-C-P--C---TPCCRA Fi3.1 ---CCPWP-CNIG-C---VPCCA Fi3.2 ---CCSKN-CAV--C---IPCCPA Fi3.3 ---CCRWP-CP-ARC---GSCCLA Fi3.4 ---CCELSRCL-G-C---VPCCTS" Fi3.5 ---CCELSKCH-G-C---VPCCIPA Ge3.1 Z--CCTF--CNFG-C---QPCCVP" Ge3.2 Z--CCTF--CNFG-C---QPCCLTA Ge3.3 Z--CCTF--CNFG-C---QPCCVPA Gm3.1 ---CCDDSECDYS-C---WPCCMF# Gm3.2 G--CCHLLACRFG-C---SPCCWA Gm3.3 ---CCSWDVCDHPSC---T-CCG# La3.1 ---CCDWP-CS-G-C---IPCCA Lp3.1 ZINCCPWP-CPST-C--RHQCEHA Lv3.1 ZINCCPWP-CPDS-C--HYQCCHA Mr3.2 ---CCRLS-CGLG-C---HPCC# Mr3.3 --ECCGSFACRFG-C---VPCCV" Mr3.4 SKQCCHLPACRFG-C---TPCCWA Mr3.5 -MGCCPFP-CKTS-C--TTLCC# Ms3.1 --ACCEQS-CTT--C---FPCC! A Nb3.1 --CCELP-CGPGFC---VPCCA Pu3.1 ---CCN-S-CYMG-C---IPCCF A Qc3.1 ZR-CCQWP-CPGS-C----RCCRT#
56 Qc3.2 ZR-CCRWP-CPGS-C----RCCRYRA Qc3.3 R--CCRYP-CPDS-C--HGSCCYK" QciiiA ---CCSQD-CLV--C---IOCCPN# QciiiB ---CCSRH-CWV--C ---IOCCPN? Ra3.1 2-TCCS-N-CGED-C---DGCCQA Scratcher I ---CCR-T-C-FG-C---TOCC# Ts3.1 ---CCH-K-CYMG-C---IPCCI A Ts3.2 K--CCRPP-CAMS-C-GMARCCYA Bt3.5 R--CCRWP-CPSI-C-GMARCCFVMITC/ Bt3.6 R--CCRWP-CP-SRC-GMARCCFVMITCA Tx3.1 F--CCDSNWCHISDC----ECCY# J10l4 ---CCHWNWCDHL-C----SCCGS" J10l7 --DCCOLPACPFG-C---NOCC # J10l9 ---CCAPSACRLG-C---ROCCR" J1020 ---CCAOSACRLG-C---ROCCRA J1022 ---CCAPSACRLG-C---RPCCRA M024 --GCCGSFACRFG-C---VOCCV" J1031 ---CCSWDVCDHPSC----TCC# M032 R--CCKFP-CPDS-C--RYLCC# Ae3.1 ---CCDDSECDYS-C ---WPCCIF# Ae3.2 ---CCNDWECDDS-C---WPCCY# At3.1 R--CCR-FPCPDT-C---RHLCC# At3.2 ---CC--MTC-FG-C---TPCC# At3.3 ---CCDDSECDYS-C---WPCCLFSA At3.4 ---CCR-LLC-LS-C---NPCC# At3.6 ---CCDDSECGYS-C---WPCCY# Au3.2 G--CCS-PPCHSI-C--AAFCC# Au3.3 ---CCRPVACAMG-C---KPCC# Au3.4 Z--CCPAVACAMG-C---EPCC# Em3.1 ---CCS-RDC-SV-C---IPCCPYGSP-
57 Ep3.1 ---CCDEDECNSS-C---WPCCW# Ep3.2 ---CCDEDECSSS-C---WPCCW# Ep3.3 ---CCPAAACAMG-C---KPCC# Om3.1 ---CCDEEECSSA-C---WPCCW# Om3.3 ---CCHLLACRFG-C---SPCCWA Sf3.1 ---CC--PRC-SE-C---NPCC# Pn3.2 -RCC--KFP-CPDS-C--KYLCC# Fd3.2 -RCC --RWP-CPSI-C-GMARCCSSA Pu3.3 --CC--KLL-CYSG-C---TPCCHI A Eb3.1 --CC--EQP-CYMG-C---IPCCFA Eb3.2 --CC--AQP-CYMG-C---IPCCFA Pu3.2 --CC--V-S-CYMG-C---IPCCFA Mf3.1 --CC--DWP-CSAG-C---YPCCFPA Mf3.2 -GCC--PPM-C-TP-C---FPCCFRA Ra3.2 RGCCAPPRK-CKDRACK-PARCCGP# Sm3.3 ZRCCNGRRG-CSSRWCRDHSRCC# Cn3.3 GRCCDVPNA-CSGRWCRDHAQCC# A3.1 -MCCGEGRKCPSYFRNSQICHCC* A3.2 --CCR--WPCPRQIDGEY-CGCCL# Bu3.5 -RCCGEGLTCPRYWKNSQICACC A Ca3.1 --CCGPGGSCPVYFRDNFICGCCA Cr3.1 RKCCGKDGPCPKYFKDNFICGCCA E3.1 --CCS--WPCPRYSNGKLVCFCCL# M3.2 --CCGPGGSCPVYFRDNFICGCCA M3.3 -MCCGESAPCPSYFRNSQICHCCA M3.4 ZKCCGPGGSCPVYFTDNFICGCEA M3.5 ZKCCGPGGSCPVYFRDNFICGCCA S3.1 ZKCCGEGSSCPKYFKNNFICGCCA UOOI ZKCCS-GGSCPLYFRDRLICPCCA J1034 ZKCCGPGASCPRYFKDNFICGCCA
58 Cn3.1 -MCCGEGAPCPSYFRNSQICHCCA A3.3 ZK--CCTGK---KGSCSGKACKNL-KCCS# A3.5 ZK--CCTGR---KGSCSGKACKNL-KCCS# Bu3.1 VTDRCCK----GKREC-GRWCRDHSRCC# Bu3.1A VGDRCCK----GKRGC-GRWCRDHSRCC# Bu3.2 VGERCCK---NGKRGC-GRWCRDHSRCC# Bu3.3 IVDRCCN-KGNGKRGC-SRWCRDHSRCC# Bu3.4 VGLYCCRPKPNGQMMC-DRWCEKNSRCC# Ca3.2 -RD-CCTPP ---KK-CKDRQCKPQ-RCCA# L3.1 GRD-CCTPP---RK-CRDRACKPQ-RCCG# L3.2 ZRL-CCGFP---KS-CRSRQCKPH-RCC# La3.2 -RD-CCTPP---KK-CRDRQCKPA-RCCG# La3.3 RPP-CCTYD---GS-CLKESCMRK-ACC# La3.3A RPP-CCTYD---GS-CLKESCKRK-ACC# Jt-GIIIA -RD-CCTOO---KK-CKDRQCKOQ-RCCA# Jt-GIIIB -RD-CCTOO---RK-CKDRRCKOM-KCCA# Jt-GIIIC -RD-CCTOO---KK-CKDRRCKOL-KCCA# Jt-PIIIA ZRL-CCGFO---KS-CRSRQCKOH-RCC# M3.1 -RD-CCTPP---KK-CKDRQCKPQ-RCCA# Mr3.1 RGG-CCTPP---RK-CKDRACKPA-RCCGP# Nb3.2 ZK--CCTGK---KGSCSGKACKNL-KCCS# Pr3.1 RGG-CCTPP---KK-CKDRACKPA-RCCGP# Pr3.2 -RG-CCTPP---RK-CKDRACKPA-RCCGP# R3.1 LOS-CCSLN---LRLCOVOACKRN-OCCT# R3.2 ZQR-CCTVK----RICOVOACRSK-OCCKS A R3.3 RGG-CCTPP---RK-CKDRACKPA-RCCGP# Sm3.1 ZK--CCTGK---KGSCSGKACKNL-KCCS# T3.1 H-G-CCKGO---EG-CSSRECROQ-HCC# T3.2 H-G-CCEGP---KG-CSSRECRPQ-HCC# Wi3.1 LPS-CCDFE----RLCWPACIRH-QCCT#
59 Om3.2 CCKYGWTCLLGCTPCDC" Om3.4 CCRYGWTCWLGCTPC GC? S3.2 Z-NCCN GG -CSSKW CR DHARC C#
Glu or y-carboxy-Giu, Gin or pyro-Giu, Pro or hydroxy-Pro, Trp or bromo-Trp, Tyr, mono-iodo-Tyr, di iodo-Tyr, 0-sulpho-Tyr or 0 phospho-Tyr, "'is free carboxyl or amidated C-terminus, preferably free carboxyl,# is free carboxyl or amidated C-terminus, preferably amidated, ? = Status ofC-term not known.
Table 3.3b Table oflJ.-conotoxins (Patent W002107678).
The lJ.-conotoxins listed in this patent are each unique and none of them have the
same sequence as nux-1. Each of the lJ.-Conotoxins have many similarities to each other
with respect to the alignment of the cysteines within the sequences and the common amino acids present between the cysteines.
The other family belonging to the M-superfarnily, \j/-conotoxin, is less studied than the lJ.-conotoxin family, because as of yet, it only has a single member, \j/-PIIIE, which was isolated from C. purpurascens. The sequence of \j/-conotoxin PillE is
HOOCCLYGKCRRYOGCSSASCCQR* (O=trans 4-hydroproline; *indicates and amidated C-terminus) (20). This peptide has three disulfide bonds, the characteristic (CC-
C-C-CC) cysteine pattern of the M-superfarnily and is structurally homologous to the sodium channel-blocking lJ.-conotoxins. However, \j/-PIIIE does not interact with the voltage-gated sodium channel; instead \j/-PIIIE binds to the muscular-subtype nicotinic acetylcholine receptor (nAChR). a-bungarotoxin and \j/-PIIIE fail to compete with each other for the binding to Torpedo (muscle-like) nAChR. \j/-PIIIE is likely a noncompetitive antagonist (20). It has been suggested that in common with the a-
60 conotoxins, the \j/-PIIIE is a direct channel-blocker and therefore it could be used as a potential probe for the ion-conducting pore of the muscle nAChR (12).
Based on the J..t-library and an extensive search of published studies on sequences belonging to the M-super family, the sequence of nux-1 has never been isolated from
Conus nux or any other species of cone snail.
61 4. CONCLUSION
This work describes for the first time the isolation and characterization of the components of the venom of Conus nux, the smallest of the Panamic cone snail species.
The venom of Conus nux is quite complex. From the initial method of separation, using
Semi-preparative RP-HPLC, a total of 103 fractions were collected (NUX_A), each having multiple components of different molecular weights. When Size Exclusion
Chromatography (second method of separation) was applied, 12 fractions were collected for each run (NUX_E). Using this technique, most of the undesirable higher molecular weight components (>6000 Da) present throughout many of the NUX_ A fractions were efficiently separated. The Semi-preparative RP-HPLC separations of each Size Exclusion
Chromatography fraction had fewer components than the initial NUX_ A chromatogram.
After applying both chromatographic techniques, each final fraction required fewer purification steps, which is important when working with limited amounts of sample. The selected fraction for sequencing, NUX _E0905 was further purified using Analytical RP
HPLC.
The sequence, RCCOOQRCSTHCRSCCG, nux- I reported in this thesis is the first sequence ever isolated from the cone snail Conus nux. nux-1 is a novel member of the M-superfarnily of conotoxins and is most likely to be Jl-conotoxin. The results of the preliminary bioassays done on the original NUX_ A sample suggest that it is a !l-
62 conotoxin, because this fraction showed the significant activity for the voltage-gated sodium channel.
!.l-conotoxins GIIIA and GIIIB are capable of blocking skeletal muscle Na+ channels by binding to tetrodotoxin/saxitoxin-binding sites through the guanidinium group of their arginine at position 13 (25). Although Nux-1 has arginine as it thirteenth amino acid, it is not positioned in the same loop as f.l-GIIIA, as shown earlier in the sequence alignment ofthe nux-1 and other !.l-conotoxins (Table3.3a).
Future work could include the synthesis and characterization of nux-1 to determine the precise bridging pattern of the disulfide bonds and electrophysiological studies to determine the specific type of sodium channels (i.e. skeletal muscle sodium channel) that it targets. Additionally, the synthesis of the analogue R13Q of nux-1 could be carried out to determine whether this amino acid is essential for the blocking capability of nux-1 on the Na+ channel.
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