Isolation and Characterization ofNovel Conopeptides from Conus brunneus
by J alidsa Pellicier
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
August 2006 Isolation and Characterization of Novel Conopeptides from Conus brunneus
by Jalidsa Pellicier
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 ofthe Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science.
I s ~~=0 ,_------Thesis Advisor
epartment of Chemistry and Biochemistry
Date
11 Acknowledgements
First and foremost, I would like to thank Dr. Frank Mari for giving me this great opportunity to work with his research group.
I would also like to thank Sanaz Rahmankhah and Dr. Carolina Moller for mentoring and teaching me all that I needed to know in order to conduct this research. Thank you also to Jose Riviera-Ortiz, Simon Bulley, Dr. Herminsul Cano, and Dr. Aldo
Franco for having directed me through all my questions and mistakes. And to the rest of Dr. Mari ' s research group for supporting me and for making my experience in the group enjoyable.
Additionally, I would like to thank Dr. Keith Brew and Dr. Jang Yen Wu for their time and efforts as members of my advisory committee.
I would also like to thank Paul for all the motivation and encouragement that he has given me through all my struggles.
Finally, I would like to thank my parents for all the support that they have given me my entire life and for showing me the importance of a good education.
111 ABSTRACT
Author: Jalidsa Pellicier
Title: Isolation and Characterization ofNovel Conopeptides from Conus brunneus
Institution: Florida Atlantic University
Thesis Advisor: Dr. Frank Mari
Degree: Master of Science
Year: 2006
Cone snails are predatory marine mollusks found in the genus Conus that use a complex cocktail of peptides to capture prey and deter predators. Most of the venom components selectively target ion channels or receptors, making them invaluable tools in neurophysiological studies. In this study, the venom of Conus brunneus, a common Panamic vermivorous cone snail species, was characterized by the use of high performance liquid chromatography, nuclear magnetic resonance, mass spectrometry, and automated Edman degradation sequencing. Three novel peptide sequences were reported: two peptides were members of theM-superfamily and one peptide was classified as an a-conotoxin. The disulfide connectivity of a previously isolated P-conotoxin was also determined. These peptides comprise a partial peptide library of Conus brunneus and may prove useful in numerous structural and neurological studies.
lV Table of Contents
List of Tables ...... vi
List of Figures ...... vii
Introduction ...... !
Venom Composition ...... 4
Pharmacology and Physiology ofVenom Components ...... 6
Conopeptides as Pharmaceutical Drugs ...... ?
Materials and Methods ...... 9
Specimen Collection ...... 9
Crude Venom Extraction ...... 9
Purification of Peptide ...... 10
Mass Spectrometry ...... 11
Nuclear Magnetic Resonance ...... 12
Reduction and Alkylation of Peptides ...... 12
Partial Reduction and Alkylation ...... 13
Peptide Sequencing ...... 13
Nomenclature ...... 14
Results and discussion ...... 15
Conclusions ...... 4 7
References ...... 49
v List of Tables
Table 1 - Targets and Therapeutic Potential of Conopeptides ...... 8
Table 2- Elution Times ofbruK Batch 1Crude Venom Fractions from Superdex 30 .... .16
Table 3- Elution Times ofbruK Batch 2 Crude Venom Fractions from Superdex 30 .... 17
Table 4- Elution Times ofbruK05 from the Semi-Preparative Column ...... 18
Table 5 -Elution Times of bruK0504 in the Analytical Column ...... 20
Table 6- Sequence Analysis ofbruK0504c ...... 23
Table 7- Elution times ofbruK07 from the Semi-Preparative Column ...... 26
Table 8- Elution Times of Reduced & Alkylated bruK0718 from the Analytical Column ...... 32
Table 9- Sequence Analysis ofbruK0718e ...... 33
Table 10- Elution Times ofbruK0714(combined fraction) from the Analytical Column ...... 35
Table 11 - Elution Times of Reduced & Alkylated bruK0714b in the Analytical Column ...... 39
Table 12- Sequence Analysis ofbruK0714b ...... 40
Table 13- Elution Times for bruK0714a from the Analytical Column ...... 41
Table 14- Sequence Analysis ofbruK0714a7 ...... 44
Table 15- Elution Times ofPartially Reduced & Alkylated bruK0714a7 ...... 46
Table 16 - Conus brunneus Partial Peptide Library ...... 4 7
VI List of Figures
Figure 1 - Structure of Typical Conus Venom Apparatus ...... 2
Figure 2- Picture of a Conus brunneus shell...... 3
Figure 3 - Classification of Conopeptides ...... 5 \ Figure 4 - Hook & Line and Net Strategies ofFish- Hunting Cone Snails ...... 7
Figure 5 - Elution Profile of Batch 1 bruK Crude Venom in Superdex 30 Column ...... 16
Figure 6 - Elution Profile of Batch 2 bruK Crude Venom in Superdex 30 Column ...... 17
Figure 7- Elution Profile ofbruK05 in the Semi-Preparative Column ...... 18
Figure 8 - TOF MS ofbruK0503 ...... 19
Figure 9- TOF MS of bruK0504 ...... 19
Figure 10 - Elution Profile ofbruK0504 in the Analytical Column ...... 20
Figure 11 - TOF MS of bruK0504c ...... 21
Figure 12- TOF MS of Reduced & Alkylated bruK0504c ...... 22
Figure 13- Elution Profile ofbruK07 in the Semi-Preparative Column ...... 25
Figure 14 - TOF MS ofbruK0713 from Batch 1 ...... 27
Figure 15- TOF MS ofbruK0713 from Batch 2 ...... 27
Figure 16- TOF MS ofbruK0714 from Batch 1 ...... 27
Figure 17- TOF MS ofbruK0714 from Batch 2 ...... 28
Figure 18-TOF MS ofbruK0718 from Batch 1 ...... 28
Figure 19- TOF MS of bruK0719 from Batch 2 ...... 28
Figure 20- TOF MS ofbruK0718 (combined fraction) ...... 29
Vll Figure 21 -1D NMR Spectra of0718 (combined fraction) ...... 30
Figure 22 - TOF MS of the Reduced and Alkylated bruK0718 ...... 31
Figure 23- Elution Profile ofReduced & Alkylated bruK0718 in the Analytical Column ...... 31
Figure 24- TOF MS of Reduced & Alkylated bruK0718e ...... 32
Figure 25- Elution Profile ofbruK0714(combined fraction) in the Analytical Column ..34
Figure 26- TOF MS ofbruK0714a ...... 35
Figure 27- TOF MS ofbruK0714b ...... 35
Figure 28- Elution Profile ofbruK0714b in the Analytical Column ...... 36
Figure 29- 1-D NMR Spectra ofbruK0714b ...... 37
Figure 30- TOF MS of Reduced & Alkylated bruK0714b ...... 38
Figure 31 -Elution Profile ofReduced & Alkylated bruK0714b in the Analytical Column ...... 38
Figure 32- TOF MS of Reduced & Alkylated bruK0714b from the Analytical Column ...... 39
Figure 33- Elution Profile ofbruK0714a in the Analytical Column ...... 41
Figure 34- TOF MS ofbruK0714a7 (combined fraction) ...... 42
Figure 35- 1-D NMR Spectra ofbruK0714a7 ...... 43
Figure 37- Elution Profile for Partially Reduced & Alkylated ofbruk0714a7 ...... 45
Vlll Introduction
Cone snails are venomous marine mollusks that belong to the superfamily Conacea and to the genus Conus. It is believed that a major radiation of cone snails occurred after the massive extinction at the K-T (Cretaceous/Tertiary) boundary; a radiation that required the evolution of unique venoms [1] . These mollusks use their venom for prey capture, defense, and competition [ 1]. More than 1000 different species of cone snails use complex venom to capture their prey [2, 3] . Cone snails typically inhabit tropical shallow water marine environments. They are divided into three major groups depending on the type of prey: vermivorous, molluscivorous, and piscivorous.
Mulluscivorous conus prey on other gastropods, vermivorous conus feed on small polycheate worms, and piscivorous conus prefer small fish. Although vermivorous cone snails are the most common, most of the research has been conducted on piscivorous cone snails because their venom has an evident effect on the mammalian system.
Cone snails initially were valued for their beautifully patterned shells, but it was quickly discovered that the snails can be deadly. Due to their lack of mobility, cone snails have developed fast acting compounds that can quickly paralyze the prey by affecting its neurophysiological system. Cone snail venom is made up of a powerful cocktail of highly specialized peptides ( conopeptides) and proteins ( conoproteins).
These compounds, most notable the conopeptides, bind to voltage and ligand gated
1 ion channels which are essential for the proper functioning of the neuromuscular system [4]. Consequently, these conotoxins have proven to be extremely useful in numerous neurophysiological studies. Moreover, it has been estimated that greater than 100,000 conopeptides exist with only - 0.2% characterized pharmacologically; so there is still lots of discovery to be done [5].
Every group of cone snails uses an extremely specialized venom apparatus to envenomate their prey [6] (Figure 1). The venom apparatus consists of a venom bulb which is a muscle that pushes the venom out, a venom duct where venom is synthesized by epithelial cells and stored, a radular sheath where hollow harpoon-like teeth are stored, and the proboscis which is used to eject the harpoon and deliver the venom to the prey.
Figure 1- Structure of a Typical Conus Venom Apparatus (Oliveira 2002) A
Radular teeth Venom bulb
Proboscis
2 Conus brunneus is a common eastern pacific vermivorous cone snail that mainly feeds on polycheate worms. Conus brunneus is found in the class Gastropoda, order
Caenogastropoda, superfamily Conacea, family Conidae and subgenera
Stephanoconus. It is the most common member of the Stephanoconus subgenera.
These snails are found in the Panamic providence ranging from Mexico to Peru and inhabit shallow to moderately deep waters.
Figure 2 - Picture of a Conus brunneus shell from www .gastropods.com/ 0/Shell 590.html
3 Venom Composition
Cone snail venom is mainly made up of highly structured peptides that can consist of multiple disulfide bonds and/or numerous post - translational modifications (PTM).
Conopeptides is a general term used for all peptides found within cone snail venom.
Conopeptides are small peptides consisting of 6-40 amino acids; yet, they have a well-defined 3D structure [7]. There are two major divisions of conopeptides: non- disulfide rich peptides and disulfide rich conotoxins (peptides with 2 or more disulfide bonds) [8]. Non-disulfide rich conopeptides consists of conophans and conantokins, which are linear peptides with no disulfide bonds and conopressins, which have one disulfide bond. The multiple disulfide bonds provide an important scaffold for stabilizing the peptide's structure [6]. Cone snail venom also has some of the highest densities of disulfides bonds in any biological system [3].
Conotoxins are grouped into superfamilies based on their conserved signal sequences.
Research on venom duct eDNA clones has shown that conopeptides are initially translated as pre-propeptide precursors [1]. The pre-region ofthe peptide precursor at theN-terminal consists of a signal sequence followed by a pro-region and finally a . mature toxin at the C-terminal [9]. This suggests that cleavage of the precursor is required to produce the functional peptide [ 1]. It has also been shown that the signal sequence of conopeptides is highly conserved and the mature toxin region is hypervariable. Yet, most secreted proteins have a highly variable signal sequence [10,
11]. It is also interesting to note that the sequence diversity ofthe mature toxin is believed to influence the folding of the peptides not the cysteine pattern [ 12, 13].
4 In addition to their highly conserved signal sequence, superfamilies also have a characteristic cysteine backbone within the mature peptide. Several superfamilies have already been identified including: 0 , M, A, S, T, P, and I superfamilies. These superfamilies are further classified based on their cysteine backbone and molecular targets (Figure 3).
Figure 3 - Classification of Conopeptides (Terlau and Oliveira 2004)
...... l ' •nll<., ' •• '" ( ... ' 1 ' ... •L . ' ""'"'f.t;..~r u ...._:t "-""'("•'• ' ' •I I" ¥T .. ...,..~., ~" 1'' • rj1-• ~ ,.. ·· ",.~·
. c.w.:. ,__,u \ I 1 I --,-- - 0 •·I 'l'· - .1_ r- LU. -:! .!... J..\i .'.).
~(( 0 ( ( ... cct LC X.C'IX .(C ( .. ~CC•. "'Ct 0 0 (>. -~ 0 '·· c. T."' I_ - • " ( ~-
~ ... \..•. >J• '" ... I ...... r,,...._ "'"' '"• ...... ""'.. ... ,. . , I ,, ~ \1 II \ ,, Ll~ ~t-0 ' I' v ..
Amazingly, extensive peptide diversity has been created by varying only a few gene superfamilies [6]. This variation of conopeptides is enhanced by post-translational modifications such as proline hydroxylation, y-carboxylation of glutamate residues, bromination of tryptophan, and L to D epimerization of pep tides. These modifications may help to retard enzymatic digestion or may optimize peptide entry into the central nervous system (CNS) or blood brain barrier (BBB) [14]. The exact reason for the hyperdivergence of the mature toxin is still unknown; however, theories include increased mutation rates, recombination events, and evolutionary selection [13].
5 Pharmacology and Physiology of Venom Components
To ensure prey capture, cone snails use multiple conopeptides in a synergistic fashion to distress their targeted prey. The term "toxin cabal" refers to a combination of conopeptides that are utilized together to meet a common goal. Two different toxin cabals have been identified in cone snails that use the hook and line strategy for prey capture: the lightning strike cabal and the motor cabal. The lightning strike cabal is involved in the initial phase of prey capture; thus, it is responsible for the immediate paralysis of the prey. The lightning strike cabal causes a massive depolarization of axons near the venom injection site by inhibiting Na channel inactivation and blocking K channels [1]. The motor cabal is part of the second phase of prey capture.
It acts at the neuromuscular junction by inhibiting Ca channels and postsynaptic nicotinic receptors and blocks Na conductance [1]. The cabals do not overlap since the motor cabal requires some time to reach the neuromuscular junction. Both the lightning strike and motor cabals act to keep the prey continuous immobilized, thereby increasing the likelihood of capture [10].
In addition to the lig~tning strike and motor cabals, a nirvana cabal is found in cone snails that utilize their false mouth to capture prey. The nirvana cabal contains conopeptides that affect the sensory circuit of their prey, making the prey more quiescent and easy to capture [1].
6 Figure 4 - Hook & Line and Net Strategies of Fish-Hunting Cone Snails (Olivera 1997).
The synergistic activity of these cabals produces a physiological effect that is more
potent than the individual activities of the peptides. The successful use of a
combinatorial peptide library by Conus may inspire the development of synergistic
drugs with enhanced target specificity.
Conopeptides as Pharmaceutical Drugs
Currently, numerous conopeptides are being developed for the treatment of
neuropathic pain and-other nervous disorder. These peptides represent an invaluable
tool for studying the complexity of voltage and ligand gated ion channels [ 15]. Due to
their high selectivity, numerous conopeptides have undergone therapeutic studies and
some are in various stages of clinical trials for the management of neurological
disorders. The most notable example of a conotoxin therapeutic agent is Ziconotide
(Prialt); a peptide isolated from the species Conus magus. Ziconotide has the ability to selectively target N-type calcium channels, which are associated with the cause of
7 pain. Ziconotide has proven to be more potent than morphine, but exhibits a low benefit to risk ratio and a narrow therapeutic window [16] . Currently, drug companies are working on ways to reduce the side effects caused by Ziconotide and to increase the drug's bioavailability. Another conotoxin that has received much attention is
Conantokin-G, isolated from Conus geographus. This peptide contains five y- carboxyglutamate residues and has been shown to be an effective anti-epileptic agent
[17]. Already numerous conopeptides with great therapeutic potentials have been identified (Table 1) and there is the possibility for conopeptides to be used as therapeutic agents for cancer, stroke, cardiovascular disease, epilepsy, and other neurological disorders.
Table 1 - Targets and Therapeutic Potential ofConopeptides (Alonso et al. 2003)
Class Target Therapeutic Potential Contulakins - Linear Neurotensin receptors Neuropathic pain Conatokins - Linear NMDA receptors Epilepsy, Parkinson's Conopressins - CC Vasopressin receptors Regulates blood pressure x-Conotoxins - CC-CPC Neuropathic pain Neuropathic pain co-Conotoxins - C-C-CC-C- Ca channels Analgesic, Stroke c K-Conotoxins - C-C-CC-C-C K channels Arrhythmia, Hypertension ~-t - Conotoxins - CC-C-C-CC Skeletal muscle Na Neuromuscular block . channels '1'-Conotoxins - CC-C-C-CC Skeletal muscle nACh Analgesic, Parkinson's, channels Hypertension a-Conotoxins - CC-C-C Skeletal muscle nACh Analgesic, Parkinson's, receptor Hypertension
8 Materials and Methods
Specimen Collection
Living specimens of Conus brunneus were collected from several locations off the
Pacific coast of Costa Rica. Conus brunneus snails tend to be distributed in shallow to moderately deep water. A shallow water dive was conducted along the Pacific coast of Costa Rica and several cone snails were obtained from rocks, coral, and sand. The specimens were placed in salt-water tanks and transported alive to the laboratory at
Florida Atlantic University. The majority of the cone snails were kept alive until dissection of the venom ducts was performed. Any cone snails that died before the extraction of the venom were stored at -80°C for separate study. These specimens tend to be large and have large amounts of venom (usually 20-70mg per specimen).
Crude Venom Extraction
Live specimens were placed on ice for about five minutes in order to increase the chance that the body.will be removed intact. A needle-like probe was inserted lengthwise between the shell and body and gently turned in the direction of the shell opening; thus, removing the body. The venom duct is located in the soft tissue of the cone snail and a dissecting probe was used to tear the tissue and remove the venom duct. The highly coiled structure and orange color ofthe venom duct made for easy identification. After dissection, the ducts were carefully uncoiled and measured. The venom ducts were placed in a small amount ofO.l% trifluoroacetic acid (TFA) and
9 frozen at -80°C. The ducts were subsequently lyophilized. The lyophilized venom ducts were homogenized with 0.1% TF A and the extracts were centrifuged at I 0,000 x g for 20 min, at 4°C. The pellets collected were washed with 0.1% TFA and re centrifuged. This procedure was repeated about three times. All the supernatants were pooled, lyophilized, and stored at -80°C.
Purification of Peptides
Size-Exclusion High Performance Liquid Chromatography CSE-HPLC)
Crude venom is first purified by a pre-equilibrated size exclusion high performance liquid chromatography (HPLC) column (Phannacia Superdex-30, 2.5 x I 00 em from
Thermo Separation Products). This column separates the venom components based on size. The mobile phase used in the column consisted of 0.1 M NH4HC03 at a flow rate of 1.5 ml/min using an isocratic gradient. A UV detector measured wavelengths at 220nm, 250nm, and 280nm. Collected fractions were lyophilized and stored at
-40 °C.
Semi-Preparative Reverse-Phase HPLC
The lyophilized samples from the SE-HPLC were subsequently subjected to separation by the C 18 semi-preparative column (Vydac, 218TP51 0, I 0 x 250 mm;
5J.lm particle diameter; 300 A pore size) of the reverse-phase HPLC. This column separates peptides according to hydrophobicity and allows for better separation of single components. The lyophilized samples were dissolved in I OOOJ.ll of 0.1% TF A solution and applied to the column. The samples were eluted with a linear gradient of
10 0.1% TF A (buffer A) and 0.1% TFA in 60% Acetonitrile (ACN) (buffer B) at a flow rate of 3.5 ml/min with a 1% buffer B increase/min. Absorbance was monitored at wavelengths of220 and 280 nm. Collected fractions are lyophilized and stored at
-40°C until further use.
Analytical Reverse-Phase HPLC
Fractions from the semi-preparative column needing further purification were subject to the analytical reverse-phase C18 column (Vydac, 238TP54, 4.6 x 250 mm; 5J..lm particle diameter; 300 A pore size). The samples were dissolved in 500f.ll of 0.1%
TFA solution and applied to the column with a flow rate of 1 ml/min. Elution was also done with the same buffers as described for the semi-preparative column, and the absorbance was similarly monitored. The samples can be eluted using either a linear or an isocratic system. Collected fractions were lyophilized and kept at -40°C.
Mass Spectrometry
Molecular mass was determined by positive ion matrix laser desorption ionization time of flight (MALDI-TOF) mass spectrometry as detected by a Voyager-DE STR
(Applied Biosystems). Samples were dissolved in 60% ACN with 0.1% TFA solution, and applied between two layers of a a-cyano-4-hydroxycinnamic acid matrix (Acros Organics) onto a magnetic plate. Spectra were acquired in either linear or reflector mode. Calmix 1 and Calmix 2 (Applied Biosystems) were used as external calibration standards.
11 Nuclear Magnetic Resonance
Pure samples were subject to one-dimensional NMR experiments to determine relative peptide concentration and to identify characteristics of the peptide. The NMR spectra were obtained by a Varian Inova 500 MHz instrument equipped with pulse field gradients, three radiofrequency channels and waveform generators. Pure peptide samples were dissolved in high purity water (Fisher) containing 38 pmol of trimethyl silyl propionic acid (TSP) (Aldrich) as an internal reference and 10% D20 (Aldrich).
The total volume of samples, 40 ).data pH of ~3.6, was placed in 1.7 mm NMR capillary tube (Wilmad), and spectra were acquired at 25°C. Samples with sufficiently high enough concentrations were subject to two dimensional NMR experiments, namely NOESY and TOCSY. 2D NMR helps to confirm the presence of certain amino acids in the sequence.
Reduction and Alkylation of Peptides
Pure samples were subject to reduction and alkylation in order to determine the number of Cysteine residues in the peptide. The lyophilized samples were dissolved in O.IM Tris-HCl (pH6.2) and reduced with 20mM dithiothreitol (OTT). The samples were incubated for 30 minutes at 60°C. For the alkylation, 5 mM iodoacetamide
(lAM) and 2j.tl ofN~OH (pH 10.5) were added to the mixture and incubated in the dark for one hour at room temperature. The reduced and alkylated peptides were recovered and purified using a pre-equilibrated Zip Tip (C18, size P10, Millipore) with a 0.1% TF A in 60% acetonitrile solution and 0.1% TF A solution.
12 Partial Reduction and Alkylation
This procedure was adapted from Hooven et al. at Wageningen University [18]. The sample is dissolved in 30J..1.l O.lM Citrate buffer at pH 3. Then, the sample was reduced with 12J..1.l offreshly prepared 0.1M tris-(2-carboxyehtyl)phosphine (TCEP) and incubated for 10-15 minutes at room temperature. After reduction, the sample was immediately alkylated with 30J..1.l of freshly prepared 0.1 M N-Ethylmaleimide
(NEM) and incubated for 30 minutes at room temperature. The sample is applied to the analytical column immediately after alkylation and run for 100 minutes using a linear gradient. Collected fractions are lyophilized, stored at -40°C and later subject to mass spectrometry.
Peptide Sequencing
The reduced and alkylated peptides are subjected to sequencing by automated Edman
Degradation. Alkylated peptides were absorbed onto Biobrene-treated glass fiber filters and sequenced by Edman degradation using an Applied Biosystems Procise model 491 A Sequencer equipped with a micro gradient delivery system, model 61 OA model 785A UV detector, and data analysis software model 140C.
Phenylisothiocyanate (PITC) reacts with an uncharged terminal amino group in basic conditions forming a phenylthiocarbamyl derivative (PTC-protein). This derivative is cleaved under acidic conditions forming another derivative, anilinothialinone derivative (AZT-amino acid) and leaving behind the next amino acid for degradation in the next cycle. The AZT-amino acid is extracted with N-butyl chloride, an organic solvent and converted to a phenylthiohydantoin derivative (PTH-amino acid) that is
13 transferred to a reverse-phase HPLC C-18 column for detection at 270 run. The PTH amino acid can be easily identified via chromatography. For cross-referencing, a standard mixture of PTH-amino acids is injected onto the column for separation and detection, providing a standard elution profile for comparison with unknowns. The sequences are then established by confirming the expected molecular weight.
Nomenclature
BrunneusK was the designation given to this batch of crude venom and bruK is the abbreviation for this batch. The nomenclature assigned from here on is as follows:
Superdex-30 fractions are labeled as numbers in numerical order such as bruKOl, bruK02, and so on. The fractions from the semi-preparative column take a number after the number from the Superdex 30 fractions such as bruKOIOl , bruK0102, bruK0103, and so on. The fractions from the analytical column take a letter after the semi-preparative column number such as bruKOIOla, bruKOlOlb, and so on.
14 Results and Discussion
Purification of Peptides
The purification of peptides requires several steps beginning with the size exclusion separation. The crude venom collected from Conus brunneus was split into 2 batches.
In order to begin the purification process, each batch of the lyophilized crude venom was dissolved in 1000J.1.L ofNH4HC03. Batch 1 was injected into the Superdex 30 column and ran for 200 minutes at a range of2 and at wavelengths of220, 250, and
280 nm. This initial separation by size yielded nine fractions at 220nm, six fractions at 250nm and three fractions at 280nm. The absorbance at 280nm suggests the presence of aromatic amino acids. Batch 2 ran for 220 minutes in the Superdex 30 column at the same absorbance as batch 1. The 220nm absorbance was set to a range of 1.5 whereas the 250nm and 280nm absorbance were at a range of 1.0. The separation produced nine fractions at 220nm, five fractions at 250nm, and five fractions at 280nm.
Each respective fraction from both batches was pooled together. The fractions were lyophilized and stored at -40° C.
15 Figure 5 - Elution Profile of Batch 1 bruK Crude Venom in Superdex 30 Column
B33 Batch 1 7 A"' 220 Range"' 1.5
2 5 6 3 8
9 •••-11.11117 ...... 0 200m in 1.383 Batch 1 A• 250 Range• 2.0 7
8
-0.1 0 200min 5.533 Batch 1 A=280 Range • 2.0
7
-0.11117 0 200m in
Table 2 - Elution Times of bruK Batch 1 Crude Venom Fractions from Superdex 30
Fraction Elution Time (minutes) I 61.22 2 76.95 . 3 96.14 4 I 07.05 5 134.84 6 142.27 7 148.41 8 185.99 9 194.72
16 Figure 6 - Elution Profile of Batch 2 bruK Crude Venom in Superdex 30 Column
Batch 2 ~=220 Range= 1.5 4
3 5 2 6
8 333 9 ...... ······ ···· ···· ·· ···- ·········· ·········· ··········· ···· ·· ··· ·· ························· ...... 0 220m in 1383 Batch 2 ~=250 Range = 1.0
4 2 3 '(j.0·······.1&7· ···················· ......
1.383 Batch 2 ~= 280 Range : 1.0
7 4 8 9 .0.107
0 220m in Table 3 - Elution Times ofbruK Batch 2 Crude Venom Fractions from Superdex 30
Fraction Elution Time {minutes) 1 60.91 2 75.44 3 91.77 4 102.93 . 5 136.28 6 142.25 7 148.45 8 188.38 9 205.61
Fraction 5 was subject to further separation by the semi-preparative column. The sample was dissolved in 1000~-tL ofO.l% TFA solution and ran for 100 minutes. The
17 run was monitored at wavelengths 220 and 280nm and a range of 2. Thirty fractions were collected at 220nm and several peaks were observed at 280nm.
Figure 7 - Elution Profile ofbruK05 in the Semi-Preparative Column
715.13 21 22 A.= 220 Range= 2.0
, ,-7,8,44 , , , I , , , , , , , , , I , , , , , , , , , I , , , , , , , , , I • , , , , , , , , I , , , , , , , , , I , , , , , , , , , I , , , , , , , , , I , , , , , , o , • I • • • • • • , • • 0 100min
).s 280 Range • 2.0
22
21
, , , , , , , , I ,, , ,,, , ,, 1 .,,,,,,, , I,,,,, , , , , I,,,, ,, ,, , I , , ,, , , , ,, I , ,, ,, , ,,, I, ,,, ,, , ,, I , , ,, ,,, , , I ,, , ,,,,,, 0 100min
Table 4 - El ution Times ofbruK05 Fractions from Semi-Preparatory Column
Fraction Elution Time Fraction Elution Time (Minutes) (Minutes) 1 3.26-5.95 16 39.41-40.03 2 26.16-27.03 17 40.03-40.65 3 . 29.53-29.73 18 40.65-41.80 4 29.73-30.61 19 41.80-42.25 5 30.61-32.03 20 42.25-42.75 6 33.22-33.85 21 42.75-43.70 7 33.85-34.10 22 43.70-45.10 8 34.10-34.51 23 45.90-46.90 9 34.51-35.03 24 46.90-47.23 10 35.03-35.85 25 47.23-49.60 11 35.85-36.80 26 49.60-50.04 12 36.80-38.10 27 50.05-50.40 13 38.10-38.76 28 56.93-57.98 14 38.76-39.1 1 29 57.98-59.01 15 39.11-39.41 30 60.06-61.16
18 Again, the samples were lyophilized and stored at -40°C. All fractions collected from bruK05 were analyzed by mass spectrometry as previously described. The samples were dissolved in 10-200 ~L of 60% ACN with 0.1% TF A solution depending on the size of each peak and 0.3 ~L of each sample was used for mass spectrometry.
Fractions 3 and 4 from the semi-preparative column were pooled together since they comprised the same peak and had similar masses as determined by mass spectrometry. The sample was renamed bruK0504.
Figure 8 - TOF MS of bruK0503 (MW = 2360.06 Da)
Figure 9 - TOF MS of bruK0504 CMW = 2360.10 Da) Vo-Speci1[1!P • %362.0, 2787] '"'" 10 ~
10 ,
- (-)
19 bruK0504 was subject to further separation by the analytical column of the reverse
phase HPLC to ensure purity of the sample. The lyophilized sample was dissolved in
500J.t.L of 0.1% TF A and injected into the analytical column. The sample was
analyzed at an isocratic gradient for 17 minutes at wavelengths of220nm and 280nm
and a range of 0.2.
Figure 10- Elution Profile ofbruK0504 in the Analytical Column
22 .13 ).= 220 Ranges 0.2
a
'0 17min ! .!53 ).•280 Range • 0.2
c
.0.807
0 17min . Table 5 - Elution Times ofbruK0504 from the Analytical Column
Fraction Elution Time (minutes) a 10.90 b 11.60 c 12.00 d 13.16
Each fraction was submitted to mass spectrometry as previously described in order to assess the purity of the fractions. Fraction c (bruK0504c) was successfully purified.
20 Figure 11 - TOF MS of bruK0504c (MW = 2359.6 Da)
Vor•u••t.PK •til" • nn o 1tOSI
1311 0413 r 11 0 2 5
)U104\l 10 0 1 r 11 0 1:, ,,., 5412 •• I I I 2313 030fl I I •• I' ,. • •• I ..'"' .. "' 'j I •• 2311!1 0613 20 ~UOOfiU ~ ~ fll5 8900 21e5 5811 lltiV 5 4'1 ,2J$8 1171 tO ;' 1 , J:lM 72&& ;lln 'OS' ,, ...... -:-,----- nn .• MauC•IJ)
BruK0504c was lyophilized and prepared for NMR experiments as previously described. A lD NMR experiment was performed on the sample; unfortunately, an
NMR spectra could not be obtained due to the low concentration of the sample.
Although the concentration of the sample was too low to perform NMR experiments, the concentration of the sample was enough for reduction and alkylation. Eighty percent of the sample was used to perform the reduction and alkylation of the sample as previously described. The complete reduction and alkylation of the sample revealed that the peptide contained 4 cysteine residues. The presence of the 4 cysteines was determined by analyzing the molecular weight increase of the sample
21 since the reduction and alkylation of each disulfide bond results in an increase of 58
Da per cysteine residue. (2593 - 2360 = 233 Da, 232/58 = 4 Cys).
Figure 12 - TOF MS of Reduced & Alkylated bruK0504c (MW = 2593.4 Da)
Voyag•r Spec •1 {8P • 25tl 4. 54JOJ 2-WlH!1 .. ,. .'...... ' ..
• ... . .,.,,1
Subsequently, the entire reduced and alkylated sample was injected into the peptide sequencer, which produced the following sequence:
22 Table 6 - Sequence Analysis of bruK0504c
Cycle# Assigned Residue 1 Thr 2 Trp 3 Asp 4 y 5 Cys 6 Cys 7 Lys 8 Asn 9 Pro 10 Ala 11 Cys 12 Arg 13 Asn 14 Asn 15 His 16 Lys 17 Asp 18 Lys 19 Cys 20 Gly
Mass Spectrometer MW = 2360 Da Calculated MW = 2361 Da
Sequence: TWDyCCKNPACRNNHKDKCG
The measured molecular weight (2360 Da) of the sample corresponds to the theoretical molecular weight (2361 Da) as determined by Protein Prospector MS-
Product software. The C-terminal ofthe peptide was determined to be non-amidated.
BruK0504c' s peptide sequence corresponds to an a-conotoxin with the framework
CC----C------C. This type of conotoxin belongs to the A superfamily and targets neuronal nicotinic acetylcholine receptors (nAChR). a-conotoxins are the largest groups ofpeptides characterized in the A-Superfamily [20].The A-Superfamily is
23 divided into two groups: peptides that selectively act on muscle nAChR and peptides
that affect neuronal nAChR [19].
bruK0504c is referred to as an a4/7 conotoxin because it contains 4 non-Cys amino
acids residues in the first loop and 7 non-Cys residues in the second loop where the
1 3 2 4 disulfide connectivity is as follows: Cys -Cys and Cys -Cys • a-conotoxins are
competitive antagonists of acetylcholine binding to nAChR and are extremely
selective inhibitors [21].
The sequence of this peptide is interesting due to its extended N-terminal andy
carboxyglutamine. y-carboxyglutamic acid (Gia) was long thought to be restricted to
vertebrates until its discovery in conotoxins. Nowadays, Gla is a relatively common
2 post-translational modifications found in various conopeptides. Gla binds Ca + and is
required for calcium-induced interactions with the membrane surfaces; Additionally,
Gla is thought to add structural rigidity to the peptide [22, 23 , 24].
bruK0504c's sequence is also very similar to the peptide, GID, isolated from Conus geographus. GID was found to have a novel four amino acid N-terminal tail along with a conserved Proline residue in loop 1 and a conserved Arginine residue in position 12. The highly conserved Proline residue is believed to stabilize the helical region and secondary structure of the peptide [25].
In a study by Nicke et al. at the University of Queensland, functional characterization of GID showed that theN-terminal and the conserved Arg residue were necessary for activity on the a4~2 subunits of nAChR. Neuronal nAChR contain a combination of a and ~ subunits [20]. The research group theorized that the y-carboxylglutamate at position 4 prevented cleavage of theN-terminal and allowed it to survive in the
24 venom; however, this theory has yet to be proven. The study also suggested that different amino acid side-chains can determine the subtype selectivity of the peptide.
Thus, bruK0504c may prove to be useful in neurophysiological studies involving acetylcholine-binding sites.
In addition to fraction 5 (bruK05) from the size exclusion HPLC, fraction 7 (bruK07) was also analyzed. The sample bruK07 was split into two batches. Each batch was dissolved in 1OOOf.lL of 0.1% TF A solution and injected into the semi-preparative column. Batch l was run for 70 minutes, whereas batch 2 ran for 100 minutes. Both batches were analyzed at wavelengths 220 and 280nm and a range of 1.
Figure 13 - Elution Profile of bruK07 in the Semi-Preparative Column
Batcl11 13,14 A.= 220 Range z 1.0 12
15.16 17,18 ~ (~------~--~------~~~ ·1ile .. 0 0 0 0 . 0 .. • • • • • ' ' ' ' • • • I ' • • • '70ri1ifi
175.78 Balcl11 :<= 280 Range= 1.0
o · ...... '70rilirl 1430 Balch 2 14,15 :<= 220 Range= 1.0 13
. 0 ..
178.78 Batch 2 :<• 280 Range= 1.0
·18.8 , , , , , , , I , , , , , , , , , I , , , , , , , , , I , , , , , , , , , I , , , , , , , , , I , , , • • • • • • I • • • • • '7drTiin' 0
25 Table 7 - Elution Times of bruK07 Fractions from the Semi-Preparative Column
Batch I
Fraction Elution Time Fraction Elution Time (Minutes) (Minutes) 1 3.69 10 37.19 2 32.29 11 37.74 3 33.29 12 38.44 4 33.85 13 39.19 5 34.61 14 39.46 6 35.09 15 41.93 7 35.50 16 43.42 8 36.12 17 44.82 9 36.78 18 45.73
Batch 2
Fraction Elution Time Fraction Elution Time (Minutes) (Minutes) l 3.59 13 40.56 2 14.96 14 41.22 3 15.74 15 41.40 4 34.28 16 44.06 5 35.17 17 45.28 6 35.86 18 47.01 7 36.58 19 47.78 8 37.14 20 51.21 9 37.56 21 58.88 10 38.12 22 61.80 11 39.26 23 62.40 12 39.85
The run for batch 1 yielded 18 fractions and the run for batch 2 yielded 23 fractions.
All of the fractions were evaluated by mass spectrometry. Several fractions from both batches were pooled together due to their similar molecular weights. Fractions 13 and
14 from each batch were pooled together and renamed bruK0714. Also, fraction 18 from batch I and fraction 19 from batch 2 were pooled together and renamed
26 bruK0718. The pooled samples were lyophilized and stored at -40°C. Figures 14-19
show the mass spectra of these samples.
Figure 14- TOF MS ofbruK0713 from Batch 1
v- SPK ~(BP • 25U.7, 1110841 "'I r 1.H+4 .. ~ .. .. f • ,..I •
Figure 15 - TOF MS ofbruK0713 from Batch 2
... f t.lf-*l ..• ..
123-4 . ,,. h t L Figure 16 - TOF MS ofbruK0714 from Batch 1 ~ Spoc ~(liP- :IS30.1. 41411 :J . j 71 1 j .. j ,.. • • 23111 .... • ,...... -- 27 28 Figure 20 - TOF MS ofbruK0718(combined fraction) (MW = 2126 Da) v.ov.- apec .-.rww· 21211 . ..u.q ,., • ,., lU1.._ • f • 11U1- Z1llW.l I ltll.O \.: ·-..... - The combined fraction bruK0718 was prepared NMR experiments where a lD- NMR spectra was obtained. 29 Figure 21 - lD NMR Spectra ofbruK0718(combined fraction) .. .... I ..~ .., I ... "! • M • ~ ~ D. .,.,u N l •u ..,.,., M N 0 .... I Illl .. 0 • -e ..:> ,.Q llo 30 After NMR analyses, 80% of the sample was used for reduction and alkylation. Unfortunately, reduction and alkylation of the sample showed that the sample was not pure. Therefore, the entire reduced and alkylated sample was combined with 400~-.tL of 0.1% TF A solution and injected into the analytical reverse phase column for purification. The sample was run for 100 minutes at wavelengths of 220 and 280nm and a range of0.5. Figure 22 - TOF MS of Reduced and Alkylated bruK0718 100 1 .., I .., J '10 10 ~ i .s "" '# .. 30 » 10 WT 1713 0 111.0 ..... (m'J:) Figure 23 - Elution Profile ofReduced & Alkylated bruK0718 in the Analytical Column 2.783 '-= 220 Range= 0.1 a h • • •,,, • • I , ,,,, • ,,, I ,,,, . ,,,, I ,,, , , ,,,, I , ,, ,,, , , , I , , ,,,,, ,, I , ,,,, , , ,, I,,, , ,, , , , I.,,,, , , , , I , ,, ,, , , , , 0 100 min 31 Table 8 - Elution Times of Reduced & Alkylated bruK0718 from the Analytical Column Fraction Elution Time (minutes)_ a 12.01 b 22.79 c 33.20 d 34.96 e 38.44 f 38.90 g 40.80 h 42.60 I 43.56 j 44.30 k 45.10 After mass spectrometry evaluation of the fractions, fraction e was shown to be the fully reduced and alkylated fraction that corresponds to bruK0718. Figure 24 - TOF MS of Reduced & Alkylated bruK0718e (MW = 2476 Da) 100.. 1.1E+4 ,. % eo ..ln1 ., .. .. ;146126n 2481 7179 '1'06 1&0 7 " 24652250 .WJ.. §;l~~~ ·~ ~1!>3~· ll· 4....- ...... ~~1~~~ 21179.8 _,.,., :rno.2 33110.& -..8 32 The reduced and alkylated peptide revealed the presence of 6 cysteine residues (24 77- 2128 = 349 Da, 349/58 = 6 Cys). The reduced and alkylated sample was sequenced and the results are shown on Table 9. Table 9- Seguence Analysis ofbruK0718e Cycle# Assigned Residue l Cys 2 Cys 3 Arg 4 Trp 5 Pro 6 Arg 7 Cy_s 8 Asn 9 Val 10 Tyr 11 Leu 12 Cys 13 Gly 14 Hyp 15 Cys 16 Cys 17 Hyp 18 Gin Mass Spectrometer MW = 2126 Da Calculated MW = 2126 Da Sequence: CCRWPRCNVYLCGOCCOQ This peptide sequence belongs to the M-superfamily with the framework CC-C-C-CC and is referred to as a mini-M conotoxin. M-superfamily conotoxins have 2 distinct structural frameworks: the maxi-M branch consisting of Jl and \jf families have a 14 2 5 3 connectivity ofCys , Cys - , and Cys -6 whereas the mini-M branch has the 1 6 24 3 5 connectivity Cys - , Cys , and Cys - . Although maxi-M and mini-M conotoxins differ in disulfide connectivity their conserved signal sequence and cysteine backbone classifies the conotoxins into the same gene superfamily [26]. The difference in 33 disulfide connectivity serves to create different folds utilizing the same cysteine backbone within the peptide [27]. Mini-M conotoxins are commonly found in worm and mollusk hunting cone snails, but have not yet been found in fish hunting cone snails. On the other hand, maxi-M conotoxins have only been found in fish hunting cone snails. Although some structural data is available for mini-M conotoxins, the molecular target of these peptides have yet to be determined [27]. Next, bruK0714 was further purified by injecting the sample into the analytical reverse phase HPLC. The sample was run for 45 minutes at wavelengths of 220nm and 280nm. The 220nm absorbance was set at a range of 0.5 while the 280nm absorbance had a range of0.2. Figure 25- Elution Profile ofbruK0714 (combined fraction) in the Analytical Column 22.348 A.• 220 Range •0.5 a , , , , , , , , I , , , , , , , , , I , , 0 , , 0 , , , I , 0 , , 0 , , , , I , , , ~~0 . m----~~------~45 min 5 587 A.• 280 Range " 0.2 a ' ' , , , , , I , e , , , , , , , I , , , , , , . , , I o , , , , , , , , I , 0 45min 34 Table 10 - Elution Times ofbruK0714 (combined fraction) from the Analytical Column Fraction Elution Time (minute~ a 18.40 b 17.33 The larger fraction was designated "a" while the smaller fraction was labeled "b". Both fractions were submitted for mass spectrometry. Figure 26 - TOF MS of bruK0714a Voyager Spec IJ1 [BP • 2534.8, 7245] 2534 8032 10Ch r 72A5.0 I() I 10 1 70 l I 10 30 1 0 4001.0 Mualnn) Figure 27 - TOF MS of bruK07 14b Voy1gerSpec: t1[BP•171U,S3232J 100 1 ·~ ~ 10 1 :1 IO j ~ ~ :l Il l ' 20 1 ~ 1!1 11 •lt2911116 ,1 ~~--~~r------~----~---- :71UL 10U 207t.l 35 bruK0714b was also subject to further purification by the analytical column ofthe reverse phase HPLC. The sample was analyzed for 25 minutes at 220nm and 280nm absorbance. The ranges were set to 0.5 for 220nm absorbance and 0.1 for the 280nm absorbance. Figure 28- Elution Profile ofbruK0714b in the Analytical Column 11.17-t A.a 220 Range a 0.5 1752 Da 0 26m in A.• 280 Range a 0.1 1752 Da .0.013 0 26m in During this run, the column exhibited several problems and several peaks appeared in at both absorbance but were more prevalent at 280nm. However, bruK0714b was still recovered at its expected elution time (13.40 min). It was lyophilized and stored at Afterward, bruK0714b was prepared for NMR experiments where a 1D NMR spectra was obtained. 36 Figure 29- ID NMR Spectra ofbruK0714b I ":... I .. • • -.•1:1' ~ •lll .,~ -.•1:1' ..I -6' 0 ..;"' •u Ill c: .. Cl) ...... t 0 • 'i! :l .Q "' 37 When the NMR experiments were completed, half of the sample was used for the reduction and alkylation of the peptide. Figure 30 - TOF MS of Reduced and Alkylated bruK0714b V0)'11g81' Spec lt1 (8P • 2085.6, 5473) 20e5enQ r N7J.O 10 Mnsfmlll Unfortunately, examination of the reduced and alkylated sample revealed that it contained some impurities. Therefore, the sample was again subjected to purification by the analytical column. Figure 31 -Elution Profile of Reduced & Alkylated bruK0714b in the Analytical Column 11.1 .. A.= 220 Range = 0.1 - 1 ~ 0 30 min 2 783 A.= 280 Range = 0.1 -0308 0 30min 38 Table 11- Elution Times of Reduced & Alkylated bruK0714b in the Analytical Column Fraction Elution Time (minutes) 13.38 0.3J..d of fraction 1 from bruk0714b was submitted for mass spectrometry in order to determine the purity of the sample. The mass spectra revealed that the sample still contained some impurities; however, half of the sample was still used for sequencing by Edman degradation. Fortunately, the impurities did not affect the sequencing of the sample. The mass spectra also revealed that the peptide contained 6 cysteine residues (2101-1752 = 349,349/58 = 6 Cys). Figure 32- TOF MS of Reduced and Alkylated bruK0714b from the Analytical Column Voyeger Spec jJ1[BP • 2102.2, 14538] 100 2102~ r 1.ae... 10 10 70 Mou(mlz) 39 Table 12- Sequence Analysis ofbruK0714b Cycle# Ass!g_ned Residue I Cys 2 Cys 3 Arg 4 Val 5 Leu 6 Cys 7 Ser 8 AI"g_ 9 Try 10 His 11 C_ys 12 Leu 13 Pro 14 Cys 15 Cys Mass Spectrometer MW = 1752.9590 Da Calculated MW = 1752.7193 Da Sequence: CCRVLCSRYHCLPCC bruK0714b was also determined to belong to theM-superfamily as part of the m-2 branch of mini-M conotoxins. Similarly, bruK0714a was subject to further purification by the analytical column of the reverse phase HPLC. The sample was run for 30 minutes at an isocratic gradient with an absorbance of 220nm and 280nm. The range for the 220nm absorbance was set to 0.75 and the range for the 280nm absorbance was 0.1. 40 Figure 33- Elution Profile ofbruK0714a in the Analytical Column 22.132 ).z220 7.8 Range z 0.75 -2.11ee ' . . . ' 0 30min 22.132 ).= 280 Range= 0.1 7,8 -2-- ' . 0 30m' in Table 13 - Elution Times ofbruK0714a from the Analytical Column Fraction Elution Time (minutes}_ 1 2.82 2 3.63 3 9.94 4 11.74 5 13.38 6 13.76 7 14.09 8 14.26 . 9 16.22 Fractions 7, 8, and 9 were combined since they were part of the same peak and renamed bruK0714a7. 0.3J.1.l ofthis sample was submitted to mass spectrometry. 41 Figure 34 - TOF MS ofbruK0714a7(combined fraction) (MW = 2534 Da) Voy._ Spoc; 11 (BP • 2535.1, 1M3l) :] -1 :j I 10 , .. IIJ toQI " • " 1734401 1147 411'0 U8881!107 Voy090f Spoc; 11(8P • 253U, 1M33J ,.. , ,. 1. .... 10 J t . j ,. j . 1 I, ..10 • • " ....• The mass spectra ofbruK0714a7 shows a mass difference of 16 Da and 40 Da, which corresponds to a hydroxyl group and potassium, respectively. Since bruK0714a7 was fairly pure, it was prepared for NMR spectrometry. A 1D NMR spectra was obtained and revealed that the sample had been previously isolated by Fred Pflueger in our laboratory. 42 Figure 35- lD NMR Spectra ofbruk0714a7 .. • ..•co i .,.. I I •.. ...": • I I : () II' "' .. "'... I i ..;"' § ...3 ... l 0 ...• e :J ,Q llo 43 The peptide contains 6 cysteines and belongs to the P-superfamily with the following peptide sequence: Table 14- Sequence Analysis ofbruK0714a7 Cycle# Assigned Residue 1 Ser 2 Cys 3 Gly 4 Gly 5 Ser 6 Cys 7 Phe 8 Gly 9 Gly 10 Cys 11 Trp 12 Hyg 13 Gly 14 C_ys 15 Ser 16 Cys 17 Tyr 18 Ala 19 Arg 20 Thr 21 Cys 22 Phe 23 Arg 24 Asp . Mass Spectrometer MW = 2534 Da Calculated MW = 2532 Da Sequence: SCGGSCFG WOGCSCY ARTCFRD The molecular target for the P-superfamily remains unknown; however, a previously identified P-superfamily conotoxin, tx9a, causes loss of motor control and seizure like symptoms in mice, leading researchers to believe that the peptide targets glycine receptors [28]. It is interesting to note that because the P-superfamily peptides have 44 no adjacent Cysteines, these peptides may be able to exhibit a greater range of structural and functional diversity [29]. Since bruK0714a7 had been previously identified, it was used in experiments to determine the disulfide connectivity of the peptide. To begin the identification of the disulfide bonds, the peptide was cleaved with chymotrypsin; however, due to the cysteine arrangement and possible connectivity it was difficult to identify if any cleavage of the molecule occurred. As a result, partial reduction and alkylation of bruK0714a7 was attempted using a method established by Hooven et al. at Wageningen University [18]. Figure 37 - Elution Profile of Partially Reduced & Alkylated bruK0714a7 5.587 --Native Cont.amonant l Contaminant l.= 220 -o.e13 Range = 0.10 38 Contaminant l.= 280 -o.e13 Range = 0.01 I , o , , , o , , , I t , , , , , o , , I , o , , , , ' ' I ' ' t ' 38 80min 45 Table 15- Elution Times of Partially Reduced & Alkylated bruK0714a7 Fraction Elution Time (minutes) Native 40.48 2 Cys 52.36 4 Cys 59.24 6 Cys 62.53 Although the partial reduction and alkylation of bruK0714a7 was successful, the amount of sample collected from each peak was extremely small. Therefore, the partial reduction and alkylation ofbruK0714a7 had to be repeated several times in order to ensure that enough sample was collected for sequencing. In order to repeat the experiment, the native peptide was recycled after every run. Once enough sample was collected, the fractions were sequence to determine the bonding of the cysteine residues. Results of the experiment revealed that bruK0714a7 had the following disulfide This pattern of disulfide connectivity has been previously reported in P- conotoxins found in Conus gloriamaris [29]. These results are extremely important in identifying the structural functionality of the peptide. 46 Conclusions This work describes the isolation and characterization of novel conopeptides from Conus brunneus, a vermivorous cone snail. Several techniques including mass spectrometry and nuclear magnetic resonance spectroscopy were utilized in the identification of these conopeptides. Three novel conopeptides were discovered from Conus brunneus, which included one a4/7 conotoxin and two mini-M conotoxins. These sequences make up the following partial peptide library: Table 16 - Conus brunneus Partial Peptide Library Name and S~uence Framework SuQerfamily Target I. bruK0504c CC-C-C A neuronal TWDyCCKNPACRNNHKDKCG a4/7 nAchR 2. bruK0718e CC-C-C-CC M unknown CCRWPRCNVYLCGOCCOQ mini-M 3. bruK0714b CC-C-C-CC M unknown CCRVLCSRYHCLPCC m-2 branch mini-M 4. bruK0714a7 . C-C-C-C-C-C p unknown SCGGSCFGGCWOGCSCY ARTCFRD Also, the disulfide connectivity of a P-conotoxin was determined. These findings are extremely useful in determining the functionality and potential of these peptides as pharmaceutical agents. The determination of the molecular targets of these peptides is very important as the specificity of these peptides may prove useful in the treatment 47 of several neurological disorders. Future work of these peptides should include the identification of molecular targets and the determination of the disulfide connectivity ofthe mini-M conotoxins. 48 References 1. Terlau, H. and B.M. Olivera. Conus Venoms: A Rich Source ofNovel Ion Channel- Targeted Peptides. Physiology Review. 84, (2004), pp.41-68. 2. Jakubowski, J.A., W.P. Kelley, J.V. Sweedler, W.F. Gilly, and J.R. Schulz. Intraspecific variation of venom injected by fish-hunting Conus snails. The Journal ofExperimental Biology. 208, (2005), pp. 2873-2883. 3. Cruz LJ, C.A. Ramilo, G.P. Corpuz and B.M. Olivera, Conus peptides: phylogenetic range ofbiological activity. Bioi. Bull. 183 (1992), pp. 159-164. 4. Newcomb R., S. Gaur, J.R. Bell and L. Cruz, Structural and biosynthetic properties of peptides in cone snail venoms. Peptides 16 (1995), pp. 1007-1017. 5. Lewis, R.J. and M.L. Garcia. Therapeutic Potentials ofVenom Peptides. Nature Reviews. 2, (2003), pp.790-802. 6. Olivera B.M. Conus Venom Peptides: Reflections from the Biology of Clades and Species. Annual Review ofEcological Systems. 33, (2002), pp. 25-47. 7. 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The FEBS Journal. 272, (2005), pp. 1727-1738. 49 13. Corticello, S.G., Y. Gilad, N. Avidan, E. Ben-Asher, Z. Levy and M. Fainzilber. Mechanism for Evolving Hypervariability: The Case ofConopeptides. Molecular Biology and Evolution. 18, (2001), pp. 120-131. 14. Jones R. and G. Bulaj, Conotoxins - new vistas for peptide therapeutics. Curr. Pharm. Des. 6 (2000), pp. 1249-1285. 15. Alonso D., Khalil Z. , Satkunanthan N., Livett BG., Drugs From the Sea: Conotoxins as Drug Leads for Neuropathic Pain and Other Neurological Conditions. Mini reviews in Medicinal Chemistry, 3, (2003), pp. 785-787. 16. Wermeling, D.P. Ziconotide,an Intrathecally Administered N-Type Calcuim Channel Antagonist for the Treatment of Chronic Pain. Pharmacotherapy. 25(8), (2005), pp. 1084-1094. 17. Mcintosh, J.M. and R.M. Jones. Cone venom- from accidental stings to deliberate injection. Toxicon. 39, (2001), pp.l447-1451. 18. Hooven H.W., Van den Burg H.A., Vossen P., Boeren S., Wit P.J.G., and Vervoort J. 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