Conus

The venom of marine gastropods belonging to the genus Conus has yielded numerous
structurally and functionally diverse peptidic components. The increase variety of
bioactive peptides identified in cone snail venoms is the product of the variety of
molecular adaptations taken by Conus species in evolving neuroactive molecules to suit
their diverse biological purposes. Toxins from cone snails are classified into two major
groups. One group consists of disulfide-rich peptides commonly termed conotoxins; the
second group comprises peptides with only one disulfide bond or none.
In this work, we present the discovery and characterization from the marine snails C.
planorbis and C. ferrugineus. Both species are commonly found in the Indo-Pacific region and are very similar and is not distinguishable by size and shape of the shell.
Novel P and T-Supefamiles were found in both species along with small linear peptides
with have a high frequency of tyrosine residues. Each chapter contains a detailed look at
the discovery process for the isolation and characterization of C. planorbis and C.
ferrugineus. At discussion part, we also compared the peptides isolated in this work with
other peptides from the literature.

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 the M-superfamily and one peptide was classified as an alpha-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.

Cone snails are predatory marine mollusks that utilize their peptide rich venom to capture prey, deter competitors and defend themselves. Each of the 1000 known species expresses over 100 conotoxins with little overlap between species. Most of these conotoxins selectively target a specific neuronal ion-channel or receptor. Because of their unprecedented diversity and specificity, they hold enormous potential as neuropharmacological agents, and as neuroscience research tools. In this study, the venom of a common shallow water cone snail that thrives in the Indo-Pacific to the Panamic region, Conus tessulatus , was analyzed; conopeptide components of the venom were isolated and investigated by high performance liquid chromatography, nuclear magnetic resonance, mass spectrometry, and automated Edman degradation sequencing. Five new peptide sequences are herein reported, among which there are three members of the M superfamily, one alpha conotoxin, and a conophan. The novel peptides comprise a partial peptide library of this particular cone.

Conus dalli is a cone snail species that preys upon mollusks (molluscivorous) and it belongs to the same clade as the better studied Conus textile. They have different biogeographical distribution: C. dalli is restricted to the Panamic area; whereas C. textile is a widespread species found from the Red Sea to Hawaii. The venom of C. textile is an extremely complex mixture of conopeptides characterized for their very high content of modified amino acids; particularly, for their high content of gamma-carboxy glutamate (Gla). Therefore, it is expected that the venom of C. dalli is equally complex and it might provide us with a library of novel conopeptides. We have collected 6 specimens of C. dalli from the Pacific coast of Panama. Their venom ducts were dissected and 40 mg of crude venom were extracted. Venom was separated using SE-HPLC and RP-HPLC and several single-component fractions with unique molecular weights have been found. 1D and 2D NMR methods in conjunction with mass spectrometry techniques have been applied to the main components of the venom. Three novel conopeptides have been isolated and characterized; dal_C1011h, dal_C0910, and dal_C0805g. dal_C1011h is a 27-residue hydrophobic conotoxin that belongs to O-superfamily, dal_C0910 is a 16-residue conotoxin that belongs to M-superfamily, and dal_C0805g is a 12-residue linear conopeptide the belong to the Conorfamide family. The details on the characterization of these conopeptides along with a comparison with previous data obtained from C. textile are presented.

Cone snails are marine 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 Panamic 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-superfamily of conotoxins.

The mam objective of the work described in this thesis is isolation and
characterization of novel neuroactive peptides from the venom of cone snail species.
The first section is an introduction about cone snails. The first chapter is dedicated to
the analysis of the milked venom obtained from three different specimens of C.
ermineus which is the only fish-hunter cone snail from the east Atlantic region.
MALDI-TOF mass spectrometry analysis of two specimens showed an identical
profile with all components of the venom being novel conopeptides. The third
specimen showed a mass spectrometry profile with molecular weights corresponding
to already reported conotoxins plus one additional new conopeptide. Ten new
conotoxins were isolated from C. ermineus; seven of them have sequences
corresponding to A-superfamily of conotoxins, specifically a-conotoxins family. Six of these seven conotoxins are the first a4/4 conotoxins isolated from the milked
venom from any fish-hunter cone snail specimens; the other one is a a4/7 conotoxin
similar sequence to the already reported a-EI from C. ermineus. Two more
conotoxins that belong to the 0-superfamily have the same amino acid sequence with
the only difference being a hydroxyproline residue instead of a proline at position 21
of the sequence. In the second chapter, four specimens of C. purpurascens, the only fish-hunter of the
Eastern Pacific region were analyzed. One of the specimens was sacrified and the
crude venom was dissected-out of the venom duct. For the three remaining live
specimens the venom was obtained by the "milking" procedure. Mass spectrometry
profiles were compared between dissected and milked venom and between milked
venom from different specimens. Analysis showed both similarities and differences in
the profiles of the dissected and the three milked venoms. A comparison of the three
milked venoms found some differences. This analysis showed that one specimen
expressed two isomorphs of a putative a4/4-conotoxin; the only difference was the
presence of proline instead of hydroxyproline at position seven in the amino acid
sequence. These a4/4-conotoxins are the second report of this sub-class of conotoxin
from the milked venom of cone snails and they have sequence homology to the a4/4
conotoxins isolated from C. ermineus. The analysis of the MALDI-TOF MS/MS
spectra of the Leu-contryphan-P conopeptide from C. purpurascens revealed that
conotoxins with a single disulfide bond in the sequence behave as a linear peptide in
the mass spectrometry experiment exhibiting a good fragmentation pattern. Using this
information by comparing the MS/MS spectra we were able to identify L-contryphan-P conopeptide lacking the first Gly residue in the sequence. In the third chapter, three conotoxins with sequence homology to the omega-superfamily
were isolated from the crude dissected venom of the worm-hunter cone snail C.
vexillum. The precursor of one of these conotoxins was already characterized by
another research group. Analysis and comparison of this precursor with already
known precursor allowed us to hypothesize that these conotoxins were ro-conotoxins.
Two of the three conotoxins have the same amino acid sequence with hydroxyproline
instead a proline in the structure. These conotoxins were the first ones isolated from
the venom duct of these cone snail species. Several conotoxins had been reported
from C. vexillum but they were isolated using eDNA cloning techniques.
Chapter four shows the analysis of the worm-hunter cone snail C. pseudoarantius
crude venom. Eight novel conotoxins were isolated from the pooled duct dissected
venom from different specimens. The first was a a4/3-conotoxin with a
carboxyglutamate residue present at position one in the sequence. Five more
conotoxins with conotoxin frameworks and sequences similar to M-superfamily of
conotoxins were also found; additionally, two more novel conotoxins with sequence
homology to o-conotoxins from the S-superfamily were isolated. All the above conotoxins were analyzed by comparison of their structures against
sequences of known conotoxins. All 23 conotoxins found in this research are novel
conopeptides isolated from cone snail specimens. Future work on the activity these
conotoxins will be important in the search for possible drugs in treatment of many
diseases.

Cone snails are predatory marine gastropods that use venom for means of predation and defense. This venom is a complex mixture of conopeptides that selectivity binds to ion channels and receptors, giving them a wide range of potential pharmaceutical applications. Conus brunneus is a wide spread Eastern Pacific cone snail species that preys upon worms (vermivorous). Vermivorous cone snails have developed very specific biochemical strategies for the immobilization of their prey and their venom has not been extensively studied to date. The main objective of this dissertation is the characterization of novel conopeptides isolated from Conus brunneus. Chapter 1 is an introduction and background on cone snails and conopeptides. Chapter 2 details the isolation and characterization of a novel P-superfamily conotoxin. Chapter 3 presents the 3D solution structure of the novel P-superfamily conotoxin. Chapter 4 details the isolation and characterization of two novel M-superfamily conotoxins. Chapter 5 covers the use of nano-NMR to characterize a novel P-superfamily conotoxin using nanomole quantities of sample. Chapter 6 is a reprint of a paper published in the Journal of the American Chemical Society in which we combined and implemented techniques developed in the previous chapters to report the presence of D-(Sd(B-Hydroxyvaline in a polypeptide chain. This dissertation contains the first reported work of a P-superfamily structure obtained directly from the crude venom therefore accurately representing native post-translational modifications. In this paper, crude cone snail venom was characterized by: high performance liquid chromatography, nuclear magnetic resonance spectroscopy, nanonuclear magnetic resonance spectroscopy, mass spectrometry, amino acid analysis, Edman degradation sequencing, and preliminary bioassays.

Cone snails are venomous marine gastropods that produce venom rich in neuroactive peptides, called conopeptides. The majority of published work on conopeptides has been from fish-hunting and mollusk-hunting cone snails. The work in this dissertation focuses on the discovery and characterization of novel conopeptides from the venom of worm-hunting cone snails. Eleven novel conopeptides have been isolated and biochemically characterized from the venom of C. nux using high performance liquid chromatography for the isolation and purification, and mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy were used for the biochemical characterization of the conopeptides. Nano-NMR spectroscopy was used as a tool to elucidate the three-dimensional structures of four conotoxins using native quantities of peptide isolated from the venom of C. nux, C. villepinii, and C. regius. In addition, the sequence-specific assignments and molecular model of a conotoxin from the venom of C. flo ridanus was also completed. The first chapter reviews the known conotoxin three-dimensional structures and cystine-constrained frameworks. The second chapter presents the mini-M conotoxins isolated from the venom of C. nux. The third chapter presents the three-dimensional NMR solution structure of a mini-M conotoxin from the venom of C. regius. The fourth chapter presents the cysteine-free conopeptides isolated from the venom of C. nux; conorfamide-nux1, a RFamide-related peptide, and nux770, a short pentapeptide. The fifth chapter presents the T-superfamily conotoxins isolated from the venom of C. nux, as well as the three-dimensional solution structure of one of the T-superfamily conotoxins. The sixth chapter presents the NMR solution structure of the first conotoxin with a cysteine-stabilized helix-loop-helix fold.