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Discovery and biological characterization of conotoxins from the venom of Conus Brunneus in Drosophila Melanogaster
Title
Discovery and biological characterization of conotoxins from the venom of Conus Brunneus in Drosophila Melanogaster.
Creator
Heghinian, Mari D., Mari, Frank, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
Cone snails are venomous marine predators whose venom is a complex mixture of modified peptides (conopeptides). Conopeptides have direct specificity towards voltage- and ligand-gated ion channels and G-protein coupled receptors. More specifically, alpha conotoxins target nicotinic acetylcholine receptors (nAChR) and are of great interest as probes for different nAChR subtypes involved in a broad range of neurological function. Typically, the amount of peptide provided directly from the cone...
Show moreCone snails are venomous marine predators whose venom is a complex mixture of modified peptides (conopeptides). Conopeptides have direct specificity towards voltage- and ligand-gated ion channels and G-protein coupled receptors. More specifically, alpha conotoxins target nicotinic acetylcholine receptors (nAChR) and are of great interest as probes for different nAChR subtypes involved in a broad range of neurological function. Typically, the amount of peptide provided directly from the cone snails (from either dissected or “milked” venom) is minimal, thus hindering the wide use of bioassay-guided approaches for compound discovery. Biochemical-based approaches for discovery by means of identification and characterization of venom components can be used due to their compatibility with the small quantities of cone snail venom available; however, no direct assessment of the bioactivity can be gleaned from these approaches. Therefore, newly discovered conotoxins must be acquired synthetically, which can be difficult due to their complicated folding motifs. The ability to test small quantities of peptide for bioactivity during the purification process can lead to the discovery of novel components using more direct approaches. Presented here is the description of use of an effective method of bioassay-guided fractionation for the discovery of novel alpha conotoxins as well as further biological characterization of other known alpha conotoxins. This method requires minimal amounts of sample and evaluates, via in vivo electrophysiological measurements, the effect of conotoxins on the functional outputs of a well-characterized neuronal circuit in Drosophila melanogaster known as the giant fiber system. Our approach uses reversed-phase HPLC fractions from venom dissected from the ducts of Conus brunneus in addition to synthetic alpha conotoxins. Fractions were individually tested for activity, re-fractionated, and re-tested to narrow down the compound responsible for activity. A novel alpha conotoxin, bru1b, was discovered via the aforementioned approach. It has been fully characterized in the giant fiber system through the use of mutant flies, as well as tested in Xenopus oocytes expressing nicotinic acetylcholine channels and against the acetylcholine binding protein. Other well-known alpha conotoxins have also been characterized in the giant fiber system.
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Date Issued
2014
PURL
http://purl.flvc.org/fau/fd/FA00004122, http://purl.flvc.org/fau/fd/FA00004122
Subject Headings
Drosophila melanogaster, Gastropoda -- venom, Peptides -- Structure, Venom
Format
Document (PDF)
Isocation and characterization of conotoxins from the venom of Conus Planorbis and Conus Ferrugineus
Title
Isocation and characterization of conotoxins from the venom of Conus Planorbis and Conus Ferrugineus.
Creator
Pak, Adriana, Mari, Frank, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
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...
Show moreThe 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.
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Date Issued
2014
PURL
http://purl.flvc.org/fau/fd/FA00004146, http://purl.flvc.org/fau/fd/FA00004146
Subject Headings
Conus, Gastropoda -- Venom, Peptides -- Structure, Venum
Format
Document (PDF)
Isolation and structure elucidation of Perthamide B, a novel peptide from the sponge Theonella sp.
Title
Isolation and structure elucidation of Perthamide B, a novel peptide from the sponge Theonella sp.
Creator
Gulavita, N. K., Pomponi, Shirley A., Wright, Amy E., Yarwood, Donna, Sills, Matthew A.
Date Issued
1994
PURL
http://purl.flvc.org/FCLA/DT/3331880
Subject Headings
Sponges, Cyclic peptides, Peptides--Structure, Spectroscopic techniques
Format
Document (PDF)
Peptidomic analysis and characterization of the venom from Conus purpurascens
Title
Peptidomic analysis and characterization of the venom from Conus purpurascens.
Creator
Rodriguez, Alena, Mari, Frank, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
The venom of cone snails is a potent cocktail of peptides, proteins, and other small molecules. Several of the peptides (conopeptides and conotoxins) target ion channels and receptors and have proven useful as biochemical probes or pharmaceutical leads. In this study, the venom of a fish-hunting cone snail, Conus purpurascens was analyzed for intraspecific variability; α-conotoxins from the venom were isolated by high performance liquid chromatography, identified by mass spectrometry and...
Show moreThe venom of cone snails is a potent cocktail of peptides, proteins, and other small molecules. Several of the peptides (conopeptides and conotoxins) target ion channels and receptors and have proven useful as biochemical probes or pharmaceutical leads. In this study, the venom of a fish-hunting cone snail, Conus purpurascens was analyzed for intraspecific variability; α-conotoxins from the venom were isolated by high performance liquid chromatography, identified by mass spectrometry and nuclear magnetic resonance, and tested in a electrophysiological assay in Drosophila melanogaster; the effects of diet change on venom composition was investigated. It has been determined that each specimen of C. purpurascens expresses a distinct venom, resulting in the expression of more than 5,000 unique conopeptides across the species. α- conotoxin PIA was shown to inhibit the Dα7 nicotinic acetylcholine receptor.
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Date Issued
2015
PURL
http://purl.flvc.org/fau/fd/FA00004403, http://purl.flvc.org/fau/fd/FA00004403
Subject Headings
Conidae -- Environmental aspects, Drosophila melanogaster, Gastropoda -- Venom, Peptides -- Structure, Venom
Format
Document (PDF)
New conopeptides from Conus jaspedius
Title
New conopeptides from Conus jaspedius.
Creator
Williams, Orette St. Aubyn, Florida Atlantic University, Mari, Frank
Abstract/Description
Cone snails (genus conus) are marine gastropods of tropical waters. They capture their prey by envenomation and for this; they have evolved a highly efficient and diverse venom cocktail of proteins and peptides. This has elicited extreme interest in venom composition and their molecular targets. In this study, the venom of Conus jaspedius, a small Atlantic cone snail was extracted, venom components isolated and analyzed, by a combination of Size exclusion and High performance liquid...
Show moreCone snails (genus conus) are marine gastropods of tropical waters. They capture their prey by envenomation and for this; they have evolved a highly efficient and diverse venom cocktail of proteins and peptides. This has elicited extreme interest in venom composition and their molecular targets. In this study, the venom of Conus jaspedius, a small Atlantic cone snail was extracted, venom components isolated and analyzed, by a combination of Size exclusion and High performance liquid chromatography, NMR, Mass spectrometry and Edman sequencing. Here, four novel conopeptide sequences are reported, namely; an alpha conotoxin, a member of the A and O superfamilies and a new family.
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Date Issued
2004
PURL
http://purl.flvc.org/fcla/dt/13303
Subject Headings
Venom, Gastropoda--Venom, Poisonous animals, Peptides--Structure
Format
Document (PDF)
Novel conopeptides from Conus tessulatus
Title
Novel conopeptides from Conus tessulatus.
Creator
Borges, Paula., Florida Atlantic University, Mari, Frank
Abstract/Description
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,...
Show moreCone 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.
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Date Issued
2005
PURL
http://purl.flvc.org/fcla/dt/13231
Subject Headings
Conus, Venom, Gastropoda--Venom, Peptides--Structure
Format
Document (PDF)
Isolation and characterization of novel conopeptides from Conus brunneus
Title
Isolation and characterization of novel conopeptides from Conus brunneus.
Creator
Pellicier, Jalidsa., Florida Atlantic University, Mari, Frank
Abstract/Description
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...
Show moreCone 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.
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Date Issued
2006
PURL
http://purl.flvc.org/fcla/dt/13365
Subject Headings
Conus, Gastropoda--Venom, Venom, Peptides--Structure
Format
Document (PDF)
Three-dimensional structure of the alpha-conotoxin EI determined by proton NMR spectroscopy
Title
Three-dimensional structure of the alpha-conotoxin EI determined by proton NMR spectroscopy.
Creator
Franco, Aldo, Florida Atlantic University, Mari, Frank
Abstract/Description
The alpha-conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4-10, 5-18) isolated from the venom of Conus ermineus. This peptide targets the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ of Torpedo. 2D-NMR methods and dynamical simulated annealing protocols have been used to determine the 3D structure of EI. 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The...
Show moreThe alpha-conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4-10, 5-18) isolated from the venom of Conus ermineus. This peptide targets the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ of Torpedo. 2D-NMR methods and dynamical simulated annealing protocols have been used to determine the 3D structure of EI. 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between T9 and M12 that is stabilized by the C4-C10 disulfide bond and turns involving C4-C5 and N14-P15. The overall fold of EI is similar to that of other alpha4/7 conotoxins (PnIA/B, MII, EpI). However, unlike these other alpha4/7 conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to the surface charge distribution among these alpha4/7 conotoxins.
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Date Issued
1998
PURL
http://purl.flvc.org/fcla/dt/15609
Subject Headings
Conus--Venom, Peptides--Structure, Nuclear magnetic resonance spectroscopy
Format
Document (PDF)
Isolation and characterization of neuroactive peptides from the venom of cone snail species
Title
Isolation and characterization of neuroactive peptides from the venom of cone snail species.
Creator
Cano, Herminsul, Florida Atlantic University, Mari, Frank
Abstract/Description
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...
Show moreThe 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.
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Date Issued
2005
PURL
http://purl.flvc.org/fcla/dt/12185
Subject Headings
Peptides--Structure, Gastropoda--Venom, Conus, Mass spectrometry--Analysis
Format
Document (PDF)