Peptides--Structure

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 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.

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 (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.

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.

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.