Isolation and characterization of neuroactive peptides from the venom of cone snail species

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.