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Bioengineering alpha conotoxin ViI : from disulfide bonds to native chemical ligation

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Item Summary

Title: Bioengineering alpha conotoxin ViI : from disulfide bonds to native chemical ligation
Authors: Cabalteja, Chino Cabasa
Keywords: peptide isomers
Issue Date: Aug 2014
Publisher: [Honolulu] : [University of Hawaii at Manoa], [August 2014]
Abstract: The paradigm of rational drug design has broadened its focus from classical small-molecule drugs and into larger biological molecules (biologics). At the forefront of this change are peptide drugs with high potency, selectivity and efficacy toward a broader range of targets. Conotoxins isolated from the venom of marine cone snails have received much attention because they are highly specific and isoform selective towards their target receptors, making them ideal resources for potential drug leads.
Members of the α-conotoxin family have been extensively studied because they are competitive antagonists of nicotinic acetylcholine receptors (nAChRs) located in the central and peripheral nervous systems. Modulation of these receptors could aid in the control of pain and other associated pathologies. The characteristic receptor-ligand interaction of these peptides is contingent on their well-defined tertiary structure, which is stabilized by several disulfide bonds. In the venom duct, native α-conotoxins enzymatically fold into the globular conformation. However, permutation of the disulfide bond connections of α-conotoxins can theoretically yield three conformations/isomers: globular, ribbon, and beaded. In vitro studies on conotoxin folding have suggested that post-translational modifications (PTM) such as hydroxylated prolines aid in the folding of conotoxins. As such, it was hypothesized that elimination of this PTM would increase the likelihood of peptide isomer formation.
To investigate this connection, the novel peptide, α-conotoxin ViI from Conus virgo was utilized as it contained two hydroxyprolines at position 6 and 13 in its sequence. α-Conotoxin ViI and its non-post-translationally modified variant Vi1.1 were synthesized via fluorenylmethoxycarbonyl solid phase peptide synthesis. Random oxidation of Vi1.1 revealed two products of identical mass later identified as the ribbon and globular isomers. These isomers exhibited a degree of nAChR inhibition in a bovine chromaffin cell assay. Subsequently, both α-conotoxin ViI and Vi1.1 were allowed to oxidize in buffers containing chaotropic agents. It was found that oxidation of Vi1.1in various chaotropic agents increased the creation of non-native isomer as compared to oxidation in ammonium bicarbonate. Furthermore, oxidations of α-conotoxin ViI revealed that some portion of the peptides remained in the reduced form. This work provides an initial investigation for a long-term study on the connection between the generation of disulfide bond isomers and hydroxyprolines.
Efforts were also undertaken to connect the N-and C-terminal peptide fragments of the spider toxin Huwentoxin I via native chemical ligation. Assembly of the complete linear sequence of Huwentoxin I was subsequently verified through electrospray ionization mass spectrometry. This work will be utilized to create an intramolecular native chemical ligation strategy for the cyclization of α-conotoxin ViI and its analogues. Like their linear counterparts, these cyclic peptides will also be subject to further oxidation studies. Cyclized conotoxins display increased structural stability and resistance to enzymatic degradation and may be used to create orally-active conotoxin therapeutics. In all, the works presented in this thesis display a progression in conotoxin bioengineering to improve their pharmacological properties so that they may be developed as therapeutics.
Description: M.S. University of Hawaii at Manoa 2014.
Includes bibliographical references.
URI/DOI: http://hdl.handle.net/10125/100541
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections:M.S. - Molecular Biosciences and Bioengineering



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