Discovery and characterization of NaV modulatory venom peptides

Wingerd, Joshua Seth (2013). Discovery and characterization of NaV modulatory venom peptides PhD Thesis, Institute for Molecular Bioscience, The University of Queensland. doi:10.14264/uql.2017.342

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Author Wingerd, Joshua Seth
Thesis Title Discovery and characterization of NaV modulatory venom peptides
Formatted title
Discovery and characterization of NaV modulatory venom peptides
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
DOI 10.14264/uql.2017.342
Publication date 2013-01-01
Thesis type PhD Thesis
Supervisor Richard Lewis
Irina Vetter
Lachlan Rash
Total pages 217
Total colour pages 41
Total black and white pages 176
Language eng
Subjects 110106 Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics)
111501 Basic Pharmacology
060112 Structural Biology (incl. Macromolecular Modelling)
Formatted abstract
Voltage-gated sodium channels (NaV) are integral membrane proteins that are responsible for the increase in sodium permeability that initiates and propagates the rising phase of action potentials, carrying electrical signals along nerve fibers and through excitable cells. NaV channels play a diverse role in neurophysiology and neurotransmission, as well as serving as molecular targets for several groups of neurotoxins that bind to different receptor sites and alter voltage-dependent activation, inactivation and conductance. There are nine NaV channel isoforms so far discovered, each of which display distinct functional profiles and tissue-specific expression patterns. The modulation of specific isoforms for therapeutic purposes has become an important research objective for the treatment of conductance diseases exhibiting phenotypes of chronic pain, epilepsy, myotonia, seizure, and cardiac arrhythmia. However, because of the high sequence similarity and structural homology between NaV channel isoforms, many current therapeutics that target NaV channels – the vast majority of which are small molecules – lack specificity between isoforms, or even other voltage-gated ion channels. The current push for greater selectivity while maintaining a relevant degree of potency has led the focus away from small molecules and towards the discovery and development of peptidic ligands for therapeutic use. Venom derived peptides have proven to be naturally potent and selective bioactive molecules, exhibiting inherent secondary structures that add stability through the formation of disulfide bonds. Organisms such as cone snails and spiders have evolved venom for the purpose of prey capture and defense, therefore many of the components exhibit paralytic qualities and specifically target NaV channels. Much of the discovery process has focused on screening crude venoms for a particular function and then isolating the molecule responsible for that function using assay guided purification.

The first section of this thesis describes the development of a cell-based, high-throughput functional assay for the detection of NaV modulating venom peptides in crude venom. This assay was successfully implemented and resulted in the isolation and sequencing of MVIA from Conus magus. Initial results and sequence similarity placed MVIA into the δ-conotoxin family, a poorly described class of peptides that inhibits fast inactivation. However, multiple solid-phase synthesis and bacterial recombinant expression methods were unsuccessful in producing enough of the extremely hydrophobic δ-MVIA for further characterization. As no more C. magus crude venom was available, this project was left until optimized methods of production for highly hydrophobic peptides could be developed.

An optimized method of bacterial recombinant expression was successful in producing large yields of another venom peptide, β/δ-TRTX-Pre1a, isolated from the spider Psalmopoeus reduncus. The same recombinant expression method was also used to produce uniformly labeled 13C/15N-peptide for determination of a heteronuclear NMR solution structure. Pre1a was revealed to be a sub-micromolar inhibitor of both NaV1.2 and NaV1.7 peak currents, yet demonstrated potent inhibition of fast inactivation of NaV1.3. This dual mode of action on different NaV isoforms had not yet been reported for any known venom peptide. Further, Pre1a exhibited structural heterogeneity as determined by analysis of rpHPLC and NMR 15N-HSQC, which was traced to the contribution of residues making up the all aromatic, hydrophobic face of the peptide.

The high sequence similarity of Pre1a to previously discovered spider venom peptides allowed comparative functional analysis and the identification of key residues contributing to NaV isoform selectivity. Mutagenesis focusing on both structural and functional aspects of Pre1a was performed incorporating both alanine and non-alanine substitutions. Through a single mutation a residue critical for the observed conformational heterogeneity was identified. This mutation served as a model for the obtainment of a high-resolution solution structure for comparison with the active Pre1a. Lastly, we identified a single residue on the C-terminus critical for NaV channel isoform selectivity. Substitution with amino acids exhibiting different properties of charge, polarity, and size could modify selectivity and in the process create a highly selective inhibitor of NaV1.2. This study not only revealed a unique mode of action for a venom peptide, but also demonstrated novelty as a proof-of-concept for the rational design and engineering of venom peptides using available information and non-standard methods of selective mutagenesis.
Keyword Spider and cone snail venom toxins
Sodium channels
Inhibitory cystine knot
Recombinant Expression
Structure activity relationships
Assay development
Two electrode voltage clamp

Document type: Thesis
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Created: Tue, 20 May 2014, 21:03:06 EST by Joshua Wingerd on behalf of Learning and Research Services (UQ Library)