Urodacus yaschenkoi, Australian scorpion: phylogeny, venom characterization and pharmacology
Loading...
Citations
Altmetric:
Altmetric::
Download
Associated Records
Authors
File Licence
Editors
Date
2014
Source Title
Journal Title
Journal ISSN
Volume Title
Publisher
Melbourne Authors
Affiliation
Pharmacology
Document Type
PhD thesis
Subject Code
Citation
Access Status
This item is currently not available from this repository
DOI
Access URL
Open Access URL
Open Access at PMC
Linked Resource URL
NHMRC Grant Code
ARC Grant Code
Description
© 2014 Dr. Karen Sofia Luna Ramirez
Abstract
Australia has over 40 species of scorpions organised in four families: Buthidae, Bothriuridae, Urodacidae and Liochelidae (old name Ischnuridae). Unlike their overseas counterparts, Australian scorpions have received little attention and there is a lack of knowledge about their venoms and phylogeny. Urodacus yaschenkoi belongs to the family Urodacidae of scorpions, the most widely distributed in Australia. In a multidisciplinary approach, the aims of this study were to: (i) verify if the Urodacus yaschenkoi scorpion is in fact one species via a phylogenetic study with molecular markers; (ii) fully understand and characterise the venom (using proteomic and genomic approaches); and (iii) determine if there are specific toxins in the venom of Urodacus yaschenkoi that may be useful as pharmacological tools or as drug leads.
The phylogenetic review of this species was conducted with 3 loci (2 mitochondrial and 1 nuclear genes) and suggested that the U. yaschenkoi population is in expansion and has given rise to at least two different clades where the Western Australian clade shows strong molecular divergence and some morphological difference. These results indicate that samples from WA need to be treated as a distinct group when performing toxinological characterisation. This study represents the first phylogenetic review of this species since Koch (1977) and the first one made with genetic analysis and not only with morphological characters.
The venom characterisation showed that U. yaschenkoi venom is as complex as other scorpion venoms; several bioactive molecules were identified and assayed. The venom profile rendered 74 fractions where approximately 250 different molecular masses were identified with molecular weights varying from 291 to 16,290 Da. The most abundant peptides were those from 1 and 4-5 KDa representing antimicrobial peptides and putative potassium channel toxins, respectively. Also, the construction of a cDNA library from a replete venom gland was performed. The average titre of the cDNA library obtained was 1 X 105 cfu/ml with 70% recombinant clones, but only 172 expressed sequence tags were analysed (based on a size >400 bp). These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins could be assorted in several groups such as precursors similar to gene products implicated in common cellular processes, others representing putative neurotoxins and antimicrobial peptides.
Further, characterisation of four antimicrobial peptides named UyCT peptides – was carried out. The activity of these peptides against Gram-positive, Gram-negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release of the fluorophore calcein from liposomes and isothermal titration calorimetry; their secondary structure was determined by circular dichroism. Three different lipid systems were used to mimic red blood cells, E. coli and S. aureus membranes. UyCT peptides exhibited broad-spectrum antimicrobial activity with low Minimal Inhibitory Concentrations for S. aureus and multi-drug resistant Gram-negative strains. Peptide combinations showed synergy enhancing their potency, but not haemolytic activity. These results suggest that UyCT peptides may be candidates for peptidomimetics to maximise their antibacterial activity.
Additionally, the first K+ channel toxin, called urotoxin, from an Australian scorpion was identified and studied. Urotoxin was tested against eight different potassium channels and proved to be a potent blocker of human Kv1.2 channels with an IC50 of 160.5 pM. Its affinity for hKv1.1 channels is in the order of 253.5 nM, while for hKv1.3 is 90.9 nM and KCa3.1 is 69.9 nM. Urotoxin has no effect on Kv1.4, Kv1.5, hERG1 and ELK2. By constructing the whole transcriptome by means of Next Generation Sequencing, six new members – isoforms – similar to urotoxin were also identified. The computer modelling of urotoxin’s 3D structure suggested the presence of the characteristic α/β-scaffold of other scorpion toxins, very likely forming a disulfide pairing similar to maurotoxin. Using molecular dynamics, a model for the binding of this peptide to human Kv1.2 and hKv1.1 channel is included. Urotoxin may still be considered a selective hKv1.2 blocker as a minimum of 100-fold difference in IC50 makes a toxin selective for a given channel. Urotoxin may be considered a leading pharmacological tool to study K+ channel subtypes.
Notes
Terms of Use
Keywords
toxinology, venom, scorpion, proteomics, cDNA library, antimicrobial, potassium channel blocker