Publications by authors named "Ai-Hua Jin"

29 Publications

  • Page 1 of 1

Globular and ribbon isomers of Conus geographus α-conotoxins antagonize human nicotinic acetylcholine receptors.

Biochem Pharmacol 2021 May 29;190:114638. Epub 2021 May 29.

Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW 2522, Australia. Electronic address:

The short disulfide-rich α-conotoxins derived from the venom of Conus snails comprise a conserved CC(m)C(n)C cysteine framework (m and n, number of amino acids) and the majority antagonize nicotinic acetylcholine receptors (nAChRs). Depending on disulfide connectivity, α-conotoxins can exist as either globular (C-C, C-C), ribbon (C-C, C-C) or bead (C-C, C-C) isomers. In the present study, C. geographus α-conotoxins GI, GIB, G1.5 and G1.9 were chemically synthesized as globular and ribbon isomers and their activity investigated at human nAChRs expressed in Xenopus oocytes using the two-electrode voltage clamp recording technique. Both the globular and ribbon isomers of the 3/5 (m/n) α-conotoxins GI and GIB selectively inhibit heterologous human muscle-type α1β1δε nAChRs, whereas G1.5, a 4/7 α-conotoxin, selectively antagonizes neuronal (non-muscle) nAChR subtypes particularly human α3β2, α7 and α9α10 nAChRs. In contrast, globular and ribbon isomers of G1.9, a novel C-terminal elongated 4/8 α-conotoxin exhibited no activity at the human nAChR subtypes studied. This study reinforces earlier observations that 3/5 α-conotoxins selectively target the muscle nAChR subtypes, although interestingly, GIB is also active at α7 and α9 α10 nAChRs. The 4/7 α-conotoxins target human neuronal nAChR subtypes whereas the pharmacology of the 4/8 α-conotoxin remains unknown.
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http://dx.doi.org/10.1016/j.bcp.2021.114638DOI Listing
May 2021

Venom chemistry underlying the painful stings of velvet ants (Hymenoptera: Mutillidae).

Cell Mol Life Sci 2021 May 10. Epub 2021 May 10.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.

Velvet ants (Hymenoptera: Mutillidae) are a family of solitary parasitoid wasps that are renowned for their painful stings. We explored the chemistry underlying the stings of mutillid wasps of the genus Dasymutilla Ashmead. Detailed analyses of the venom composition of five species revealed that they are composed primarily of peptides. We found that two kinds of mutillid venom peptide appear to be primarily responsible for the painful effects of envenomation. These same peptides also have defensive utility against invertebrates, since they were able to incapacitate and kill honeybees. Both act directly on cell membranes where they directly increase ion conductivity. The defensive venom peptides of Dasymutilla bear a striking similarity, in structure and mode of action, to those of the ant Myrmecia gulosa (Fabricius), suggesting either retention of ancestral toxins, or convergence driven by similar life histories and defensive selection pressures. Finally, we propose that other highly expressed Dasymutilla venom peptides may play a role in parasitisation, possible in delay or arrest of host development. This study represents the first detailed account of the composition and function of the venoms of the Mutillidae.
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http://dx.doi.org/10.1007/s00018-021-03847-1DOI Listing
May 2021

Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from .

Mar Drugs 2021 Jan 26;19(2). Epub 2021 Jan 26.

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4072, Australia.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I-subfamily was identified as the major component of the predatory venom of piscivorous . Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded β-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.
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http://dx.doi.org/10.3390/md19020060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912261PMC
January 2021

Pharmacological activity and NMR solution structure of the leech peptide HSTX-I.

Biochem Pharmacol 2020 11 7;181:114082. Epub 2020 Jun 7.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia; National Cancer Institute, National Institutes of Health, Frederick, MD 21702, United States. Electronic address:

The role of voltage-gated sodium (Na) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms Na1.8 and Na1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat Na1.8 and mouse Na1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant Na isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human Na1.8 and Na1.9, and lacked analgesic efficacy in a murine model of inflammatory pain.
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http://dx.doi.org/10.1016/j.bcp.2020.114082DOI Listing
November 2020

Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of Na1.7.

Biomedicines 2020 Feb 19;8(2). Epub 2020 Feb 19.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (Na). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula that modulate Na channels. Pme1a is a 35 residue peptide that inhibits Na1.7 peak current (IC 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits Na1.7 peak current (EC > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at Na1.7 (IC 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at Na channels in the future.
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http://dx.doi.org/10.3390/biomedicines8020037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167818PMC
February 2020

Conotoxins: Chemistry and Biology.

Chem Rev 2019 11 21;119(21):11510-11549. Epub 2019 Oct 21.

Institute for Molecular Bioscience , The University of Queensland , Brisbane Queensland 4072 , Australia.

The venom of the marine predatory cone snails (genus ) has evolved for prey capture and defense, providing the basis for survival and rapid diversification of the now estimated 750+ species. A typical venom contains hundreds to thousands of bioactive peptides known as conotoxins. These mostly disulfide-rich and well-structured peptides act on a wide range of targets such as ion channels, G protein-coupled receptors, transporters, and enzymes. Conotoxins are of interest to neuroscientists as well as drug developers due to their exquisite potency and selectivity, not just against prey but also mammalian targets, thereby providing a rich source of molecular probes and therapeutic leads. The rise of integrated venomics has accelerated conotoxin discovery with now well over 10,000 conotoxin sequences published. However, their structural and pharmacological characterization lags considerably behind. In this review, we highlight the diversity of new conotoxins uncovered since 2014, their three-dimensional structures and folds, novel chemical approaches to their syntheses, and their value as pharmacological tools to unravel complex biology. Additionally, we discuss challenges and future directions for the field.
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http://dx.doi.org/10.1021/acs.chemrev.9b00207DOI Listing
November 2019

Novel conorfamides from Conus austini venom modulate both nicotinic acetylcholine receptors and acid-sensing ion channels.

Biochem Pharmacol 2019 06 24;164:342-348. Epub 2019 Apr 24.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia; School of Pharmacy, The University of Queensland, Wooloongabba, Queensland 4102, Australia. Electronic address:

Conorfamides are a poorly studied family of cone snail venom peptides with broad biological activities, including inhibition of glutamate receptors, acid-sensing ion channels, and voltage-gated potassium channels. The aim of this study was to characterize the pharmacological activity of two novel linear conorfamides (conorfamide_As1a and conorfamide_As2a) and their non-amidated counterparts (conopeptide_As1b and conopeptide_As2b) that were isolated from the venom of the Mexican cone snail Conus austini. Although As1a, As2a, As1b and As2b were identified by activity-guided fractionation using a high-throughput fluorescence imaging plate reader (FLIPR) assay assessing α7 nAChR activity, sequence determination revealed activity associated with four linear peptides of the conorfamide rather than the anticipated α-conotoxin family. Pharmacological testing revealed that the amidated peptide variants altered desensitization of acid-sensing ion channels (ASICs) 1a and 3, and the native lysine to arginine mutation differentiating As1a and As1b from As2a and As2b introduced ASIC1a peak current potentiation. Surprisingly, these conorfamides also inhibited α7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. This is the first report of conorfamides with dual activity, with the nAChR activity being the most potent molecular target of any conorfamide discovered to date.
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http://dx.doi.org/10.1016/j.bcp.2019.04.025DOI Listing
June 2019

Transcriptomic-Proteomic Correlation in the Predation-Evoked Venom of the Cone Snail, .

Mar Drugs 2019 Mar 19;17(3). Epub 2019 Mar 19.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia.

Individual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between individual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of . Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly diverse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to diversifying selection.
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http://dx.doi.org/10.3390/md17030177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471084PMC
March 2019

Venomics Reveals Venom Complexity of the Piscivorous Cone Snail, .

Mar Drugs 2019 Jan 21;17(1). Epub 2019 Jan 21.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4068, Australia.

The piscivorous cone snail has evolved a net-hunting strategy, akin to the deadly , and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two specimens revealed striking differences in conopeptide expression levels (2.5-fold) between individuals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, venom comprised both paralytic (putative ion channel modulating α-, ω-, μ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of
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http://dx.doi.org/10.3390/md17010071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356538PMC
January 2019

Hypoglycemic property of soy isoflavones from hypocotyl in Goto-Kakizaki diabetic rats.

J Clin Biochem Nutr 2018 Mar 12;62(2):148-154. Epub 2017 Dec 12.

Department of Biochemistry and Molecular Biology, Yanbian University Medical College, Yanji 133002, China.

The present study was carried out to investigate the hypoglycemic effect of soy isoflavones from hypocotyl in GK diabetic rats. A single administration and long-term administration tests were conducted in GK diabetic rats to test the hypoglycemic effect of soy isoflavones. At the end of long-term administration trial, blood protein, cholesterol, triglyceride, glycosylated serum protein, C-reactive protein, insulin, aminotransferase, lipid peroxide, interleukin-6, tumor necrosis factor-α were estimated. Inhibition of soy isoflavones against α-amylase and α-glucosidase, as well as on glucose uptake into brush border membrane vesicles or Caco-2 cells were determined . In single administration experiment, soy isoflavones reduced postprandial blood glucose levels in GK rats. In long-term administration, hypoglycemic effect of soy isoflavones was first observed at week 12 and maintained till week 16. A significant reduction in fasting blood glucose, C-reactive protein, and lipid peroxide was noted at week 16. However, there was no significant treatment effect on blood insulin. Furthermore, soy isoflavone administration resulted in significant decreases in glycosylated serum protein, tumor necrosis factor-α, and interleukin-6. Other biochemical parameters, such as protein, cholesterol, triglyceride and aminotransferases were not modified, however. The results showed that soy isoflavones showed a potent inhibitory effect on intestinal α-glucosidase, but not on pancreatic α-amylase. Soy isoflavones also decreased glucose transport potency into brush border membrane vesicles or Caco-2 cells. It is concluded that soy isoflavones from hypocotyl, performs hypoglycemic function in GK rats with type 2 diabetes, maybe via suppression of carbohydrate digestion and glucose uptake in small intestine.
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http://dx.doi.org/10.3164/jcbn.17-68DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874235PMC
March 2018

[Rhaponticum uniflorum inhibits H2O2-induced apoptosis of liver cells via JNK and NF-κB pathways].

Zhongguo Zhong Yao Za Zhi 2017 Mar;42(6):1189-1193

Medical College, Yanbian University, Yanji 133002, China.

To study the inhibitory effect of Rhaponticum uniflorum on apoptosis induced by H2O2 in HepG2 cells. Human HepG2 cells injury models were established by H2O2, then cell survival rate was assayed by MTT method; levels of LDH, ALT, and AST were detected by chemical colorimetric method;SOD activity was detected by xanthine oxidase method; GSH content was detected by dithio-bis-nitrobenzoic acid(DTNB); MDA level was detected by thiobarbituric acid (TBA) method;and the relative activities of Caspase-3, 8 and 9 were measured by Colorimetry. The expression levels of Cleaved Caspase-3(Casp-3), cytochrome(Cyto c), NF-κB, ERK, JNK, p38 MAPK, as well as the phospharylated proteins were determined with Western blotting method. The results showed that R. unifloru had no significant effect on cell viabilities of HepG2 cells at the concentrations of 25-400 mg•L⁻¹. However, H2O2decreased the cell viabilities, increased the cellular oxidative stress, and up-regulated the protein expressions of Casp-3, cytoplasmic Cyto c, p-JNK and nuclear NF-κB. As compared with the model group,R. unifloru could increase the cell viability, reduce LDH, ALT and AST leakage, reduce the MDA formation, increase the SOD and GSH levels,reduce the relative activities of Caspase-3, 8 and 9, down-regulated the protein expressions of Casp-3 and cytoplasmic Cyto c, and down-regulate the p-JNK and nuclear NF-κB levels.The results indicated that R. unifloru had the inhibitory effect on apoptosis induced by H2O2in HepG2 cells, and the mechanism maybe associated with inhibiting JNK activation and NF-κB nuclear translocation.
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http://dx.doi.org/10.19540/j.cnki.cjcmm.20170121.013DOI Listing
March 2017

Conotoxin Φ-MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti-Apoptotic Activity.

Angew Chem Int Ed Engl 2017 11 24;56(47):14973-14976. Epub 2017 Oct 24.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, 4072, Australia.

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC 17.85 μm) while inhibiting apoptosis (EC 2.2 μm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors.
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http://dx.doi.org/10.1002/anie.201708927DOI Listing
November 2017

Isolation and characterization of a structurally unique β-hairpin venom peptide from the predatory ant Anochetus emarginatus.

Biochim Biophys Acta 2016 11 28;1860(11 Pt A):2553-2562. Epub 2016 Jul 28.

VenomeTech, 473 Route des Dolines, 06560 Valbonne, France.

Background: Most ant venoms consist predominantly of small linear peptides, although some contain disulfide-linked peptides as minor components. However, in striking contrast to other ant species, some Anochetus venoms are composed primarily of disulfide-rich peptides. In this study, we investigated the venom of the ant Anochetus emarginatus with the aim of exploring these novel disulfide-rich peptides.

Methods: The venom peptidome was initially investigated using a combination of reversed-phase HPLC and mass spectrometry, then the amino acid sequences of the major peptides were determined using a combination of Edman degradation and de novo MS/MS sequencing. We focused on one of these peptides, U1-PONTX-Ae1a (Ae1a), because of its novel sequence, which we predicted would form a novel 3D fold. Ae1a was chemically synthesized using Fmoc chemistry and its 3D structure was elucidated using NMR spectroscopy. The peptide was then tested for insecticidal activity and its effect on a range of human ion channels.

Results: Seven peptides named poneritoxins (PONTXs) were isolated and sequenced. The three-dimensional structure of synthetic Ae1a revealed a novel, compact scaffold in which a C-terminal β-hairpin is connected to the N-terminal region via two disulfide bonds. Synthetic Ae1a reversibly paralyzed blowflies and inhibited human L-type voltage-gated calcium channels (CaV1).

Conclusions: Poneritoxins from Anochetus emarginatus venom are a novel class of toxins that are structurally unique among animal venoms.

General Significance: This study demonstrates that Anochetus ant venoms are a rich source of novel ion channel modulating peptides, some of which might be useful leads for the development of biopesticides.
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http://dx.doi.org/10.1016/j.bbagen.2016.07.027DOI Listing
November 2016

Transcriptome and proteome of Conus planorbis identify the nicotinic receptors as primary target for the defensive venom.

Proteomics 2015 Dec 17;15(23-24):4030-40. Epub 2015 Nov 17.

The Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia.

Most venomous predators have evolved complex venom primarily to immobilize their prey and secondarily to defend against predators. In a new paradigm, carnivorous marine gastropods of the genus Conus were shown to rapidly and reversibly switch between two types of venoms in response to predatory or defensive stimulus, suggesting that the defensive use of venom may have a more important role in venom evolution and specialization than previously thought. To further investigate this phenomenon, the defensive repertoire of a vermivorous species, Conus planorbis, was deciphered using second-generation sequencing coupled to high-throughput proteomics. The venom gland transcriptome of C. planorbis revealed 182 unique conotoxin precursors from 25 gene superfamilies, with superfamily T dominating in terms of read and paralog numbers. Analysis of the defense-evoked venom revealed that this vermivorous species uses a similarly complex arsenal to deter aggressors as more recently evolved fish- and mollusk-hunting species, with MS/MS validating 23 conotoxin sequences from six superfamilies. Pharmacological characterization of the defensive venom on human receptors identified the nicotinic acetylcholine receptors as a primary target. This work provides the first insights into the composition and biological activity of specifically evolved defensive venoms in vermivorous cone snails.
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http://dx.doi.org/10.1002/pmic.201500220DOI Listing
December 2015

Comparative Venomics Reveals the Complex Prey Capture Strategy of the Piscivorous Cone Snail Conus catus.

J Proteome Res 2015 Oct 10;14(10):4372-81. Epub 2015 Sep 10.

Institute for Molecular Bioscience, The University of Queensland , Brisbane, 4072 Queensland, Australia.

Venomous marine cone snails produce a unique and remarkably diverse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.
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http://dx.doi.org/10.1021/acs.jproteome.5b00630DOI Listing
October 2015

δ-Conotoxin SuVIA suggests an evolutionary link between ancestral predator defence and the origin of fish-hunting behaviour in carnivorous cone snails.

Proc Biol Sci 2015 Jul;282(1811)

The Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia Institut des Biomolécules Max Mousseron, UMR 5247, Université Montpellier - CNRS, Place Eugène Bataillon, Montpellier Cedex 5 34095, France

Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition.
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http://dx.doi.org/10.1098/rspb.2015.0817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528551PMC
July 2015

α-Conotoxin MrIC is a biased agonist at α7 nicotinic acetylcholine receptors.

Biochem Pharmacol 2015 Mar 31;94(2):155-63. Epub 2015 Jan 31.

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia; School of Pharmacy, The University of Queensland, Woolloongabba, Queensland 4102, Australia. Electronic address:

MrIC is a recently described selective agonist of endogenously expressed α7 nAChR. In this study, we further characterize the pharmacological activity of MrIC using Ca(2+) imaging approaches in SH-SY5Y cells endogenously expressing α7 nAChR and demonstrate that MrIC exclusively activates α7 nAChR modulated by type II positive allosteric modulators, including PNU120596. MrIC was a full agonist at PNU120596-modulated α7 nAChR compared with choline, albeit with slower kinetics, but failed to elicit a Ca(2+) response in the absence of PNU120596. Interestingly, the NMR structure of MrIC showed a typical 4/7 α-conotoxin fold, indicating that its unusual pharmacological activity is likely sequence-dependent. Overall, our results suggest that MrIC acts as a biased agonist that can only activate α7 nAChR modified by type II positive allosteric modulators, and thus represents a valuable tool to probe the pharmacological properties of this important ion channel.
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http://dx.doi.org/10.1016/j.bcp.2015.01.011DOI Listing
March 2015

Cone snail venomics: from novel biology to novel therapeutics.

Future Med Chem 2014 Oct;6(15):1659-75

Centre for Pain Research, Institute for Molecular Bioscience, University of Queensland, Queensland, Australia.

Peptide neurotoxins from cone snails called conotoxins are renowned for their therapeutic potential to treat pain and several neurodegenerative diseases. Inefficient assay-guided discovery methods have been replaced by high-throughput bioassays integrated with advanced MS and next-generation sequencing, ushering in the era of 'venomics'. In this review, we focus on the impact of venomics on the understanding of cone snail biology as well as the application of venomics to accelerate the discovery of new conotoxins. We also discuss the continued importance of medicinal chemistry approaches to optimize conotoxins for clinical use, with a descriptive case study of MrIA featured.
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http://dx.doi.org/10.4155/fmc.14.99DOI Listing
October 2014

Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose.

Toxicon 2014 Dec 7;91:135-44. Epub 2014 Oct 7.

Institute for Molecular Bioscience, The University of Queensland, 4072 Queensland, Australia.

Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have been used to determine animal LD50 and to aid the isolation of several potent paralytic toxins. However, not only is the composition of injected venoms known to differ significantly from that in dissected venom glands, but also to vary according to predatory or defensive stimuli. Therefore, to study the venom that is directly relevant to human envenomation, the defense-evoked venom of several specimens of C. geographus was collected and analyzed by standard LC-MS methods. The molecular composition of individual defense-evoked venom showed significant intraspecific variations, but a core of paralytic conotoxins including α-GI, α-GII, μ-GIIIA, ω-GVIA and ω-GVIIA was always present in large amounts, consistent with the symptomology and high fatality rate in humans. Differences between injected and dissected venoms obtained from the same specimen were also evident. Interestingly, an apparent linear correlation between the dry weight/volume of injected venom and the size of the shell allowed extrapolation to a human lethal dose (0.038-0.029 mg/kg) from an historic fatal case of C. geographus envenomation, which may help in the management of future victims.
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http://dx.doi.org/10.1016/j.toxicon.2014.09.011DOI Listing
December 2014

Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails.

Nat Commun 2014 Mar 24;5:3521. Epub 2014 Mar 24.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, 4072 Queensland, Australia.

Venomous animals are thought to inject the same combination of toxins for both predation and defence, presumably exploiting conserved target pharmacology across prey and predators. Remarkably, cone snails can rapidly switch between distinct venoms in response to predatory or defensive stimuli. Here, we show that the defence-evoked venom of Conus geographus contains high levels of paralytic toxins that potently block neuromuscular receptors, consistent with its lethal effects on humans. In contrast, C. geographus predation-evoked venom contains prey-specific toxins mostly inactive at human targets. Predation- and defence-evoked venoms originate from the distal and proximal regions of the venom duct, respectively, explaining how different stimuli can generate two distinct venoms. A specialized defensive envenomation strategy is widely evolved across worm, mollusk and fish-hunting cone snails. We propose that defensive toxins, originally evolved in ancestral worm-hunting cone snails to protect against cephalopod and fish predation, have been repurposed in predatory venoms to facilitate diversification to fish and mollusk diets.
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http://dx.doi.org/10.1038/ncomms4521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973120PMC
March 2014

MrIC, a novel α-conotoxin agonist in the presence of PNU at endogenous α7 nicotinic acetylcholine receptors.

Biochemistry 2014 Jan 30;53(1):1-3. Epub 2013 Dec 30.

Institute for Molecular Bioscience, The University of Queensland , Brisbane, QLD 4072, Australia , and Discipline of Pharmacology, The University of Sydney , Sydney, NSW 2006, Australia.

α-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Their high selectivity and affinity for the various subtypes of nAChRs have led to significant advances in our understanding of the structure and function of these key ion channels. Here we report the discovery of a novel 4/7 α-conotoxin, MrIC from the venom duct of Conus marmoreus, which acts as an agonist at the endogenous human α7 nAChR in SH-SY5Y cells pretreated with PNU120596 (PNU). This unique agonist activity of MrIC at α7 nAChRs may guide the development of novel α7 nAChR modulators.
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http://dx.doi.org/10.1021/bi400882sDOI Listing
January 2014

Systematic interrogation of the Conus marmoreus venom duct transcriptome with ConoSorter reveals 158 novel conotoxins and 13 new gene superfamilies.

BMC Genomics 2013 Oct 16;14:708. Epub 2013 Oct 16.

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.

Background: Conopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining.

Results: Here we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies.

Conclusions: Taken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.
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http://dx.doi.org/10.1186/1471-2164-14-708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853152PMC
October 2013

Transcriptomic messiness in the venom duct of Conus miles contributes to conotoxin diversity.

Mol Cell Proteomics 2013 Dec 16;12(12):3824-33. Epub 2013 Sep 16.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia.

Marine cone snails have developed sophisticated chemical strategies to capture prey and defend themselves against predators. Among the vast array of bioactive molecules in their venom, peptide components called conotoxins or conopeptides dominate, with many binding with high affinity and selectivity to a broad range of cellular targets, including receptors and transporters of the nervous system. Whereas the conopeptide gene precursor organization has a conserved topology, the peptides in the venom duct are highly processed. Indeed, deep sequencing transcriptomics has uncovered on average fewer than 100 toxin gene precursors per species, whereas advanced proteomics has revealed >10-fold greater diversity at the peptide level. In the present study, second-generation sequencing technologies coupled to highly sensitive mass spectrometry methods were applied to rapidly uncover the conopeptide diversity in the venom of a worm-hunting species, Conus miles. A total of 662 putative conopeptide encoded sequences were retrieved from transcriptomic data, comprising 48 validated conotoxin sequences that clustered into 10 gene superfamilies, including 3 novel superfamilies and a novel cysteine framework (C-C-C-CCC-C-C) identified at both transcript and peptide levels. A surprisingly large number of conopeptide gene sequences were expressed at low levels, including a series of single amino acid variants, as well as sequences containing deletions and frame and stop codon shifts. Some of the toxin variants generate alternative cleavage sites, interrupted or elongated cysteine frameworks, and highly variable isoforms within families that could be identified at the peptide level. Together with the variable peptide processing identified previously, background genetic and phenotypic levels of biological messiness in venoms contribute to the hypervariability of venom peptides and their ability to evolve rapidly.
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http://dx.doi.org/10.1074/mcp.M113.030353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861727PMC
December 2013

Deep venomics reveals the mechanism for expanded peptide diversity in cone snail venom.

Mol Cell Proteomics 2013 Feb 14;12(2):312-29. Epub 2012 Nov 14.

The Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia.

Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.
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http://dx.doi.org/10.1074/mcp.M112.021469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567856PMC
February 2013

ConoServer: updated content, knowledge, and discovery tools in the conopeptide database.

Nucleic Acids Res 2012 Jan 3;40(Database issue):D325-30. Epub 2011 Nov 3.

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

ConoServer (http://www.conoserver.org) is a database specializing in the sequences and structures of conopeptides, which are toxins expressed by marine cone snails. Cone snails are carnivorous gastropods, which hunt their prey using a cocktail of toxins that potently subvert nervous system function. The ability of these toxins to specifically target receptors, channels and transporters of the nervous system has attracted considerable interest for their use in physiological research and as drug leads. Since the founding publication on ConoServer in 2008, the number of entries in the database has nearly doubled, the interface has been redesigned and new annotations have been added, including a more detailed description of cone snail species, biological activity measurements and information regarding the identification of each sequence. Automatically updated statistics on classification schemes, three-dimensional structures, conopeptide-bearing species and endoplasmic reticulum signal sequence conservation trends, provide a convenient overview of current knowledge on conopeptides. Transcriptomics and proteomics have began generating massive numbers of new conopeptide sequences, and two dedicated tools have been recently implemented in ConoServer to standardize the analysis of conopeptide precursor sequences and to help in the identification by mass spectrometry of toxins whose sequences were predicted at the nucleic acid level.
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http://dx.doi.org/10.1093/nar/gkr886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245185PMC
January 2012

Molecular engineering of conotoxins: the importance of loop size to alpha-conotoxin structure and function.

J Med Chem 2008 Sep;51(18):5575-84

Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072 Australia.

Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.
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http://dx.doi.org/10.1021/jm800278kDOI Listing
September 2008

Structure of alpha-conotoxin BuIA: influences of disulfide connectivity on structural dynamics.

BMC Struct Biol 2007 Apr 20;7:28. Epub 2007 Apr 20.

Institute for Molecular Bioscience, Australian Research Council Special Research Centre for Functional and Applied Genomics, The University of Queensland, Brisbane QLD, Australia.

Background: Alpha-conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in alpha-conotoxins is variable, leading to the classification of sub-families. BuIA is the only alpha-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin.

Results: In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer.

Conclusion: These findings are opposite to the general trends observed for alpha-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure.
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http://dx.doi.org/10.1186/1472-6807-7-28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865545PMC
April 2007

[CDNA cloning of human leptin and its expression].

Sheng Wu Gong Cheng Xue Bao 2003 Jul;19(4):476-9

Zhejiang Academy of Medical Sciences, Hangzhou 310013, China.

To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.
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July 2003

[Practicability study on a group of vigilant chemical compounds including chlorheridine diacetate].

Zhonghua Nan Ke Xue 2002 ;8(5):329-31

Family Planning Research Institute, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, China.

Objectives: To test in vitro the spermatozocidine drug which can also prevent sex transmitting diseases (STD) pathogens.

Methods: Chlorheridine diacetate and other three chemical compounds were applied in vitro spermatozocidine and sperm inhibitting tests.

Results: The lowest concentrations of chlorheridine diacetate and p-nitrophenol which can inhibit human sperm in 20 seconds were 1.25 mg/ml. The minimal inhibitory concentration and minimal bactericidal concentration of chlorheridine diacetate and p-nitrophenol on Streptococcus albus Stemberg were 0.125 to 0.50 mg/ml and 0.25 to 1.00 mg/ml.

Conclusions: Chlorheridine diacetate and p-uitrophenol have strong spermatozocidine and antibacteria effects.
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January 2003