ω-Lsp-IA, a novel modulator of P-type Ca2+ channels

Pluzhnikov, Kirill A.; Vassilevski, Alexander A.; Korolkova, Yuliya V.; Fisyunov, Alexander; Iegorova, Olena; Krishtal, O. A.; Grishin, Eugene V.

doi:10.1016/j.toxicon.2007.07.004

Cited by 30 publications

(23 citation statements)

References 37 publications

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“…The selective elution of monomeric fusion proteins using IMAC offers an easy solution for isolation of correctly folded material. The yield of HwTx-IV(acid) in this system was 0.9mg/L, which is similar to other ICKs expressed in recombinant systems and purified to obtain a homogeneous product [10,11,14,15]. However, the folding propensity of each ICK is different and will affect the total yield.…”

Section: Discussionsupporting

confidence: 52%

“…During the preparation of this manuscript, two reports of ICKs expressed in an E. coli strain engineered to enable cytoplasmic disulfide-bond formation were published [11,23]. Pluzhnikov et al used Origami B to express ω-Lsp-IA as a fusion to thioredoxin in order to increase the yield of folded ICK [11].…”

Section: Discussionmentioning

confidence: 99%

“…Pluzhnikov et al used Origami B to express ω-Lsp-IA as a fusion to thioredoxin in order to increase the yield of folded ICK [11]. Nozach et al have compared the expression of six ICKs in SHuffle T7 Express lysY and Origami B(DE3)pLysS using various fusion partners, including DsbC and thioredoxin [23].…”

Section: Discussionmentioning

confidence: 99%

“…Structurally, HwTx-IV is very similar to HwTx-I, but whereas HwTx-I seems to fold readily in several recombinant systems [9,10,14,17], HwTx-IV does not (unpublished findings). E. coli strains engineered for cytoplasmic expression and folding of disulfide-rich proteins, like Origami B and SHuffle, are another alternative to express folded ICKs [11,23–26]. …”

Section: Introductionmentioning

confidence: 99%

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Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV

Sermadiras

Revell

Linley³

et al. 2013

PLoS ONE

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Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express lysY Escherichia coli strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Nav1.7 in a dose dependent manner (IC50 = 463-727 nM). In comparison to HwTx-IV(amide) (IC50 = 11 ± 3 nM), the carboxylate was ~50 fold less potent on Nav1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IVG36(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Nav1.7 in comparison to HwTx-IV(acid) (IC50 = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.

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Section: Discussionsupporting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV

Sermadiras

Revell

Linley³

et al. 2013

PLoS ONE

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“…Therefore, these homologs can be considered as references for the function prediction of the toxins from L. vittata. For example, the toxins in the families F and G show high sequence identity with u-agatoxin-1A and u-LSP-1A, two blockers of VGCCs, respectively (Pluzhnikov et al, 2007;Scott et al, 1990), indicating their functions might be potentially correlated with VGCC blockage. This prediction was consistent with the data that the venom was able to suppress the Ca 2þ currents in rat DRG neurons.…”

Section: Transcriptomic Analysis Of Venom Peptides From L Vittatamentioning

confidence: 99%

A survey of the venom of the spider Lycosa vittata by biochemical, pharmacological and transcriptomic analyses

Zhang

Liu

Tan

et al. 2015

Toxicon

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Lycosa vittata, mainly distributed in the southwest of China, is a medium-sized and venomous spider, whose venom remains unexplored so far. This study aims to present an overview of the venom. It mainly consisted of diverse peptides and exhibited inhibitory effects on voltage-gated ion channels in rat dorsal root ganglia neurons, with a strongest inhibition on tetrodotoxin-sensitive and tetrodotoxin-resistant voltage-gated Na(+) channels. Interestingly, it exerted cytotoxicity to cancer cells, with approximately 10-fold selectivity on PC-3 over others, implying the existence of selective anti-PC-3 agents in the venom. Moreover, 51 toxin-like peptides were deduced from the venom gland transcriptome. Bioinformatic analyses suggested their structures might have some distinguished properties and their predicted functions were consistent with the venom activities. This study suggests that the venom is an attractive source of neurotoxins with therapeutic significance, and provides references for the structure and function investigation of specific toxins in the future.

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Scorpion toxins interact with nicotinic acetylcholine receptors

et al. 2019

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Neurotoxins are among the main components of scorpion and snake venoms. Scorpion neurotoxins affect voltage‐gated ion channels, while most snake neurotoxins target ligand‐gated ion channels, mainly nicotinic acetylcholine receptors (nAChRs). We report that scorpion venoms inhibit α‐bungarotoxin binding to both muscle‐type nAChR from Torpedo californica and neuronal human α7 nAChR. Toxins inhibiting nAChRs were identified as OSK‐1 (α‐KTx family) from Orthochirus scrobiculosus and HelaTx1 (κ‐KTx family) from Heterometrus laoticus, both being blockers of voltage‐gated potassium channels. With an IC50 of 1.6 μm, OSK1 inhibits acetylcholine‐induced current through mouse muscle‐type nAChR heterologously expressed in Xenopus oocytes. Other well‐characterized scorpion toxins from these families also bind to Torpedo nAChR with micromolar affinities. Our results indicate that scorpion neurotoxins present target promiscuity.

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ω-Lsp-IA, a novel modulator of P-type Ca2+ channels

Cited by 30 publications

References 37 publications

Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV

Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV

A survey of the venom of the spider Lycosa vittata by biochemical, pharmacological and transcriptomic analyses

Scorpion toxins interact with nicotinic acetylcholine receptors

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