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Recombinant expression and in vitro characterisation of active Huwentoxin-IV.

ABSTRACT: 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-IV(G36)(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.

SUBMITTER: Sermadiras I

PROVIDER: S-EPMC3855799 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Recombinant expression and in vitro characterisation of active Huwentoxin-IV.

Sermadiras Isabelle I Revell Jefferson J Linley John E JE Sandercock Alan A Ravn Peter P

PloS one 20131206 12

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 T ...[more]

PMID: 24324842

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Project description:Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider Cyriopagopus schmidti, has been reported to be a potent antinociceptive compound due to its action on the genetically-validated NaV1.7 pain target. Using this peptide for antinociceptive applications in vivo suffers from one major drawback, namely its negative impact on the neuromuscular system. Although studied only recently, this effect appears to be due to an interaction between the peptide and the NaV1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) NaV1.7/NaV1.6 selectivity ratio of 23. Nineteen HwTx-IV analogues were chemically synthesized and tested for their blocking effects on the Na+ currents flowing through these two channel subtypes stably expressed in cell lines. Dose-response curves for these analogues were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure-activity relationship (SAR) studies. Among the analogues tested, the potency of HwTx-IV E4K was significantly improved for hNaV1.6, leading to a decreased hNaV1.7/hNaV1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed for HwTx-IV analogues that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV E1G/E4G and HwTx-IV E4K/R26Q). In contrast, increased selectivity ratios (>46) were obtained if the E4K mutation was combined to an additional double substitution (R26A/Y33W) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNaV1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analogue for hNaV1.6 and/or hNaV1.7 subtypes. In addition, selective analogues for hNaV1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.

Project description:Venom-derived peptides have attracted much attention as potential lead molecules for pharmaceutical development. A well-known example is Huwentoxin-IV (HwTx-IV), a peptide toxin isolated from the venom of the Chinese bird-eating spider Haplopelma schmitdi. HwTx-IV was identified as a potent blocker of a human voltage-gated sodium channel (hNaV1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at NaV1.7 (IC50 ~26 nM) and selectivity over the cardiac NaV subtype (NaV1.5). Mutagenesis studies aimed at optimising the potency of the peptide resulted in the development of a triple-mutant of HwTx-IV (E1G, E4G, Y33W, m3-HwTx-IV) with significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. The activity of m3-HwTx-IV against other NaV subtypes was, however, not investigated. Similarly, the structure of the mutant peptide was not characterised, limiting the interpretation of the observed increase in potency. In this study we produced isotope-labelled recombinant m3-HwTx-IV in E. coli, which enabled us to characterise the atomic-resolution structure and dynamics of the peptide by NMR spectroscopy. The results show that the structure of the peptide is not perturbed by the mutations, whilst the relaxation studies reveal that residues in the active site of the peptide undergo conformational exchange. Additionally, the NaV subtype selectivity of the recombinant peptide was characterised, revealing potent inhibition of neuronal NaV subtypes 1.1, 1.2, 1.3, 1.6 and 1.7. In parallel to the in vitro studies, we investigated NaV1.7 target engagement of the peptide in vivo using a rodent pain model, where m3-HwTx-IV dose-dependently suppressed spontaneous pain induced by the NaV1.7 activator OD1. Thus, our results provide further insight into the structure and dynamics of this class of peptides that may prove useful in guiding the development of inhibitors with improved selectivity for analgesic NaV subtypes.

Project description:BACKGROUND:Human metapneumovirus (HMPV) has been shown to cause respiratory infection, accounting for approximately 7% of all such disease, and contributes to the development of asthma in humans. HMPV has a worldwide distribution with infectivity rates approaching 100%, and immunocompromised patients are particularly at risk from viral exposure. No anti-HMPV vaccine is available and diagnosis is primarily based on in-house molecular or serological tests, in part due to limited availability of recombinant HMPV antigens. OBJECTIVE:To generate a panel of HMPV-derived recombinant antigens, develop standardised ELISA systems for HMPV IgG detection and explore the nature of B cell memory against HMPV to underpin future vaccine studies. STUDY DESIGN:HMPV viral RNA was isolated from a clinical specimen and RT-PCR was conducted. The HMPV M and P genes were cloned and expressed in Escherichia coli. The HMPV N gene was cloned and expressed in insect cells using the baculovirus expression system. Each purified recombinant antigens was subsequently employed in HMPV-specific ELISA. RESULTS:High-level expression, and purification, of both HMPV matrix (M) (10 mg/g cells) and phosphoprotein (P) (3.82 mg/g cells) were achieved in an E. coli expression system. Recombinant HMPV (N) was successfully expressed in, and purified from the baculovirus expression system. Overall, a 99% HMPV IgG seroprevalence was observed (n = 96) using HMPV M-, N- and P-ELISA, respectively. The M antigen proved to be the most diagnostically useful with 99% of specimens tested exhibiting anti-M protein reactivity. A high correlation was observed between anti-M and N IgG reactivity (r = 0.96), with significant correlation also evident for anti-N and P IgG reactivity (r = 0.74). Lowest correlation was evident for anti-M and P IgG reactivity (r = 0.57). Finally, the first demonstration of HMPV-specific B cell memory (ranging 1-15 spot forming cells (SFC)/million cells) was achieved against M and P antigens in 40% of individuals tested. CONCLUSION:This work describes robust diagnostic systems for HMPV and new insight into antigen-specific B cell memory against HMPV.

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Recombinant expression and in vitro characterisation of active Huwentoxin-IV.

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Recombinant expression and in vitro characterisation of active Huwentoxin-IV.

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