Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry

Abstract

Background: Numerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spider Selenocosmia huwena. Results: HWTX-IV was first lightly labeled with biotin under the optimized mild experimental conditions and the toxin labeled with a single biotin group (monobiotinylated HWTX-IV) was demonstrated by electrophysiological experiments to retain its original bioactivity and was used in combination with far-western blotting to detect its interacting proteins. Comparative experiments indicated that some membrane proteins from rat neuromuscular junction preparations bind to monobiotinylated HWTX-IV after being transferred onto a PVDF membrane from the SDS-gel. With capillary high performance liquid chromatography-tandem mass spectrometry, several membrane proteins with which HWTX-IV potentially interacted were identified from the preparations and then bioinformatically analyzed.

Direct evidence for high affinity blockade of NaV1.6 channel subtype by huwentoxin-IV spider peptide, using multiscale functional approaches

“…the relative high affinity of HwTx-IV for Na V 1.6 and/or Na V 1.4 channel subtypes. It is worth noting that HwTx-IV was previously reported to completely block the conduction of isolated mouse phrenic nerve-hemidiaphragm preparations, by acting on multiple ion channel proteins, especially the Ca V channels(Yu et al, 2014). However, this is not supported by the present results showing a…”

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“…[7] Spider venom toxin peptides interact with ligand-gated channels and modulate the activity of neuronal ion channels and receptors located on cell membrane. Spider toxins show inhibition of voltage-gated ion channels, [8] and especially target vertebrate or invertebrate voltage-gated potassium (Kv), calcium (Cav), or sodium (Nav) channels. They also target specific Na (V) channel subtypes and show analgesic effects.…”