title: Native pyroglutamation of huwentoxin-IV: a post-translational modification that increases the trapping ability to the sodium channel.
abstract: Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC50 nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.
authors.affiliation: The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, China.
authors.forename: Mingqiang
authors.initials: M
authors.lastname: Rong
authors.forename: Zhigui
authors.initials: Z
authors.lastname: Duan
authors.forename: Juliang
authors.initials: J
authors.lastname: Chen
authors.forename: Jianglin
authors.initials: J
authors.lastname: Li
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Xiao
authors.forename: Songping
authors.initials: S
authors.lastname: Liang
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005727:Ganglia, Spinal / Q000187:drug effects / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015222:Sodium Channels / Q000378:metabolism*; D013111:Spider Venoms / Q000378:metabolism* / Q000494:pharmacology; D013112:Spiders; D053719:Tandem Mass Spectrometry
chemical_list: D015222:Sodium Channels; D013111:Spider Venoms; C470632:huwentoxin IV, Selenocosmia huwena; D011761:Pyrrolidonecarboxylic Acid