Determination of disulfide bridges of two spider toxins: hainantoxin-III and hainantoxin-IV

Abstract

Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40°C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

“…Peptide toxins are usually highly bridged proteins with multiple pairs of intrachain disulfide bonds. The analysis of disulfide connectivity is important in protein structure determination [ 42 ]. The disulfide pattern in the venom peptides of C. taeniatus was estimated directly by LCMS-IT-TOF without venom fractionation.…”

Deadly Proteomes: A Practical Guide to Proteotranscriptomics of Animal Venoms

“…Since its introduction, this method has been used to resolve the structures of many spider-venom toxins. [151][152][153][154] In one case, partial reduction and alkylation was used to solve the connectivity of a peptide toxin from the Blue Mountains funnel-web spider H. versuta, which revealed an extremely rare vicinal disulfide bond that proved to be critical for insecticidal activity. [153] Han et al [155] used a similar method to demonstrate that the M3 family of conotoxins has different connectivity to the M2 or M4 families.…”