The Structure And Function Of Spider Peptide Toxins

A neurotoxic peptide (HNTX-IV) was purified from the venom of the spider Selenocosmia hainana with combination of ion-exchange and reverse-phase HPLC. The complete amino acid sequence (35 residues) was obtained by using automated Edman degradation, which is agreement with the molecular mass 3 988.70Da by mass spectrometry and the amino acid analysis. The amino acid sequence of HNTX-IV is similar to that of HWTX-IV except only seven residues. HNTX-IV was partially reduced by TCEP at pH 3, and the intermediates were isolated by revers-phase HPLC. Alkylation of free thiols, followed by sequencer analysis, enabled all three bridges to be identified (Cys2-Cys 17, Cys9-Cys24 and Cysl6-Cys31). HNTX-IV was synthesized by using Fmoc-chemistry methodology and the renatured peptide had the same biology activity as the native toxin. HNTX-IV indues lethal activity on rats but has no effect on cockroches. The LD50 of rats is (0.20 ± 0.07) mg/kg. HNTX-IV courses enhancement of the twitch response induced by electronical stimulation of vas deferens and then inhibition completely. HNTX-IV only inhibits the constraction of phrenic nerve hemidiaphragm preparations. HNTX-IV has no effect on the rhythmic contraction of vas deferens triggered by adding noradrenaline and the twitch response of hemidiaphragm induced by directly stimulating muscale, which indicats that is a nerve specific toxin. Electrophysiological studies showed that HNTX-IV inhibits TTX-S sodium current completely in NG108-15 cellsand has no effect on the activation and inactivation of sodium current, which suggests HNTX-lV is a site-1 toxin affecting the sodium channel through a mechanism quite similar to that of TTX. The three-dimensional structure of HNTX-IV has been determined by 2DTSJMR. The resulting structure is composed of a double-stranded antiparallel (3-sheet (L22-S25 and W30-Y33) and three turns (E4-K7, PI 1-D14, K18-K21 and R26-R29) and belongs to the inhibitor cystine knot structural family. Comparison with u-conotoxin GIIIA and HWTX-IV shows that the positively charged residues of loop IV (residue 26-29), especially residue Lys 27, must play a crucial role in its binding toneuronal tetrodotoxin-sensitive voltage-gated sodium channel.Two peptide components, named m-HWTX-III and SHL-II, were isolated from the crude venom of the spider Selenocosmia huwena. The sequences of the two peptides are similar to these of HWTX-III and SHL-I besides lack of the C-terminal tryptophan residue repectively. m-HWTX-III neither causes paralysis in cockroaches (p,americana) nor enhances electrically induced contraction of rat vas deferens, while as HWTX-III does. The result indicates that Trp33 is a key residue related to the biological function of HWTX-III. SHL-II and SHL-I, However, have the same haemagglutination activity to each other, which suggests that the C-terminal of SHL-I is not important to its haemagglutination activity.