Structure And Function Of Spider Peptide Toxins

The Chinese bird spider Selenocomia huwena is one of the most venomous spiders in China, which distributes in southwestern hilly area of China and habitually lives in the holes underground. The venom of this spider ontains a mixture of compounds with different types of biological activity. Many kinds of neurotoxic toxins such as HWTX-I, HWTX-II, HWTX-III, and HWTX-IV etc have been isolated by combination of ion exchange chromatography and reverse phase high performance liquid chromatography.A neurotoxic peptide (named huwentoxin-V) was purified from the venom of the spider by a combination of ion exchange chromatography and reverse phase HPLC. HWTX-V has 35 amino acid residues, with the molecular mass 4111.4Da. The amino acid sequence has been determined as NH2-ECRWYLGGCSQDGDCCKHLQCHSN-YEWCVWDGTFS-COOH, which consists of 6 Cys, formed three pairs of disulfide bridges. A natural mutant of the toxin (called mHuwentoxin-V) was also isolated from the venom. The complete amino acid sequence is NH2.ECRWYLGGCSQDGDCCKHLQCHSNYE -WCVWDGT-COOH determined by automated Edman degradation. mHWTX-V was only truncated two amino acid residues from the C-terminus of HWTX-V, and its molecular weight is 3877.1 Da determined by mass spectrometry. There are three disulfide bridges in each of two peptides demonstrated by mass spectrometry. According to the cDNA of HWTX-V, HWTX-V and mHWTX-V might be come from the same gene. The difference of two toxins may be result from post-translation modification.HWTX-V can reversibly paralyze locusts and cockroaches for several hours with an ED50 value as 16+5ug/g to locusts, and largerdose of the toxin can cause death. The neurotoxic symptoms inducedby intra-abdominal injection of Huwentoxin-V were paralysis, immobilization and last gradually recovery. HWTX-V was not shown to block neuromuscular transmission in isolated mouse phrenic nerve diaphragm preparation. HWTX-V also had not evidently effect on mice by intra-abdominal injection with high dose of 400ug/g. We can conclude that Huwentoxin-V is a potent insecticidal toxin, without no verbrate toxicity. However, mHWTX-V shows no significant effect on locusts and cockroaches. The structure-activity relationship indicates that the residues Phe34 and Ser35 in the C-terminus of HWTX-V are the key residues of the biological activity.To assign the disulfide bonds of HWTX-V, we employ the method of partial reduction using TCEP. The labeled thiols were identified by Edman degradation. The results indicated specific location of three disulfide bonds. The three-disulfide linkage of HWTX-V has been assigned as Cys2-Cysl6, Cys9-Cys21, and Cysl5-Cys28. The primarystructure of two toxins exhibited sequence identity with other spidertoxins such as ProTx-I (65%), a toxin from the venom the tarantula spider Thrixopelma pruriens, SGTx (57%), a toxin from the spider Scodra grisepes, SNX-482 (55%), a toxin from the tarantula spider Hysterocrates gigas and HaTxl (54%) /HaTx2 (54%) from the spider Grammostola spatulata. According to the sequences similarity and the same disulfide linkage of HWTX-V family with other toxins with known 3D structures, we can postulate that HWTX-V may belong to ICK (Inhibitory Cysteine Knot) motif.