Mutant The. Huwentoxin - I (of Hwtx-¢ñ) And Natural ¦Ø-conotoxin Mviia The Synthesis And Activity Identification

Huwentoxin-I (HWTX-I) is a polypeptide neurotoxin isolated from the crude venom of Chinese bird spider Selenocosmia huwena, a Chinese bird -hunting spider defined as a new species of the family Selenocosmia .Its molecular weight is 3750 Dalton. The toxin contains 33 amino acid residues and three disulfide bonds whose linkage pattern is 1-4(Cys-Cys) , 2-5 ( Cys-Cys), 3-6 ( Cys-Cys). Solution conformation analysis indicated that the molecule of HWTX-I adopts a compact and stable structure consisting of a small triple stranded anti-parallel -sheet and five -turns characterized by "inhibitor cystine knot motif'(ICK) which is also widely found in other protease in nature. The toxin can reversible block the neuromuscular transmission in an isolated mouse phrenic nerve-diaphragm preparation and also is a neurotoxin (N-type Ca2+ channels inhibitor) which binds to the postsynaptic nicotinic acetylcholine receptor(nAChR). Its action site is located on the postsynaptic membrane and the biological activity of HWTX-I is located in the fifth P -turn containing Lys25 and Lys 27. to -conotoxin MVIIA is a neurotoxin belonging to one of N-Type Ca2+ channels inhibitors, purified from the venom of the conus marine snails, contains 25 amino acid residues with three disulfide binds. It adopts the pattern of ICK and its linkage of the disulfide binds and space conformation are highly similar to HWTX-I. The structure motif is considered a proper model molecule suitable for protein engineering.In order to prove HWTX-I that can be used as one of natural scaffolds for protein engineering .We have synthesized two multiple mutants: HW-MA and RGD-MA and natural co-conotoxin MVIIA by using solid phase method with Fmoc chemistry on peptide synthesizer and by hand respectively. At first , the systematic studies was made on the mechanisms and optimal conditions of denaturation and renaturation of MVIIA which lay the groundwork of renaturationof chimera of HWTX-I. The results indicated that the synthetic MVIIA was reduced and oxidized in a buffer containing acetamide / guanidine hydrochloride by air oxidization in the room temperature and obtained a relatively well oxidization result, the synthetic MVIIA showed 100 % of physiological activity of native MVIIA, but obtained 1 % product of synthetic crude peptide. Then formation of three disulfide binds of HW-MA and RGD-HW was monitored by reversed-phase HPLC using air oxidization or reduced and oxidized glutathione respectively. The synthetic peptide was purified by double reversed-phase HPLC . Mass spectrometry of synthetic HW-MA and RGD-HW are in full agreement with those speculated theoretically, which proves the success of peptide synthesis and refold .On isolated mouse phrenic nerve-diaphragm preparations, HW-MA can block the neuromuscular transmission in 35 minutes or so(l 10-5 g/ml),its biological activity shows 73 % decrease comparing with biological activity of native HWTX-I. It proves that the protein engineering of synthetic chimera HWTX-I has gained success to some extent, although it did not achieve our expectations. Thus it proved that HWTX-I can be using as natural scaffold for protein engineering .and also emphasized the importance of "local stereo circumstances" of activity site when the foreign activity site was transferred into a natural scaffold. Another chimera RGD-HW did not showed activity of platelet adhesion and aggregation that RGD sequence is thought to possess after complete refolding and purification, which explained that the resign and the progress of refolding of chimera RGD-HW was required improvement further. Protein engineering studies ,taking HWTX-I adopting ICK Structure model as a model molecule, provide important value of basic research and possess a widely applied prospect for other polypeptide neurotoxin according to document reports else.