The Huwentoxin - ¢ñ And ¦Ø-conotoxin Mviia Chimera Synthesis And Identification Of Its Activity

Huwentoxin-I (HWTX-I) is a polypeptide neurotoxin isolated from the crude venom of Chinese bird spider Selenocosmia huwena. Its molecular weight is 3750 Dalton. The toxin contains 33 amino acid residues and three disulfide bonds. On isolated mouse phrenic nerve-diaphragm preparations, 1 x l(T5g/mL HWTX-I can reversibly block the neuromuscular transmission in 13.4 min. And it is a neurotoxin(N-type Ca channels inhibitor) that binds to the presynaptic nicotinic acetylcholine receptor (nAChR).Structure analysis indicated that the molecule of HWTX-I consists of a small triple stranded anti-parallel (3 -sheet and five (3 -turns. The N-terminal, C-terminal and most basic amino acid residues are located in the surface of the molecule. The key residues for the activity of this toxin are K25, H26, K27 and K30 in the fifth 0 -turn.oo-Conotoxin MVIIA (SNX-111) is one of the co-conotoxins purified from the venom of Conus magus. It has 25 animo acid residues with a net charge of+6 and pi 11.2, containing three disulfide binds. MVIIA is also an N-type Ca2+ channels inhibitor. Its key residues for the activity include Lys2, Arg10, Leu11, Tyr13 and Arg21 near the N-terminal.The space conformation of co-Ctx MVIIA and HWTX-I is highly similar. Both molecules contain the typical "Inhibitor cystine knot motif'(ICK), which is a proper molecular model for protein engineering.Based on the former works in our laboratory, a series of chimera peptides of HWTX-I and co - MVIIA were designed. Two of them, HM-1227 and HM-XW, were synthesized by SPPS and then refolded byair oxidization. We also synthesized w -Ctx MVIIA and studied its renaturation under air oxidizing and GSH-GSSG condition. We researched the physiological activities of the renatured peptides on isolated mouse phrenic nerve-diaphragm preparations or on isolated mouse spermaduct preparations.The results showed that all peptides were successfully synthesized and completely folded. After renaturation, the biological activity of MVIIA is nearly 100% of that of natural one; the activity of HM-1227 is less than t|iat of HWTX-I; however, HM-XW cannot block the neuromuscul ar transmission in an isolated mouse phrenic nerve-diaphragm preparation. The research of the renaturation and biological activity of the chimeras confirmed the active sites of HWTX-I and MVIIA further. It also indicates that the structure of ICK is stable and is an ideal model molecule for protein engineering.