Structure-function relationship of lapemis toxin: A synthetic approach

Abstract

The synthetic approach to the structure-function relationship of lapemis toxin has been very useful in clarifying the important binding regions. To identify the neurotoxic binding domain(s) of lapemis toxin, several peptides were synthesized using the 9-fluorenylmethoxycarbonyl protocols. These peptides were based on the sequence of lapemis toxin, a 60-amino-acid, shortchain postsynaptic neurotoxin found in sea snake ( Lapemis hardwickii) venom. The peptides were purified using high-performance liquid chromatography and sequenced to verify the correct synthesis, isolation, and purity. The synthetic peptide names and single letter sequences were Peptide A1 (15 mer) CCNQQSSQPKTTTNC Peptide B1 (18 mer) CYKKTWSDHRGTRIERGC Peptide B2 (16 mer) YKKTWSDHRGTRIERG Peptide C1 (12 mer) CPQVKPGIKLEC Peptide NS (20 mer) EACDFGHIKLMNPQRSTVWY. The peptide NS (nonsense peptide) sequence was arbitrarily determined and used as a control peptide. Biological activities of the synthetic peptides were determined by in vivo as well as by in vitro assay methods. For the in vivo assay, lethality was determined by intravenous injection in mice (Swiss Webster). For the in vitro assay, peptide binding to the Torpedo californica nicotinic acetylcholine receptor was determined. The peptides were found to be nontoxic at approximately 114 times the known LD 50 of lapemis toxin. Binding studies with 125-Iradiolabeled lapemis toxin and tyrosine-containing peptides indicated that lapemis toxin and peptide Bl bound the receptor, while the other peptides had no detectable binding. The central loop domain of lapemis toxin (peptide B1) plays a dominate role in the toxin's binding ability to the receptor. These results and the hydrophilicity analysis predict peptide B1 may serve as an antagonist or antigen to neutralize the neurotoxin effects in vivo.