lambda -Conotoxins, a New Family of Conotoxins with Unique Disulfide Pattern and Protein Folding. ISOLATION AND CHARACTERIZATION FROM THE VENOM OF CONUS MARMOREUS (original) (raw)

2000, Journal of Biological Chemistry

Conotoxins are multiple disulfide-bonded peptides isolated from marine cone snail venom. These toxins have been classified into several families based on their disulfide pattern and biological properties. Here, we report a new family of Conus peptides, which have a novel cysteine motif. Three peptides of this family (CMrVIA, CMrVIB, and CMrX) have been purified from Conus marmoreus venom, and their structures have been determined. Their amino acid sequences are VCCGYK-LCHOC (CMrVIA), NGVCCGYKLCHOC (CMrVIB), and GICCGVSFCYOC (CMrX), where O represents 4-transhydroxyproline. Two of these peptides (CMrVIA and CMrX) have been chemically synthesized. Using a selective protection and deprotection strategy during disulfide bond formation, peptides with both feasible cysteine-pairing combinations were generated. The disulfide pattern (C 1-C 4 , C 2-C 3) in native toxins was identified by their co-elution with the synthetic disulfideisomeric peptides on reverse-phase high pressure liquid chromatography. Although cysteine residues were found in comparable positions with those of ␣-conotoxins, these toxins exhibited a distinctly different disulfide bonding pattern; we have named this new family "-conotoxins." CMrVIA and CMrX induced different biological effects when injected intra-cerebroventricularly in mice; CMrVIA induces seizures, whereas CMrX induces flaccid paralysis. The synthetic peptide with-conotoxin folding is about 1150-fold more potent in inducing seizures than the mispaired isomer with ␣-conotoxin folding. Thus it appears that the unique disulfide pattern, and hence the "ribbon" conformation, in-conotoxins is important for their biological activity. Conotoxins are biologically active peptide toxins isolated from venomous marine cone snails. They are typically small disulfide-rich peptides containing 11-30 amino acid residues. Conotoxins can be classified into several families based on the number and pattern of disulfide bonds and biological activities (1, 2). Members of a single family of conotoxins share similar protein folding but in some cases exhibit different biological activities (3-5). These differences in biological activities are due to their ability to bind with specific ion channels or receptors (6). Some of these conotoxins are used as tools in investigating receptor structure and function (2) and ion channel geometry (7). The structurally constrained scaffolds of conotoxins are utilized as a template for protein engineering to create chimeric proteins (8, 9). Conotoxins, like toxins from other venoms, evolve rapidly by positive Darwinian selection (10). The specificity of conotoxins is due to their disulfide bonding framework and specific amino acids in inter-cysteine loops. The high density of disulfides in conotoxins plays a vital role in their stability and imposes a distinct protein folding with a specific orientation of hypervariable loop regions. The diversity in conotoxins is also achieved through disulfide pairings (see "Discussion").