Identification, by molecular cloning, of a novel type of I2-superfamily conotoxin precursor and two novel I2-conotoxins from the worm-hunter snail Conus spurius from the Gulf of México

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, México. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I2-conotoxins and three of I2-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I2) or 23- residue signal peptide (I2-like), a 31-residue “pro” region (I2-like), and a 32-residue mature toxin region (I2 and I2-like). In addition, all the precursors have a 13-residue “post” region which contains a γ-carboxylation recognition sequence that directs the γ-carboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a “post” region has been found in precursors of I-conotoxins that also contain a “pro” region. The “post” peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that γ-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I2-like-conotoxins of C. spurius are more related to I2-conotoxins than to I1- and I3-conotoxins from other species, and that they might represent a new subgroup of the I2-superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes.