Hydrophobic amino acids define the carboxylation recognition site in the precursor of the gamma-carboxyglutamic-acid-containing conotoxin epsilon-TxIX from the marine cone snail Conus textile

To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the Venom of the marine snail Conus textile, th e cDNA encoding this peptide was cloned from a C. textile venom duct librar y. The cDNA of the precursor form of epsilon-TxTX encodes a 67 amino acid p recursor peptide, including an N-terminal prepro-region, the mature peptide , and four residues posttranslationally cleaved from the C-terminus. To det ermine the role of the propeptide in gamma-carboxylation, peptides were des igned and synthesized based on the propeptide sequence of the Gla-containin g conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent ga mma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy p eptide epsilon-TxIX was a high K-M substrate for the gamma-carboxylase. Syn thetic peptides based on the precursor epsilon-TxIX were low K-M substrates (5 mu M) if the peptides included at least 12 residues of propeptide seque nce, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leu cine-8 and leucine-4 contribute to the interaction of the pro-conotoxin wit h carboxylase since their replacement by aspartic acid increased the K-M of the substrate peptide. Although the Conus propeptide and the propeptides o f the mammalian vitamin K-dependent proteins show no obvious sequence homol ogy, synthetic peptides based upon the structure of pro-epsilon-TxIX were i ntermediate K-M substrates for the bovine carboxylase. The propeptide of ep silon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that play s the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation rec ognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.