Precursors of Novel Gla-Containing Conotoxins Contain a Carboxy-Terminal Recognition Site That Directs γ-Carboxylation^,

Abstract

Vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamyl residues to gamma-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the gamma-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel gamma-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs gamma-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue "postpeptide" from the Gla-TxXI precursor reduced the K(m) for the reaction of the Conus gamma-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it was positioned at the N- or C-terminal end of the mature toxin. Comparison of the postpeptides to propeptides from other conotoxins suggested some common elements, and amino acid substitutions of these residues perturbed gamma-carboxylation of the Gla-TxXI peptide. The demonstration of a functional and transferable C-terminal postpeptide in these conotoxins indicates the presence of the gamma-carboxylation recognition site within the postpeptide and defines a novel precursor structure for vitamin K-dependent polypeptides. It also provides the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.

A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus

“…Framework 12 (C-C-C-C-CC-C-C) conotoxins have been previously described (Brown et al, 2005;Hansson et al, 2004), but the relevant peptides (GlaMrII and GlaTxX) bear little similarity to Cal12 cDNAs, other than the predicted framework (Fig.3). Peptides cal12a and cal12b (corresponding to cDNAs Cal12.1.1a and Cal12.1.1b, respectively) are the first framework 12 conotoxins for which a high-affinity target has been identified.…”

“…Cal12.2d 12 peptide, GlaTxX (Brown et al, 2005), but it has no similarity with the leader of the Cal12 members. However, significant identity does exist in the N-terminal leader sequence between Cal12.1 and Cal12.2 members and a number of framework 6/7 (C-C-CC-C-C) conotoxins (Fig.10A).…”

“…These Cal12.1 cDNAs predict an unusual 8-Cys peptide framework (C-C-C-C-CC-C-C) that has been designated as framework 12 (Brown et al, 2005). This framework clearly differs from that of other conotoxins that target voltage-gated Na + channels, all of which are 6-Cys peptides of the O-(C-C-CC-C-C) or Msuperfamily (CC-C-C-CC) (Terlau and Olivera, 2004).…”

“…Alignment of sequences for the toxin-coding regions of these peptides with that of Cal12.1.1a (Fig.3B) shows that the intra-cysteine loops have essentially no amino acid identity and sizeable differences in length. Sequences of the leader and propeptide regions for GlaMrII are unavailable, but these regions of GlaTxX (Brown et al, 2005) bear little resemblance to those of Cal12.1 (Fig.3A). Functional properties of the Gla peptides have not been reported.…”

“…These peptides have an unusual type 12 Cys-framework (Brown et al, 2005), and molecular cloning and mass spectrometry (MS) yielded their complete sequences and PTMs. At least 15 homologues of these toxins were found by molecular cloning, and constitute the Cal12.1 family of cDNAs.…”