Description:

The Vitamin K-dependent gamma-glutamyl carboxylase post-translationally modifies glutamyl residues into gamma-carboxyglutamyl residues (Gla). In vertebrates, substrates for the enzyme include proteins involved in blood coagulation, bone mineralization, and signal transduction. The only invertebrate so far shown to synthesize Gla is a marine invertebrate, the cone snail, which synthesizes Gla-containing ion channel blockers known as conotoxins. The gamma-carboxylation reaction occurs in the endoplasmic reticulum on proteins that carry the adequate recognition sequence, which is typically located within a cleavable propeptide preceding the targeted glutamyl residues.

Progress:

Recently, we reported the purification and cDNA cloning of novel conotoxins that differ from other vitamin K-dependent polypeptides in having precursors in which the gamma-carboxylation recognition site is located within a C-terminal extension rather than at the N-terminus. The identification of these conotoxins has defined a novel precursor structure for vitamin K-dependent polypeptides and provided the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.

We have cloned the gene for the gamma-glutamyl carboxylase from the cone snail and have expressed it in Sf 21 (insect) cells. Several stably transfected cell lines expressing mutated cone snail and bovine carboxylase constructs have been established for enzymological studies. The cell lines were generated and are maintained in the NCRR supported BioCurrents Research Center at the MBL.