Expression and characterization of recombinant vitamin K‐dependent γ‐glutamyl carboxylase from an invertebrate, Conus textile

Open Access
Publisher Website
Google Scholar
Abstract
The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, γ-carboxyglutamic acid, have been identified. To examine its biosynthesis of γ-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and ≈ 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin K-dependent epoxidase activity. The recombinant carboxylase has a molecular mass of ≈ 130 kDa. The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues −18 to +10 of human proprothrombin and proFactor IX with Km values of 420 µm, 1.7 µm and 6 µm, respectively; the Km for vitamin K is 52 µm. The Km values for peptides based on the sequence of the conotoxin ε-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 µm, 75 µm and 74 µm, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent γ-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of γ-carboxyglutamic acid synthesis in diverse biological systems.

This publication has 52 references indexed in Scilit: