title: Precursors of novel Gla-containing conotoxins contain a carboxy-terminal recognition site that directs gamma-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.
authors.affiliation: Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Brown
authors.forename: Gail S
authors.initials: GS
authors.lastname: Begley
authors.forename: Eva
authors.initials: E
authors.lastname: Czerwiec
authors.forename: Leisa M
authors.initials: LM
authors.lastname: Stenberg
authors.forename: Margaret
authors.initials: M
authors.lastname: Jacobs
authors.forename: Dário E
authors.initials: DE
authors.lastname: Kalume
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
authors.forename: Johan
authors.initials: J
authors.lastname: Stenflo
authors.forename: Barbara C
authors.initials: BC
authors.lastname: Furie
authors.forename: Bruce
authors.initials: B
authors.lastname: Furie
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020916:Conotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D008969:Molecular Sequence Data; D008974:Mollusca / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D020916:Conotoxins; C059360:Conus textile toxin; D018076:DNA, Complementary; D015055:1-Carboxyglutamic Acid; C055407:conotoxin GV