Precursors of novel Gla-containing conotoxins contain a carboxy-terminal recognition site that directs gamma-carboxylation

Biochemistry. 2005 Jun 28;44(25):9150-9. doi: 10.1021/bi0503293.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 1-Carboxyglutamic Acid / analysis
  • 1-Carboxyglutamic Acid / chemistry
  • 1-Carboxyglutamic Acid / metabolism*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Conotoxins / chemistry*
  • Conotoxins / genetics
  • Conotoxins / isolation & purification
  • Conotoxins / metabolism*
  • DNA, Complementary / genetics
  • Molecular Sequence Data
  • Mollusca / genetics
  • Mollusca / metabolism
  • Protein Processing, Post-Translational*
  • Sequence Alignment
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Substrate Specificity

Substances

  • Conotoxins
  • Conus textile toxin
  • DNA, Complementary
  • 1-Carboxyglutamic Acid
  • conotoxin GV

Associated data

  • GENBANK/AY856069
  • GENBANK/AY856070