ABSTRACT
Conantokin-G isolated from the marine snail Conus geographus is a 17-amino acid gamma-carboxyglutamate (Gla)-containing peptide that inhibits the N-methyl-D-aspartate receptor. We describe the cloning and sequence of conantokin-G cDNA and the possible role of the propeptide sequence. The cDNA encodes a 100amino acid peptide. The N-terminal 80 amino acids constitute the prepro-sequence, and the mature peptide is derived from the remaining C-terminal residues after proteolysis, C-terminal amidation, and a unique post-translational modification, gamma-carboxylation of glutamate residues to Gla. Mature conantokin-G peptide containing Glu residues (E.Con-G) in place of Gla is a poor substrate for the vitamin K-dependent gamma-glutamyl carboxylase (apparent Km = 3.4 mM). Using peptides corresponding to different segments of the propeptide we investigated a potential role for the propeptide sequences in gamma-carboxylation. Propeptide segment -20 to -1 covalently linked to E.Con-G or the synthetic pentapeptide FLEEL increased their apparent affinities 2 orders of magnitude. These substrates are not efficiently carboxylated by the bovine microsomal gamma-glutamyl carboxylase, suggesting differences in specificities between the Conus and the mammalian enzyme. However, the role of propeptide in enhancing the efficiency of carboxylation is maintained.
Subject(s)
Carbon-Carbon Ligases/metabolism , Conotoxins , Mollusk Venoms/chemistry , Peptides, Cyclic/chemistry , Protein Precursors/chemistry , 1-Carboxyglutamic Acid/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , Excitatory Amino Acid Antagonists/chemistry , Excitatory Amino Acid Antagonists/pharmacology , Glutamic Acid/metabolism , Kinetics , Microsomes/enzymology , Molecular Sequence Data , Mollusk Venoms/toxicity , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptides, Cyclic/toxicity , Protein Precursors/physiology , Protein Processing, Post-Translational/physiology , Sequence Analysis, DNA , Snails/enzymology , Substrate SpecificityABSTRACT
The Ca2+ channel blocking neurotoxin, omega-conotoxin GVIA, is a 27-amino acid peptide with three disulfide bonds. We have determined the precursor structure of this peptide by analyzing a cDNA clone obtained from a Conus geographus venom duct library. The omega-conotoxin GVIA prepropeptide is 73 amino acids in length comprising a 22 amino acid signal sequence, an intervening region of 23 amino acids, and finally, a 27 amino acid toxin region. A C-terminal glycine residue is later processed to a C-terminal amide moiety. The omega-conotoxin GVIA precursor exhibits regions of strong homology to the previously characterized King-Kong peptide precursor, but shows surprising divergence as well. The possible significance of the precursor organization is discussed.
