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Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to gamma-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a K(D) of 0.63 mM. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.

Original publication

DOI

10.1074/jbc.M313825200

Type

Journal article

Journal

J Biol Chem

Publication Date

30/07/2004

Volume

279

Pages

32453 - 32463

Keywords

1-Carboxyglutamic Acid, Amino Acid Sequence, Animals, Base Sequence, Calcium, Calcium Channels, Cations, Chromatography, High Pressure Liquid, DNA, Complementary, Disulfides, Dose-Response Relationship, Drug, Electrophysiology, Hydrolysis, Ions, Kinetics, Molecular Sequence Data, Mollusk Venoms, Peptide Biosynthesis, Peptides, Peptides, Cyclic, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Snails, Spectrometry, Fluorescence, Spectrometry, Mass, Electrospray Ionization, Time Factors, Tryptophan