Abstract
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.
Highlights
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
The vitamin K-dependent ␥-glutamyl carboxylase catalyzes the post-translational conversion of glutamyl residues to ␥-carboxyglutamyl (Gla)1 residues in precursor proteins that contain the appropriate ␥-carboxylation recognition site within the propeptide of the precursor [1,2,3,4]
The sequence shows high similarity to the amino acid sequences earlier described for peptides of the contryphan family of conotoxins (Table I)
Summary
Gla, ␥-carboxyglutamic acid; HPLC, high performance liquid chromatography; NanoESI-MS, nanoelectrospray ionization mass spectrometry. Glacontryphan-M, an L-type Ca2ϩ Ion Channel Antagonist rous [31, 32], molluscivorous [33], and vermivourous [34] cone snails are distinct for their unusual density of post-translational modifications. Cloning of the cDNA of glacontryphan-M showed a high density of arginyl and lysyl residues in the propetide region compared with the propeptide regions of the earlier described contryphans. Like in other neuro- and endocrine cells [38] exocytosis from pancreatic B-cells is determined by calcium influx through voltage-dependent Ca2ϩ channels [39] This is the first Gla-containing conopeptide reported to modulate the activity of this type of calcium ion channels
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