The First γ-Carboxyglutamic Acid-containing Contryphan

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 ␥-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 calciumdependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.

The Metal-free and Calcium-bound Structures of a γ-Carboxyglutamic Acid-containing Contryphan from Conus marmoreus, Glacontryphan-M

“…Following the synthesis and initial purification of glacontryphan-M, the Cys 5 -Cys 11 disulfide bond was formed by oxidative refolding. The HPLC elution profiles of the natural and synthetic glacontryphan-M peptides were identical and when co-injected, the two peptides co-migrated (11).…”

“…The primary sequence and the identification of post-translational modifications of native glacontryphan-M (Table I), which was isolated and purified from the venom of C. marmoreous, were determined by automated Edman degradation and NanoESI mass spectrometry (11). Following the synthesis and initial purification of glacontryphan-M, the Cys 5 -Cys 11 disulfide bond was formed by oxidative refolding.…”

“…The masses of the peptides were confirmed by NanoElectro Spray Ionization (NanoESI)-mass spectrometry on an API QSTAR Pulsar-I quadrapole TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada), and the peptides were sequenced by amino acid sequencing as described previously (11).…”

“…Peptide Synthesis-Glacontryphan-M and the glutamic acid-containing analogue, glucontryphan-M, were synthesized as described by Hansson et al (11). The masses of the peptides were confirmed by NanoElectro Spray Ionization (NanoESI)-mass spectrometry on an API QSTAR Pulsar-I quadrapole TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada), and the peptides were sequenced by amino acid sequencing as described previously (11).…”

“…In the accompanying article (11) we describe the purification, primary structure, and functional characterization of a novel contryphan from the venom of Conus marmoreous, glacontryphan-M (11). Glacontryphan-M, with the sequence N␥S␥CP(D-W)HPWC-NH 2 (␥-carboxyglutamic acid (Gla) 1 residue; NH 2 , C-terminal amidation), is the first contryphan found to contain Gla, a post-translationally modified glutamic acid residue (Table I).…”