The first gamma-carboxyglutamic acid-containing contryphan. A selective L-type calcium ion channel blocker isolated from the venom of Conus marmoreus.
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.
Hansson
Karin
K
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, S-205 02 Malmo, Sweden. Karin.Hansson@klkemi.mas.lu.se
Ma
Xiaosong
X
Eliasson
Lena
L
Czerwiec
Eva
E
Furie
Bruce
B
Furie
Barbara C
BC
Rorsman
Patrik
P
Stenflo
Johan
J
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002412:Cations; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry* / Q000378:metabolism*; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012908:Snails; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D015220:Calcium Channels; D002412:Cations; D018076:DNA, Complementary; D004220:Disulfides; D007477:Ions; D008978:Mollusk Venoms; D010455:Peptides; D010456:Peptides, Cyclic; C102355:contryphan; C489147:glacontryphan M; D015055:1-Carboxyglutamic Acid; D014364:Tryptophan; D002118:Calcium