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J. Biol. Chem., Vol. 279, Issue 31, 32453-32463, July 30, 2004

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The First -Carboxyglutamic Acid-containing Contryphan

A SELECTIVE L-TYPE CALCIUM ION CHANNEL BLOCKER ISOLATED FROM THE VENOM OF CONUS MARMOREUS^*

Karin Hansson, Xiaosong Ma¶, Lena Eliasson¶, Eva Czerwiec||, Bruce Furie||**, Barbara C. Furie||**, Patrik Rorsman¶, and Johan Stenflo

From the Department of Clinical Chemistry, Lund University, University Hospital, Malmö, S-205 02 Malmö, Sweden, the ^¶Department of Physiological Sciences, Lund University, SE-221 84 Lund, Sweden, the ^||Marine Biological Laboratory, Woods Hole, Massachusetts 02543, the ^**Center for Hemostasis and Thrombosis, and Vascular Biology, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, and The Oxford Center for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Headington, Oxford OX3 7LJ, United Kingdom

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

Received for publication, December 17, 2003 , and in revised form, March 5, 2004.

^* This work was supported by Grants K2001-03X-04487-27A and K2001-03GX-04487-27, 08647, 13147 from the Swedish Medical Research Council, the European Union Cono-Euro-Pain (QLK3-CT-2000-00204), the Swedish Foundation of Strategic Research, the Kock Foundation, the Pålsson Foundations, the Foundation of University Hospital, Malmö, the Craafordska stiftelsen, and the Royal Physiografic Society in Lund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY485226.

To whom correspondence should be addressed: Dept. of Clinical Chemistry, Lund University, University Hospital, Malmö, S-205 02 Malmö, Sweden. Tel.: 46-40-337226; Fax: 46-40-929023; E-mail: Karin.Hansson{at}klkemi.mas.lu.se.

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