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

Grant, Marianne A.; Hansson, Karin; Furie, Barbara C.; Furie, Bruce; Stenflo, Johan; Rigby, Alan C.

doi:10.1074/jbc.m313826200

Cited by 26 publications

(28 citation statements)

References 35 publications

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“…It is thus possible that binding of Ca 2ϩ to the Gla residues in glacontryphan-M contributes to its ability to adopt a defined conformation, and this divalent cation-dependent conformation is necessary for the interaction with the Ca 2ϩ ion channels. The calcium-induced structural perturbations of glacontryphan-M that are functionally necessary have been further investigated and are discussed in Grant et al (47). In a very recent report K ϩ channels were demonstrated as the molecular target of Contryphan-Vn (37).…”

Section: Discussionmentioning

confidence: 99%

“…The calcium-dependent intrinsic fluorescence quench was clearly impaired in glucontryphan-M implying the Gla residues in glacontryphan-M to be involved in the calcium interaction. In order to further elucidate the role of Gla in glacontryphan-M and the role of calcium binding to the peptide, the high resolution three-dimensional structure of the peptide in the presence and absence of metal ions has been determined (47).…”

Section: Discussionmentioning

confidence: 99%

“…Thus it can be suggested that the Ca 2ϩ -bound form of glacontryphan-M acts as an antagonist on the Ca 2ϩ channel activity whereas the Ca 2ϩ -unbound form of glacontryphan-M has no or minor stimulatory effect on the channel activity. The structural investigation of glacontryphan-M described in the accompanying report (47) revealed calcium-induced conformational changes that might be necessary for the antagonistic effect.…”

Section: Effects Of Glacontryphan-m On Inward Voltage-dependent Ca 2ϩmentioning

confidence: 99%

“…5C). In the accompanying report (47) the structure of glacontryphan-M in the absence and presence of calcium ions was determined using two-dimensional 1 H NMR spectroscopy (47). A comparison of the two structures revealed subtle calcium-induced perturbations that reorder and reposition the Gla-containing N-terminal stretch of residues.…”

Section: Purification and Characterization Of Glacontryphan-m-mentioning

confidence: 99%

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The First γ-Carboxyglutamic Acid-containing Contryphan

Hansson

Eliasson

et al. 2004

Journal of Biological Chemistry

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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 ␥-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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Effects Of Glacontryphan-m On Inward Voltage-dependent Ca 2ϩmentioning

confidence: 99%

Section: Purification and Characterization Of Glacontryphan-m-mentioning

confidence: 99%

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The First γ-Carboxyglutamic Acid-containing Contryphan

Hansson

Eliasson

et al. 2004

Journal of Biological Chemistry

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“…Characteristic features of the GT from C. marmoreus are a conserved disulfide-bonded loop, the presence of a D-tryptophan, a histidine (H) within the intercystine loop, and two γ-carboxyglutamic acid residues (13).…”

Section: Discussionmentioning

confidence: 99%

Peptide toxin glacontryphan-M is present in the wings of the butterfly Hebomoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Bae

Lödl

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Protein profiling has revealed the presence of glacontryphan-M, a peptide toxin identified only in the sea snail genus Conus, in the wings of Hebomoia glaucippe (HG). The wings and body of HG were homogenized and the proteins were extracted and analyzed by 2D gel electrophoresis with subsequent in-gel digestion. Posttranslational protein modifications were detected and analyzed by nano-LC-MS/MS. An antibody was generated against glacontryphan-M, and protein extracts from the wings of HG samples from Malaysia, Indonesia, and the Philippines were tested by immunoblotting. Glacontryphan-M was unambiguously identified in the wings of HG containing the following posttranslational protein modifications: monoglutamylation at E55, methylation at E53, quinone modification at W61, cyanylation at C56, and amidation of the C terminus at G63. Immunoblotting revealed the presence of the toxin in the wings of HG from all origins, showing a single band for glacontryphan-M in HG samples from Malaysia and Philippines and a double band in HG samples from Indonesia. Intriguingly, sequence analysis indicated that the Conus glacontryphan is identical to that of HG. The toxin may function as a defense against diverse predators, including ants, mantes, spiders, lizards, green frogs, and birds. mass spectrometry | venom A lthough a large number of low molecular weight deterrents and toxins have been demonstrated to exist in Lepidoptera (1), information on the peptide or protein sequences is limited to a few of these toxins.The cloning and purification of pierisin-2, an apoptosis-inducing protein from Pieris brassicae, was reported in 2000 (2). The protein shows homology with other ADP ribosylating toxins, including the A subunit of cholera toxin and pierisin-1. Pierisin-1-induced apoptosis is mediated by mono-ADP ribosylation of DNA (3). This toxin, isolated from the cabbage butterfly Pieris rapae, was found to induce mutations of the hypoxanthine-phosphoribosyltransferase (4). The toxin is highly expressed during the larval stage and rapidly decreases before pupation to the very low levels maintained in adults (5). Another protein, papiliocin, is a 37-residue cecropin-like peptide isolated from the larvae of the swallowtail butterfly Papilio xuthus (6). This protein shows low toxicity toward mammalian cells but demonstrates antimicrobial activity (7).In conducting a proteomic profile of the wings of Hebomoia glaucippe (HG) (8, 9), we observed a protein that was unambiguously identified as glacontryphan-M (GT). Study of this protein toxin was motivated by the result that GT, intriguingly, so far only been reported in the sea snail Conus marmoreus, was observed by 2D gel electrophoresis (2DE) in butterfly wing protein samples at relatively high abundance but was not detected in samples from the body. Furthermore, we were interested in studying Lepidoptera toxins because of the potential medical relevance of these toxins in causing human disease (10, 11). The aim of the present study was to characterize GT using a combination of full-leng...

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Biopharmaceuticals: Post‐Translational Modification Carboxylation and Hydroxylation

Brown

Stenberg

2009

Post‐translational Modification of Protein Biopharmaceuticals

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

Abstract: Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca

Cited by 26 publications

References 35 publications

The First γ-Carboxyglutamic Acid-containing Contryphan

The First γ-Carboxyglutamic Acid-containing Contryphan

Peptide toxin glacontryphan-M is present in the wings of the butterfly Hebomoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Biopharmaceuticals: Post‐Translational Modification Carboxylation and Hydroxylation

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