The Experts below are selected from a list of 1137 Experts worldwide ranked by ideXlab platform
Bruce Furie - One of the best experts on this subject based on the ideXlab platform.
-
REVIEW ARTICLE Vitamin K-Dependent Biosynthesis of g-Carboxyglutamic Acid
2016Co-Authors: Bruce Furie, Beth A. Bouchard, Barbara C. FurieAbstract:VITAMIN K, AN ESSENTIAL vitamin, is a cofactor fora single known enzymatic reaction: the conversion of glutamic Acid to g-Carboxyglutamic Acid in vitamin K-dependent proteins during their biosynthesis. Since the discovery of vitamin K and its association with blood coagula-tion,1 many milestones have been passed on the road to understanding the biological role of vitamin K. Important early landmarks include the discovery of vitamin K antagonists and their introduction as pharmacologic agents for anticoagulation2; the discovery of g-Carboxyglutamic Acid in blood clotting proteins3,4; the identification of g-Carboxyglutamic Acid as a metal binding amino Acid that confers metal binding properties on proteins, a requirement for protein-membrane interaction5,6; the detection of an enzymatic activity (ie, the vitamin K-dependent g-glutamyl-carboxylase) that catalyzes the incor-poration of CO2 into glutamic Acid5; the identification of a
-
novel gamma Carboxyglutamic Acid containing peptides from the venom of conus textile
FEBS Journal, 2006Co-Authors: Bruce Furie, Barbara C. Furie, Eva Czerwiec, Dario E Kalume, Peter Roepstorff, Bjorn Hambe, Johan StenfloAbstract:The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-Carboxyglutamic Acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino Acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino Acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.
-
the metal free and calcium bound structures of a γ Carboxyglutamic Acid containing contryphan from conus marmoreus glacontryphan m
Journal of Biological Chemistry, 2004Co-Authors: Marianne A Grant, Johan Stenflo, Alan C. Rigby, Bruce Furie, Barbara C. Furie, Karin M HanssonAbstract:Abstract Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca2+ channels expressed in mouse pancreatic β-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453–32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a d-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two γ-Carboxyglutamic Acid (Gla) residues, formed by post-translational modification of glutamic Acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional 1H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys11-Cys5-Pro6 region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla2 and Ser3, moves away from the His8 and Trp10 aromatic rings and the alignment of the d-Trp7 and His8 aromatic rings with respect to the Trp10 rings is altered. The blockage of L-type voltage-gated Ca2+ channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone Cα conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 A, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation.
-
vitamin k dependent biosynthesis of γ Carboxyglutamic Acid
Blood, 1999Co-Authors: Bruce Furie, Beth A. Bouchard, Barbara C. FurieAbstract:V ITAMIN K, AN ESSENTIAL vitamin, is a cofactor for a single known enzymatic reaction: the conversion of glutamic Acid to γ-Carboxyglutamic Acid in vitamin K-dependent proteins during their biosynthesis. Since the discovery of vitamin K and its association with blood coagulation,[1][1] many
-
role of gamma Carboxyglutamic Acid in the calcium induced structural transition of conantokin g a conotoxin from the marine snail conus geographus
Biochemistry, 1997Co-Authors: Alan C. Rigby, James D. Baleja, Bruce Furie, Leping Li, Lee G PedersenAbstract:Conantokin G is a γ-Carboxyglutamic Acid- (Gla-) containing conotoxin isolated from the venom of the marine cone snail Conus geographus. This 17-residue polypeptide, which contains five γ-carboxygl...
Alan C. Rigby - One of the best experts on this subject based on the ideXlab platform.
-
the metal free and calcium bound structures of a γ Carboxyglutamic Acid containing contryphan from conus marmoreus glacontryphan m
Journal of Biological Chemistry, 2004Co-Authors: Marianne A Grant, Johan Stenflo, Alan C. Rigby, Bruce Furie, Barbara C. Furie, Karin M HanssonAbstract:Abstract Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca2+ channels expressed in mouse pancreatic β-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453–32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a d-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two γ-Carboxyglutamic Acid (Gla) residues, formed by post-translational modification of glutamic Acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional 1H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys11-Cys5-Pro6 region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla2 and Ser3, moves away from the His8 and Trp10 aromatic rings and the alignment of the d-Trp7 and His8 aromatic rings with respect to the Trp10 rings is altered. The blockage of L-type voltage-gated Ca2+ channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone Cα conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 A, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation.
-
the ω loop region of the human prothrombin γ Carboxyglutamic Acid domain penetrates anionic phospholipid membranes
Journal of Biological Chemistry, 2001Co-Authors: Lisa A. Falls, Margaret Jacobs, Alan C. RigbyAbstract:Abstract The hydrophobic ω-loop within the prothrombin γ-Carboxyglutamic Acid-rich (Gla) domain is important in membrane binding. The role of this region in membrane binding was investigated using a synthetic peptide, PT-(1–46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1–46)F4W and PT-(1–46) bind calcium ions and phospholipid membranes, and inhibit the prothrombinase complex. PT-(1–46)F4W, but not PT-(1–46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1–46)F4W, but not PT-(1–46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1–46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles. Fluorescence quenching of membrane-bound PT-(1–46)F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 is inserted 5 to 7 A into the bilayer. This report demonstrates that the ω-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.
-
role of gamma Carboxyglutamic Acid in the calcium induced structural transition of conantokin g a conotoxin from the marine snail conus geographus
Biochemistry, 1997Co-Authors: Alan C. Rigby, James D. Baleja, Bruce Furie, Leping Li, Lee G PedersenAbstract:Conantokin G is a γ-Carboxyglutamic Acid- (Gla-) containing conotoxin isolated from the venom of the marine cone snail Conus geographus. This 17-residue polypeptide, which contains five γ-carboxygl...
-
role of gamma Carboxyglutamic Acid in the calcium induced structural transition of conantokin g a conotoxin from the marine snail conus geographus
Biochemistry, 1997Co-Authors: Alan C. Rigby, James D. Baleja, Bruce Furie, Lee G PedersenAbstract:Conantokin G is a gamma-Carboxyglutamic Acid- (Gla-) containing conotoxin isolated from the venom of the marine cone snail Conus geographus. This 17-residue polypeptide, which contains five gamma-Carboxyglutamic Acid residues, is a N-methyl-d-aspartate- (NMDA-) type glutamate receptor antagonist. To investigate the role of gamma-Carboxyglutamic Acid in the calcium-induced structural transition of conantokin G, we determined the three-dimensional structure of the conantokin G/Ca2+ complex by two-dimensional 1H NMR spectroscopy and compared it to the high-resolution structure of conantokin G in the absence of metal ions [Rigby et al. (1997) Biochemistry 36, 6906]. Complete resonance assignments were made by two dimensional 1H NMR spectroscopy at pH 5.6 in the presence of saturating amounts of Ca2+. Distance geometry and simulated annealing methods were used to derive 23 convergent structures from a set of 302 interproton distance restraints and two torsion angle measurements. A high-resolution structure, with the backbone root mean square deviation to the geometric average of the 23 structures of 0.6 +/- 0.1 A, contains a linear alpha-helix from Gla 3 to Lys 15. Gla residues 3, 7, 10, and 14 are aligned in a linear array on one face of the helix. A genetic algorithm was applied to determine the calcium positions in conantokin G, and the conantokin G/Ca2+ complex refined by molecular simulation. Upon binding of Ca2+ to gamma-Carboxyglutamic Acid, conantokin G undergoes a conformational transition from a distorted curvilinear 310 helix to a linear alpha-helix. Occupancy of the metal binding sites, defined by gamma-Carboxyglutamic Acids, results in formation of a calcium-carboxylate network that linearizes the helix and exposes the hydrophobic amino Acids on the opposite face of the helix.
-
Three-dimensional structure of a gamma-Carboxyglutamic Acid-containing conotoxin, conantokin G, from the marine snail Conus geographus: the metal-free conformer.
Biochemistry, 1997Co-Authors: Alan C. Rigby, James D. Baleja, Bruce FurieAbstract:: Conantokin G is a gamma-Carboxyglutamic Acid-containing conotoxin from the venom of the marine cone snail Conus geographus. The 17-residue peptide, which contains five gamma-Carboxyglutamic Acid (Gla) residues and an amidated C-terminal asparagine amide, was synthesized chemically in a form identical to the natural conantokin G. To gain insight into the role of gamma-Carboxyglutamic Acid in the structure of this peptide, we determined the three-dimensional structure of conantokin G by 1H NMR and compared its structure to other conotoxins and to the gamma-Carboxyglutamic Acid-containing regions of the vitamin K-dependent blood-clotting proteins. Complete resonance assignments were made by two-dimensional 1H NMR spectroscopy in the absence of metal ions. NOE cross-peaks d(alphaN), d(NN), and d(betaN) provided interproton distance information, and vicinal spin-spin coupling constants 3J(HN alpha) were used to calculate phi torsion angles. Distance geometry and simulated annealing methods were used to derive 20 convergent structures from a set of 227 interproton distance restraints and 13 torsion angle measurements. The backbone rmsd to the geometric average for 20 final structures is 0.8 +/- 0.1 A. Conantokin G consists of a structured region commencing at Gla 3 and extending through arginine 13. This structure includes a partial loop centered around Gla 3 and Gla 4, a distorted type I turn between glutamine 6 and glutamine 9, and two type I turns involving Gla 10, leucine 11, and isoleucine 12 and arginine 13. Together, these two turns define approximately 1.6 turns of a distorted 3(10) helix. The observed structure possesses structural elements similar to those seen in the disulfide-linked conotoxins.
James D. Baleja - One of the best experts on this subject based on the ideXlab platform.
-
role of gamma Carboxyglutamic Acid in the calcium induced structural transition of conantokin g a conotoxin from the marine snail conus geographus
Biochemistry, 1997Co-Authors: Alan C. Rigby, James D. Baleja, Bruce Furie, Leping Li, Lee G PedersenAbstract:Conantokin G is a γ-Carboxyglutamic Acid- (Gla-) containing conotoxin isolated from the venom of the marine cone snail Conus geographus. This 17-residue polypeptide, which contains five γ-carboxygl...
-
role of gamma Carboxyglutamic Acid in the calcium induced structural transition of conantokin g a conotoxin from the marine snail conus geographus
Biochemistry, 1997Co-Authors: Alan C. Rigby, James D. Baleja, Bruce Furie, Lee G PedersenAbstract:Conantokin G is a gamma-Carboxyglutamic Acid- (Gla-) containing conotoxin isolated from the venom of the marine cone snail Conus geographus. This 17-residue polypeptide, which contains five gamma-Carboxyglutamic Acid residues, is a N-methyl-d-aspartate- (NMDA-) type glutamate receptor antagonist. To investigate the role of gamma-Carboxyglutamic Acid in the calcium-induced structural transition of conantokin G, we determined the three-dimensional structure of the conantokin G/Ca2+ complex by two-dimensional 1H NMR spectroscopy and compared it to the high-resolution structure of conantokin G in the absence of metal ions [Rigby et al. (1997) Biochemistry 36, 6906]. Complete resonance assignments were made by two dimensional 1H NMR spectroscopy at pH 5.6 in the presence of saturating amounts of Ca2+. Distance geometry and simulated annealing methods were used to derive 23 convergent structures from a set of 302 interproton distance restraints and two torsion angle measurements. A high-resolution structure, with the backbone root mean square deviation to the geometric average of the 23 structures of 0.6 +/- 0.1 A, contains a linear alpha-helix from Gla 3 to Lys 15. Gla residues 3, 7, 10, and 14 are aligned in a linear array on one face of the helix. A genetic algorithm was applied to determine the calcium positions in conantokin G, and the conantokin G/Ca2+ complex refined by molecular simulation. Upon binding of Ca2+ to gamma-Carboxyglutamic Acid, conantokin G undergoes a conformational transition from a distorted curvilinear 310 helix to a linear alpha-helix. Occupancy of the metal binding sites, defined by gamma-Carboxyglutamic Acids, results in formation of a calcium-carboxylate network that linearizes the helix and exposes the hydrophobic amino Acids on the opposite face of the helix.
-
Three-dimensional structure of a gamma-Carboxyglutamic Acid-containing conotoxin, conantokin G, from the marine snail Conus geographus: the metal-free conformer.
Biochemistry, 1997Co-Authors: Alan C. Rigby, James D. Baleja, Bruce FurieAbstract:: Conantokin G is a gamma-Carboxyglutamic Acid-containing conotoxin from the venom of the marine cone snail Conus geographus. The 17-residue peptide, which contains five gamma-Carboxyglutamic Acid (Gla) residues and an amidated C-terminal asparagine amide, was synthesized chemically in a form identical to the natural conantokin G. To gain insight into the role of gamma-Carboxyglutamic Acid in the structure of this peptide, we determined the three-dimensional structure of conantokin G by 1H NMR and compared its structure to other conotoxins and to the gamma-Carboxyglutamic Acid-containing regions of the vitamin K-dependent blood-clotting proteins. Complete resonance assignments were made by two-dimensional 1H NMR spectroscopy in the absence of metal ions. NOE cross-peaks d(alphaN), d(NN), and d(betaN) provided interproton distance information, and vicinal spin-spin coupling constants 3J(HN alpha) were used to calculate phi torsion angles. Distance geometry and simulated annealing methods were used to derive 20 convergent structures from a set of 227 interproton distance restraints and 13 torsion angle measurements. The backbone rmsd to the geometric average for 20 final structures is 0.8 +/- 0.1 A. Conantokin G consists of a structured region commencing at Gla 3 and extending through arginine 13. This structure includes a partial loop centered around Gla 3 and Gla 4, a distorted type I turn between glutamine 6 and glutamine 9, and two type I turns involving Gla 10, leucine 11, and isoleucine 12 and arginine 13. Together, these two turns define approximately 1.6 turns of a distorted 3(10) helix. The observed structure possesses structural elements similar to those seen in the disulfide-linked conotoxins.
-
three dimensional structure of a gamma Carboxyglutamic Acid containing conotoxin conantokin g from the marine snail conus geographus the metal free conformer
Biochemistry, 1997Co-Authors: Alan C. Rigby, James D. Baleja, Bruce FurieAbstract:Conantokin G is a γ-Carboxyglutamic Acid-containing conotoxin from the venom of the marine cone snail Conus geographus. The 17-residue peptide, which contains five γ-Carboxyglutamic Acid (Gla) residues and an amidated C-terminal asparagine amide, was synthesized chemically in a form identical to the natural conantokin G. To gain insight into the role of γ-Carboxyglutamic Acid in the structure of this peptide, we determined the three-dimensional structure of conantokin G by 1H NMR and compared its structure to other conotoxins and to the γ-Carboxyglutamic Acid-containing regions of the vitamin K-dependent blood-clotting proteins. Complete resonance assignments were made by two-dimensional 1H NMR spectroscopy in the absence of metal ions. NOE cross-peaks dαN, dNN, and dβN provided interproton distance information, and vicinal spin−spin coupling constants 3JHNα were used to calculate φ torsion angles. Distance geometry and simulated annealing methods were used to derive 20 convergent structures from a set of...
-
Structure of the calcium ion-bound gamma-Carboxyglutamic Acid-rich domain of factor IX.
Biochemistry, 1995Co-Authors: Steven J Freedman, Bruce Furie, James D. BalejaAbstract:: We have determined the Ca(II)-bound structure of factor IX, residues 1-47, by nuclear magnetic resonance (NMR) spectroscopy. The amino-terminal 47 residues include the gamma-Carboxyglutamic Acid-rich and aromatic amino Acid stack domains, and this region is responsible for Ca(II)-dependent phospholipid binding in factor IX. Protons in the 1-47 amino Acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. A total of 851 distance restraints and 57 torsion angle restraints were used to generate 17 final structures by distance geometry and simulated annealing methods. The backbone RMSD to the geometric average is 0.6 +/- 0.1 A. The Ca(II)-bound structure is substantially more ordered with increased helical content compared to the apo-factor IX (1-47) structure. The global fold is similar to the crystal structure of the Ca(II)-bound Gla domain of prothrombin fragment I from residues 12 to 47 (RMSD approximately 1.3 A), but the backbone conformation differs in the first 11 residues, particularly between residues 3 and 6. The amino-terminal nine Gla residues are oriented to the interior of the protein and suggest an internal Ca(II) binding pocket. The carboxyl-terminal three Gla residues are exposed to solvent. The majority of hydrophobic residues are required to stabilize a globular core in the carboxyl-terminal three-quarters of the molecule. However, a hydrophobic surface patch in the amino-terminal region may represent a phospholipid binding site in factor IX.
Alma L Burlingame - One of the best experts on this subject based on the ideXlab platform.
-
mass spectrometric based revision of the structure of a cysteine rich peptide toxin with γ Carboxyglutamic Acid txviia from the sea snail conus textile
Protein Science, 2008Co-Authors: Takemichi Nakamura, Zhonghua Yu, Mike Fainzilber, Alma L BurlingameAbstract:A mollusk-specific toxin, TxVIIA, having potent paralytic activity was isolated from the venom of sea snail Conus textile (Fainzilber M et al., 1991, Eur J Biochem 202:589-595). The structure reported above was based upon amino Acid analysis and the Edman degradation. We have recently reinvestigated this toxin employing some of the most novel techniques in mass spectrometry. We now report a revised structure based primarily on high-energy collision-induced dissociation analysis of the two Asp17-N peptides of the reduced, pyridinylethyl derivative representing the entire sequence using matrix-assisted laser desorption ionization (MALDI) as CGGYSTYC gamma VDS gamma CCSDNCVRSYCTLF-NH2 (gamma, gamma-Carboxyglutamic Acid or Gla). The N-terminus of the previous sequence was incorrect, apparently due to a side reaction of reduction and alkylation, which led to the erroneous assignment of Trp for the N-terminal residue. In addition, the last two C-terminal amino Acids and the C-terminal amidation had not been detected. Also, a combination of electrospray ionization mass spectrometry and positive and negative ion MALDI mass spectrometry provided information on the molecular weights of the native and derivatized toxin and presence of two Gla residues. Thus, TxVIIA does not have an "unusual" sequence as previously reported, but in fact belongs to the conserved Cys framework for omega- and delta-conotoxins. However, the four net negative charges with the cysteine-rich structure of this revised sequence is highly unusual for conopeptides.
-
mass spectrometric based revision of the structure of a cysteine rich peptide toxin with gamma Carboxyglutamic Acid txviia from the sea snail conus textile
Protein Science, 1996Co-Authors: Takemichi Nakamura, Mike Fainzilber, Alma L BurlingameAbstract:A mollusk-specific toxin, TxVIIA, having potent paralytic activity was isolated from the venom of sea snail Conus textile (Fainzilber M et al., 1991, Eur J Biochem 202:589-595). The structure reported above was based upon amino Acid analysis and the Edman degradation. We have recently reinvestigated this toxin employing some of the most novel techniques in mass spectrometry. We now report a revised structure based primarily on high-energy collision-induced dissociation analysis of the two Asp17-N peptides of the reduced, pyridinylethyl derivative representing the entire sequence using matrix-assisted laser desorption ionization (MALDI) as CGGYSTYC gamma VDS gamma CCSDNCVRSYCTLF-NH2 (gamma, gamma-Carboxyglutamic Acid or Gla). The N-terminus of the previous sequence was incorrect, apparently due to a side reaction of reduction and alkylation, which led to the erroneous assignment of Trp for the N-terminal residue. In addition, the last two C-terminal amino Acids and the C-terminal amidation had not been detected. Also, a combination of electrospray ionization mass spectrometry and positive and negative ion MALDI mass spectrometry provided information on the molecular weights of the native and derivatized toxin and presence of two Gla residues. Thus, TxVIIA does not have an "unusual" sequence as previously reported, but in fact belongs to the conserved Cys framework for omega- and delta-conotoxins. However, the four net negative charges with the cysteine-rich structure of this revised sequence is highly unusual for conopeptides.
Johan Stenflo - One of the best experts on this subject based on the ideXlab platform.
-
novel gamma Carboxyglutamic Acid containing peptides from the venom of conus textile
FEBS Journal, 2006Co-Authors: Bruce Furie, Barbara C. Furie, Eva Czerwiec, Dario E Kalume, Peter Roepstorff, Bjorn Hambe, Johan StenfloAbstract:The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-Carboxyglutamic Acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino Acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino Acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.
-
the metal free and calcium bound structures of a γ Carboxyglutamic Acid containing contryphan from conus marmoreus glacontryphan m
Journal of Biological Chemistry, 2004Co-Authors: Marianne A Grant, Johan Stenflo, Alan C. Rigby, Bruce Furie, Barbara C. Furie, Karin M HanssonAbstract:Abstract Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca2+ channels expressed in mouse pancreatic β-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453–32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a d-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two γ-Carboxyglutamic Acid (Gla) residues, formed by post-translational modification of glutamic Acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional 1H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys11-Cys5-Pro6 region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla2 and Ser3, moves away from the His8 and Trp10 aromatic rings and the alignment of the d-Trp7 and His8 aromatic rings with respect to the Trp10 rings is altered. The blockage of L-type voltage-gated Ca2+ channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone Cα conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 A, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation.
-
Calcium-dependent interaction between gamma-Carboxyglutamic Acid-containing and N-terminal epidermal growth factor-like modules in factor X.
Journal of Biological Chemistry, 1994Co-Authors: Carmen Valcarce, A Holmgren, Johan StenfloAbstract:Abstract The N-terminal epidermal growth factor (EGF)-like module in factor X binds a single Ca2+ with low affinity (Kd = 2.2 mM). When it is linked to the gamma-Carboxyglutamic Acid (Gla)-containing module, however, the affinity increases approximately 20-fold (Kd = 120 microM), indicating an interaction between the two modules and making the site in the N-terminal EGF-like module essentially saturated at physiological Ca2+ concentrations. We have now used the thioredoxin system to probe Ca(2+)-induced conformational changes and interaction between modules in the light chain of factor X. Thioredoxin, in conjunction with thioredoxin reductase and NADPH, allows direct measurements of the rate and extent of disulfide bond reduction. Most disulfide bonds accessible to the reducing agent were found to be located in the light chain of the protein. Moreover, those disulfide bonds that were resistant to reduction by thioredoxin in the presence of Ca2+, but were readily reduced in the absence of the metal ion, were located in the N-terminal EGF-like module and in the Gla module, whereas disulfide bonds in the C-terminal EGF-like module appeared to be equally accessible whether Ca2+ was present or not. Comparison of the rate of disulfide bond reduction in the isolated modules with that in mixtures of modules indicated that a Ca(2+)-dependent interaction occurred between the Gla and the N-terminal EGF-like module. This interaction was mediated by the C-terminal alpha-helical part of the Gla module. The affinity between the modules was low and could not be determined accurately owing to competing equilibria, presumably Ca(2+)-dependent aggregation of the isolated Gla module. By comparing the rates of disulfide bond reduction in Gla module-containing fragments before and after decarboxylation of Gla, we could demonstrate that Ca2+ binding to sites in the Gla module as well as to the single site in the EGF-like module contribute to the interaction between the two modules.
-
effects of gamma Carboxyglutamic Acid and epidermal growth factor like modules of factor ix on factor x activation studies using proteolytic fragments of bovine factor ix
Journal of Biological Chemistry, 1992Co-Authors: Jan Astermark, Philip J Hogg, Ingemar Bjork, Johan StenfloAbstract:Factor IX is a vitamin K-dependent zymogen of a serine protease. The NH2-terminal half of the molecule consists of a Ca(2+)-binding gamma-Carboxyglutamic Acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. To elucidate the role of these non-catalytic modules of factor IXa beta in factor X activation, we have isolated and characterized fragments of bovine factor IX, containing one or both of the EGF-like modules as well as these modules linked to the Gla module. The fragments were used as inhibitors of factor IXa beta-mediated factor X activation in a plasma clotting system and in systems with purified components of the Xase complex. Fragments consisting of either the two EGF-like modules of factor IX linked together or the NH2-terminal EGF-like module alone were found to inhibit factor Xa generation both in the presence and absence of the cofactor, factor VIIIa. Moreover, a fragment consisting of the corresponding modules of factor X had a similar effect. We therefore propose that factor IXa beta and factor X interact directly through their EGF-like modules on or in the vicinity of a phospholipid surface. We have also found that the isolated Gla module of factor IX inhibits the formation of factor Xa both in the presence and absence of phospholipid but not in the absence of factor VIIIa. Our results are compatible with a model of the Xase complex, in which both the serine protease part and the Gla module of factor IXa beta interact with factor VIIIa.