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Francis J. Castellino - One of the best experts on this subject based on the ideXlab platform.
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Properties of a recombinant chimeric protein in which the Gamma-Carboxyglutamic Acid and helical stack domains of human anticoagulant protein C are replaced by those of human coagulation factor VII.
Thrombosis and Haemostasis, 1997Co-Authors: Jie-ping Geng, Francis J. CastellinoAbstract:: A chimeric cDNA, encoding residues 1-46 (the Gamma-Carboxyglutamic Acid module and its trailing helical stack) of human coagulant factor (f) VII, bound to residues 47-419 of human anticoagulant protein C (PC), was constructed and expressed. The resulting protein, r-[delta GD-HSPC/[symbol: see text] GD-HSfVII]PC, was properly processed with regard to signal/propeptide release, cleavage of the K156R dipeptide, Gla and Hya contents, and the presence of glycosylation. The mutant protein displayed normal dependencies on Ca2+ for adoption of its metal ion-dependent conformation and for binding to Acidic phospholipid vesicles. The chimera failed to recognize a monoclonal antibody (MAb) specific for the Ca(2+)-induced conformation of the Gla domain (GD) of PC, but did react with another MAb directed in part to the Ca(2+)-dependent conformation of the GD of fVII. Further, this chimeric protein possessed similar steady state constants as wild-type r-PC toward activation by thrombin and thrombin/thrombomodulin. The activated form of the chimera was very similar to that of its wild-type counterpart in its whole plasma anticoagulant activity, as well as its activity toward inactivation of coagulation factor VIII. The chimeric protein did not bind to the fVII cofactor, tissue factor, showing that the GD/HS domain region of fVII is insufficient for that particular interaction. The results demonstrate that the GD/HS of fVII, when present in the PC and APC background, serves to maintain the Ca2+/PL-related functions of these latter proteins, and suggest that the Ca2+ and PL-dependent interactions of the GD-HS of PC are sufficiently general in nature such that the GD-HS regions of other proteins of this type can satisfy most of the requirements of PC and APC. The data presented also offer support for the independent nature of the domain unit consisting of the GD/HS module.
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calcium binding properties of synthetic gamma carboxyglutamic Acid containing marine cone snail sleeper peptides conantokin g and conantokin t
Biochemistry, 1996Co-Authors: Mary Prorok, Scott E Warder, Tamas Blandl, Francis J. CastellinoAbstract:Total chemical synthesis of two Conus-derived peptides, conantokin-G (con-G), a 17-residue polypeptide containing five residues of Gamma-Carboxyglutamic Acid (Gla), and conantokin-T (con-T), a 21-residue polypeptide possessing four residues of Gla, was accomplished. Calcium binding isotherms were obtained for each peptide, and these differed considerably from each other. The binding isotherm for con-G was complex and could only be fit to degenerate models involving multiple Ca2+ binding sites. The data for Ca2+ binding to con-T was uniquely fit to a simple one-site model. In the case of con-G, circular dichroism (CD) studies revealed a polypeptide without observable alpha-helicity in the absence of Ca2+ and a dramatic shift to a high degree of alpha-helix at saturating Ca2+ concentrations. In contrast, apo-con-T possessed significant alpha-helical structure, and saturation with Ca2+ produced a less substantial change in its alpha-helical content. Titrations with Ca2+ of the change in alpha-helical content of con-T produced a C50 value for Ca2+ that was essentially the same as its Kd from direct binding studies, demonstrating that occupancy of the single macroscopic binding site resulted in the conformational change. Similar titrations with con-G provided a C50 value in concert with the Kd for binding of Ca2+ to this peptide. Moreover, in agreement with these particular Ca(2+)-induced structural changes, gel filtration analyses demonstrated significantly reduced hydrodynamic volumes of both of these polypeptides after saturation of their apo forms with Ca2+, with con-G showing a more pronounced change than con-T. One-dimensional H-NMR spectra showed both line broadening and changes in chemical shifts of several peptide amide proton resonances after addition of Ca2+ to con-G, again suggestive of a large Ca(2+)-induced conformational change in this polypeptide. A derivative of con-G was synthesized with all amino Acids present in the D-configuration (D-con-G). This variant peptide displayed Ca2+ binding isotherms nearly identical to those of con-G and underwent a Ca(2+)-induced conformational change very similar to that of con-G. Intracranial injections of con-G and con-T in young (< 2 weeks) and older (3-4 weeks) mice produced the expected "sleep-like" and hyperactive effects, respectively. The variant, D-con-G, was inactive in these assays. These studies demonstrate that synthetic con-G and con-T possess their expected bioactivities and undergo large and defined conformational alterations in the presence of Ca2+. We propose that binding of Ca2+ to these polypeptides contributes to their ability to adopt a defined conformation, and this divalent cation-dependent conformation is necessary for their neuroactivities.
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Binding of calcium to individual Gamma-Carboxyglutamic Acid residues of human protein C.
Biochemistry, 1995Co-Authors: Tracey L. Colpitts, Maryfrances Prorok, Francis J. CastellinoAbstract:: Selectively labeled polypeptides comprising the Gamma-Carboxyglutamic Acid (Gla) domain (GD) and helical stack (HS) regions of human protein C (PC), and consisting of amino Acid residues 1-47, have been chemically synthesized and their Ca2+ binding properties assessed by [13C]-NMR methods. A total of nine such polypeptides have been studied, each containing one of the Gla residues fully enriched with [13C] at its two gamma-carboxylate carbon atoms. Additions of Ca2+ resulted in readily measurable [13C] chemical shifts, titrations of which were used to obtain apparent dissociation constants for each Gla residue in the presence of all other such residues. The Ca2+ titration data obtained on each of the nine polypeptides showed that Gla residues 6, 16, 25, and 26 were involved in the higher affinity Ca2+ binding sites, whereas the remaining Gla residues, viz., 7, 14, 19, 20, and 29, coordinated Ca2+ more weakly. The results are consistent with conclusions drawn from functional studies obtained with site-directed mutations of individual Gla residues and with the structural model of the GD/HS of human PC. In these cases, Gla residues 6, 16, and 26 served as coordination loci for internally located Ca2+ ions, and GD-related Ca(2+)- and PL-dependent properties of PC and activated PC were dependent on the integrity of these Gla residues.
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Functions of individual Gamma-Carboxyglutamic Acid (Gla) residues of human protein c. Determination of functionally nonessential Gla residues and correlations with their mode of binding to calcium.
Biochemistry, 1994Co-Authors: William T. Christiansen, Alexander Tulinsky, Francis J. CastellinoAbstract:: Previous studies from this laboratory have been directed toward elucidation of the roles of individual Gamma-Carboxyglutamic Acid (Gla) residues in Gla domain-related Ca(2+)-directed properties of human protein C (PC) and activated protein C (APC). On the basis of results using recombinant variants of PC containing highly conservative (Asp) mutations of individual Gla residues, it was previously proposed that Gla6, Gla14, and Gla19 may not be essential for properties associated with the Ca(2+)-dependent conformation of the Gla domain of these proteins. In this study, we have demonstrated that radical mutations to Val of Gla residues 14 and 19 resulted in 94% and 82%, respectively, of the Gla domain-related, Ca(2+)- and phospholipid- (PL-) dependent anticoagulant (APTT) activity of wild-type recombinant (wtr) APC, while [Gla6-->Val]r-APC showed a complete loss of this same activity. The more conservative mutant [Gla6-->Gln]r-APC possessed 4% of the APTT activity of wtr-APC, whereas [Gla6-->Asp]r-APC was nearly fully active. As with wtr-PC, both [Gla6-->Val]r-PC and [Gla6-->Gln]r-PC displayed Ca(2+)-dependent intrinsic fluorescence quenching, suggesting that they adopted a Ca(2+)-induced conformation. However, Ca2+ titration data suggested that these conformations were not identical to that undergone by wtr-PC. In addition, the Ca(2+)-mediated binding parameters of [Gla6-->Val]r-PC and [Gla6-->Gln]r-PC to Acidic PL vesicles were found to be defective. These data were interpreted at the molecular level using a model for the Gla domain of PC based on the X-ray crystal structure of the Ca2+/bovine prothrombin fragment 1 complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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Calcium and phospholipid binding properties of synthetic Gamma-Carboxyglutamic Acid-containing peptides with sequence counterparts in human protein C.
Biochemistry, 1994Co-Authors: Tracey L. Colpitts, Francis J. CastellinoAbstract:: Two peptides with counterpart sequences in the Gamma-Carboxyglutamic Acid (Gla) domain of human protein C (PC) have been synthesized and characterized. One peptide contained 38 amino Acids (38-mer) and spanned the region from the amino terminus of the protein to the DNA splice junction between the Gla domain and the following short helical stretch, and the second peptide (48-mer) included a 10 amino Acid extension that has been designed to incorporate the exon for the helical segment that is thought to play a role in stabilizing the Ca(2+)-dependent conformation of the Gla domain of proteins of this class. The peptides were synthesized by solid-phase methodology, then oxidized to allow disulfide pairing, and finally purified by FPLC methodology. Chemical characterization showed that each peptide contained its full complement of Gla residues. Two types of Ca(2+)-binding sites were found in these peptides, tighter sights (2-3) with Kd values of 60-370 microM and a weaker set of sites (7-10) with a range of Kd values from 0.8 to 3.1 mM. In general, the 48-mer interacted with Ca2+ more tightly than the 38-mer. As revealed by circular dichroism analysis, and by reactivity with monoclonal antibodies that recognize both the unfolded form of the Gla domain as well as the Ca(2+)-dependent conformation of this same domain, the 38-mer and 48-mer underwent the Ca(2+)-induced conformational changes characteristic of the intact protein. Both peptides displayed Ca(2+)-dependent binding to negatively charged phospholipid vesicles (PL).(ABSTRACT TRUNCATED AT 250 WORDS)
Johan Stenflo - One of the best experts on this subject based on the ideXlab platform.
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Novel Gamma-Carboxyglutamic Acid-containing peptides from the venom of Conus textile
FEBS Journal, 2006Co-Authors: Eva Czerwiec, Barbara C. Furie, Bruce Furie, Dario E. Kalume, Peter Roepstorff, Björn 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 Conus gamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the K-m was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 mu M when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the K-m value tenfold. (Less)
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Isolation and characterization of three novel Gla-containing Conus marmoreus venom peptides, one with a novel cysteine pattern.
Biochemical and Biophysical Research Communications, 2004Co-Authors: Karin M Hansson, Barbara C. Furie, Bruce Furie, Johan StenfloAbstract:: One defining characteristic of Conus venom peptides is the high frequency of posttranslational modifications found. We report the discovery and initial characterization of three novel Gamma-Carboxyglutamic Acid (Gla)-containing conotoxins, Gla-MrII, Gla-MrIII, and Gla-MrIV, isolated from the venom of the mollusc-hunting cone snail Conus marmoreus. Peptide Gla-MrII, a 50 amino Acid residue peptide, carries eight cysteine residues arranged in a novel cysteine pattern, and five Gamma-Carboxyglutamic Acid residues. The monoisotopic molecular mass was determined by electrospray ionization mass spectrometry to 5860.23 Da, consistent with the peptide having the cysteine residues disulphide-bonded and having a free Acid C-terminus. Peptides Gla-MrIII and Gla-MrIV each contain two Gamma-Carboxyglutamic Acid residues and share little sequence similarity to previously identified conotoxins. Both peptides contain four cysteine residues that are positioned in the linear sequence in a manner reminiscent of conotoxins belonging to cysteine scaffold superfamily T (scaffold T-1). Determination of the monoisotopic molecular masses revealed that Gla-MrIII is amidated at its C-terminus while Gla-MrIV has a free C-terminal Acid.
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Detection of vitamin K-dependent proteins in venoms with a monoclonal antibody specific for Gamma-Carboxyglutamic Acid.
Toxicon, 2002Co-Authors: Mark A. Brown, Barbara C. Furie, Bruce Furie, Johan Stenflo, Björn Hambe, Leisa M. StenbergAbstract:Gamma-Carboxyglutamic Acid (Gla) is an unusual amino Acid that is synthesized post-translationally from glutamate in a vitamin K-dependent reaction. The dicarboxylic side chain of Gla chelates Ca(2+), a property important for the biological activity of vitamin K-dependent proteins. To date, Gla-containing polypeptides have been identified in venom from two groups of organisms: elapid snakes, and snails of the genus Conus. In certain elapid snakes, a gamma-carboxylated coagulation factor Xa-like protein is a component of the venom whereas cone snails utilize Gla in a range of peptide neurotoxins. Using a monoclonal antibody that specifically recognizes Gla residues, venom samples from various organisms were screened by western blotting and immunofluorescence assays. Amino Acid analyses were also performed on most samples. A survey of 21 snake species from 12 genera detected gamma-carboxylated polypeptides only in venom of snakes from the elapid subfamily Acanthophiinae. Gla-containing polypeptides were also observed in cone snail venom but not in venom or toxic salivary secretions from several other organisms. The Gla-specific antibody used here provides a simple immunochemical means to detect gamma-carboxylated polypeptides in venom and may allow new species to be identified that utilize Gla in the biosynthesis of toxic polypeptides. (Less)
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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: C 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.
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The Gamma-Carboxyglutamic Acid and epidermal growth factor-like modules of factor IXa beta. Effects on the serine protease module and factor X activation
Journal of Biological Chemistry, 1994Co-Authors: Jan Astermark, Philip J. Hogg, Johan StenfloAbstract:Blood coagulation factors IX and X are two serine proteases with a similar modular structure. The non-catalytic part of each protein consists of a Gamma-Carboxyglutamic Acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. We have now found that the NH2-terminal EGF-like module of both factors IX and X inhibits factor Xa formation in a Gla-independent manner, both in the presence and absence of phospholipid and the cofactor, factor VIIIa. In contrast, the COOH-terminal EGF-like module has no such effect. Our data indicate that the NH2-terminal EGF-like module of factor IXa beta interacts either with the corresponding module or with the serine protease module in the substrate, factor X, without affecting the hydrolysis of low molecular weight substrates. Using antibodies as structural probes, we found that Ca2+ binding to the Gla module of factor IXa beta induces a conformational transition in the serine protease module. No evidence was found for a direct interaction between the Gla module and factor VIIIa. We therefore propose that the Gla module in factor IXa beta is indirectly involved in the cofactor interaction, in that Ca2+ binding to sites in this module induces a conformation in the serine protease module that is commensurate with factor VIIIa interaction. In addition, the immunochemical approach revealed a Gla-independent Ca2+ binding site in the serine protease module (apparent Kd of approximately 120 microM) that also might influence its conformation. Antibodies against the EGF-like modules of factor IX were used to probe Ca2+ binding to these modules in intact and in Gla-domainless factor IXa beta. The data indicate a Ca2+ binding site with an apparent Kd of approximately 50 microM in the NH2-terminal EGF-like module of both factor IX species.
Barbara C. Furie - One of the best experts on this subject based on the ideXlab platform.
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Novel Gamma-Carboxyglutamic Acid-containing peptides from the venom of Conus textile
FEBS Journal, 2006Co-Authors: Eva Czerwiec, Barbara C. Furie, Bruce Furie, Dario E. Kalume, Peter Roepstorff, Björn 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 Conus gamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the K-m was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 mu M when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the K-m value tenfold. (Less)
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Isolation and characterization of three novel Gla-containing Conus marmoreus venom peptides, one with a novel cysteine pattern.
Biochemical and Biophysical Research Communications, 2004Co-Authors: Karin M Hansson, Barbara C. Furie, Bruce Furie, Johan StenfloAbstract:: One defining characteristic of Conus venom peptides is the high frequency of posttranslational modifications found. We report the discovery and initial characterization of three novel Gamma-Carboxyglutamic Acid (Gla)-containing conotoxins, Gla-MrII, Gla-MrIII, and Gla-MrIV, isolated from the venom of the mollusc-hunting cone snail Conus marmoreus. Peptide Gla-MrII, a 50 amino Acid residue peptide, carries eight cysteine residues arranged in a novel cysteine pattern, and five Gamma-Carboxyglutamic Acid residues. The monoisotopic molecular mass was determined by electrospray ionization mass spectrometry to 5860.23 Da, consistent with the peptide having the cysteine residues disulphide-bonded and having a free Acid C-terminus. Peptides Gla-MrIII and Gla-MrIV each contain two Gamma-Carboxyglutamic Acid residues and share little sequence similarity to previously identified conotoxins. Both peptides contain four cysteine residues that are positioned in the linear sequence in a manner reminiscent of conotoxins belonging to cysteine scaffold superfamily T (scaffold T-1). Determination of the monoisotopic molecular masses revealed that Gla-MrIII is amidated at its C-terminus while Gla-MrIV has a free C-terminal Acid.
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Detection of vitamin K-dependent proteins in venoms with a monoclonal antibody specific for Gamma-Carboxyglutamic Acid.
Toxicon, 2002Co-Authors: Mark A. Brown, Barbara C. Furie, Bruce Furie, Johan Stenflo, Björn Hambe, Leisa M. StenbergAbstract:Gamma-Carboxyglutamic Acid (Gla) is an unusual amino Acid that is synthesized post-translationally from glutamate in a vitamin K-dependent reaction. The dicarboxylic side chain of Gla chelates Ca(2+), a property important for the biological activity of vitamin K-dependent proteins. To date, Gla-containing polypeptides have been identified in venom from two groups of organisms: elapid snakes, and snails of the genus Conus. In certain elapid snakes, a gamma-carboxylated coagulation factor Xa-like protein is a component of the venom whereas cone snails utilize Gla in a range of peptide neurotoxins. Using a monoclonal antibody that specifically recognizes Gla residues, venom samples from various organisms were screened by western blotting and immunofluorescence assays. Amino Acid analyses were also performed on most samples. A survey of 21 snake species from 12 genera detected gamma-carboxylated polypeptides only in venom of snakes from the elapid subfamily Acanthophiinae. Gla-containing polypeptides were also observed in cone snail venom but not in venom or toxic salivary secretions from several other organisms. The Gla-specific antibody used here provides a simple immunochemical means to detect gamma-carboxylated polypeptides in venom and may allow new species to be identified that utilize Gla in the biosynthesis of toxic polypeptides. (Less)
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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, Barbara C. Furie, 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.
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Structure of the calcium ion-bound Gamma-Carboxyglutamic Acid-rich domain of factor IX.
Biochemistry, 1995Co-Authors: Steven J. Freedman, Barbara C. Furie, 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.
Bruce Furie - One of the best experts on this subject based on the ideXlab platform.
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Novel Gamma-Carboxyglutamic Acid-containing peptides from the venom of Conus textile
FEBS Journal, 2006Co-Authors: Eva Czerwiec, Barbara C. Furie, Bruce Furie, Dario E. Kalume, Peter Roepstorff, Björn 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 Conus gamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the K-m was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 mu M when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the K-m value tenfold. (Less)
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Isolation and characterization of three novel Gla-containing Conus marmoreus venom peptides, one with a novel cysteine pattern.
Biochemical and Biophysical Research Communications, 2004Co-Authors: Karin M Hansson, Barbara C. Furie, Bruce Furie, Johan StenfloAbstract:: One defining characteristic of Conus venom peptides is the high frequency of posttranslational modifications found. We report the discovery and initial characterization of three novel Gamma-Carboxyglutamic Acid (Gla)-containing conotoxins, Gla-MrII, Gla-MrIII, and Gla-MrIV, isolated from the venom of the mollusc-hunting cone snail Conus marmoreus. Peptide Gla-MrII, a 50 amino Acid residue peptide, carries eight cysteine residues arranged in a novel cysteine pattern, and five Gamma-Carboxyglutamic Acid residues. The monoisotopic molecular mass was determined by electrospray ionization mass spectrometry to 5860.23 Da, consistent with the peptide having the cysteine residues disulphide-bonded and having a free Acid C-terminus. Peptides Gla-MrIII and Gla-MrIV each contain two Gamma-Carboxyglutamic Acid residues and share little sequence similarity to previously identified conotoxins. Both peptides contain four cysteine residues that are positioned in the linear sequence in a manner reminiscent of conotoxins belonging to cysteine scaffold superfamily T (scaffold T-1). Determination of the monoisotopic molecular masses revealed that Gla-MrIII is amidated at its C-terminus while Gla-MrIV has a free C-terminal Acid.
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Detection of vitamin K-dependent proteins in venoms with a monoclonal antibody specific for Gamma-Carboxyglutamic Acid.
Toxicon, 2002Co-Authors: Mark A. Brown, Barbara C. Furie, Bruce Furie, Johan Stenflo, Björn Hambe, Leisa M. StenbergAbstract:Gamma-Carboxyglutamic Acid (Gla) is an unusual amino Acid that is synthesized post-translationally from glutamate in a vitamin K-dependent reaction. The dicarboxylic side chain of Gla chelates Ca(2+), a property important for the biological activity of vitamin K-dependent proteins. To date, Gla-containing polypeptides have been identified in venom from two groups of organisms: elapid snakes, and snails of the genus Conus. In certain elapid snakes, a gamma-carboxylated coagulation factor Xa-like protein is a component of the venom whereas cone snails utilize Gla in a range of peptide neurotoxins. Using a monoclonal antibody that specifically recognizes Gla residues, venom samples from various organisms were screened by western blotting and immunofluorescence assays. Amino Acid analyses were also performed on most samples. A survey of 21 snake species from 12 genera detected gamma-carboxylated polypeptides only in venom of snakes from the elapid subfamily Acanthophiinae. Gla-containing polypeptides were also observed in cone snail venom but not in venom or toxic salivary secretions from several other organisms. The Gla-specific antibody used here provides a simple immunochemical means to detect gamma-carboxylated polypeptides in venom and may allow new species to be identified that utilize Gla in the biosynthesis of toxic polypeptides. (Less)
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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, Barbara C. Furie, 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.
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Structure of the calcium ion-bound Gamma-Carboxyglutamic Acid-rich domain of factor IX.
Biochemistry, 1995Co-Authors: Steven J. Freedman, Barbara C. Furie, 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.
Leonard J. Deftos - One of the best experts on this subject based on the ideXlab platform.
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Serum markers of bone formation in parenteral nutrition patients
Calcified Tissue International, 1990Co-Authors: Edward W. Lipkin, Gordon L. Klein, Leonard J. DeftosAbstract:Bone Gamma-Carboxyglutamic Acid containing protein (BGP) has been utilized effectively as a serum marker of bone turnover in healthy normals and in individuals with a variety of metabolic bone disorders including postmenopausal osteoporosis and Paget's disease. The utility of this serum marker in other bone disorders, including that associated with the maintenance of patients on long-term parenteral nutrition, still requires definition. Because of our interest in this clinical syndrome and the availability of serum and of bone formation rates (BFR) measured directly from double tetracycline labeling in 11 long-term parenteral nutrition patients, we measured BGP levels in these patients and attempted to correlate this measure with BFR. Serum vitamin D metabolites, immunoreactive parathyroid hormone (PTH), and alkaline phosphatase (alk phos) were also measured. Serum BGP was only weakly and not significantly correlated (r=0.24, p=NS) with bone formation rate for the group as a whole. However, in a subgroup of 10 patients without hyperparathyroidism, there was strong and significant correlation (r=0.81, P
