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| 2. |
- Hansson, Karin M, et al.
(författare)
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The first gamma-carboxyglutamic acid-containing contryphan - A selective L-type calcium ion channel blocker isolated from the venom of conus marmoreus
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:31, s. 32453-32463
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Tidskriftsartikel (refereegranskat)abstract
- 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 KD 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.
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| 3. |
- Astermark, Jan, et al.
(författare)
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Baculovirus-mediated expression of the epidermal growth factor-like modules of human factor IX fused to the factor XIIIa transamidation site in fibronectin. Evidence for a direct interaction between the NH2-terminal epidermal growth factor-like module of factor IXa beta and factor X
- 1994
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 269:5, s. 3690-3697
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Tidskriftsartikel (refereegranskat)abstract
- Factor IX is a vitamin K-dependent procoagulant zymogen of a serine protease. In the presence of Ca2+ the active form of factor IX (factor IXa beta) forms a complex with factor VIIIa on suitable phospholipid surfaces such as aggregated platelets. This macromolecular complex rapidly activates factor X. We have previously provided data that suggest an interaction between the NH2-terminal epidermal growth factor (EGF)-like module of factor IXa beta and the substrate factor X. In an alternative approach to study this protein-protein interaction, we have expressed three recombinant baculovirus constructs encoding the EGF-like modules of human factor IX and a truncated form of fibronectin in a system based on the infection of insect cells (Spodoptera frugiperda 21). This strategy allows a simple one-step purification of the recombinant proteins on a gelatin-Sepharose column, followed by removal of the gelatin-binding part derived from fibronectin by proteolytic cleavage. The fusion proteins were isolated at yields of 20-50 micrograms/ml culture medium. The recombinant EGF-like modules contained 0.2-0.4 mol of erythro-beta-hydroxyaspartic acid/mol of protein, i.e. similar to the amount found in factor IX from human plasma, and appeared to be glycosylated at Ser-53. The NH2-terminal EGF-like module, which contained a transamidation acceptor site derived from fibronectin, was cross-linked by factor XIIIa in solution to intact and Gla-domainless factor X. There was no evidence of cross-linking to activated factor X or to factor X fragments containing only the gamma-carboxyglutamic acid module and the two EGF-like modules. The cross-linking results suggest a specific interaction between the NH2-terminal EGF-like module of factor IXa beta and the heavy chain of unactivated factor X. This interaction, albeit weak as judged by competition experiments, may be important for the targeting of factor X to the factor IXa beta-factor VIIIa complex on biological membranes and for the subsequent dissociation of factor Xa from the complex after activation.
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| 4. |
- Astermark, Jan, et al.
(författare)
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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
- 1992
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 267:5, s. 3249-3256
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Astermark, Jan, et al.
(författare)
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The epidermal growth factor-like domains of factor IX. Effect on blood clotting and endothelial cell binding of a fragment containing the epidermal growth factor-like domains linked to the gamma-carboxyglutamic acid region
- 1991
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 266:4, s. 2438-2443
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Tidskriftsartikel (refereegranskat)abstract
- The binding of factor IX to cultured bovine endothelial cells was characterized using isolated domains of bovine factor IX. An NH2-terminal fragment that consists of the gamma-carboxyglutamic acid (Gla) region linked to the two epidermal growth factor (EGF)-like domains bound to the endothelial cells with the same affinity as intact factor IX, indicating that the serine protease part of factor IX is not involved in binding. This fragment also inhibited the factor IXa beta'-induced clotting of plasma at a concentration that would suggest a competition for phospholipid binding sites. However, after proteolytic removal of the Gla region from the fragment, the two EGF-like domains inhibited clotting almost as effectively, suggesting a direct interaction between this part of the molecule and the cofactor, factor VIIIa. Using affinity-purified Fab fragments against the Gla region, the EGF-like domains, and the serine protease part, it was observed that the serine protease part of the molecule undergoes a large conformational change upon activation, whereas the Gla region and the EGF-like domains appear to be unaffected. All three classes of Fab fragments were equally efficient as inhibitors of the factor IXa beta'-induced clotting reaction. Part of factor Va and factor VIIIa have significant sequence homology to a lectin. We therefore investigated the effect on in vitro clotting of the recently identified unique disaccharide Xyl alpha 1-3Glc, that is O-linked to a serine residue in the NH2-terminal EGF-like domain of human factor IX (Hase, S., Nishimura, H., Kawabata, S.-I., Iwanaga, S., and Ikenaka, T. (1990) J. Biol. Chem. 265, 1858-1861). However, no effect on blood clotting was observed in the assay system used. Our results are compatible with a model in which the serine protease part provides the specificity of the binding of factor IXa to factor VIIIa-phospholipid, but that the EGF-like domain(s) also contributes to the interaction of the enzyme with its cofactor.
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| 6. |
- Astermark, Jan, et al.
(författare)
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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
- 1994
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 269:5, s. 3682-3689
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Hjalmarsson, Claes, et al.
(författare)
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Activated Protein C-Protein C Inhibitor Complex, Activation Peptide of Carboxypeptidase B and C-Reactive Protein as Predictors of Severe Acute Pancreatitis.
- 2009
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Ingår i: Pancreatology. - : Elsevier BV. - 1424-3903. ; 9:5, s. 700-707
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: The concentration of carboxypeptidase B activation peptide (CAPAP) is proposed to be a predictor of severe acute pancreatitis. The activated protein C (APC)-protein C inhibitor (PCI; APC-PCI) complex in plasma could be useful in detecting the hypercoagulative condition in severe acute pancreatitis. Method: In this prospective study, mild (n = 50) and severe (n = 9) cases of acute pancreatitis were compared with respect to levels of CAPAP and APC-PCI, and sorted in time intervals from onset of symptoms to sampling. The peak values of the C-reactive protein (CRP) within the 1st week were also compared. Results: CRP detected the severe cases with a sensitivity of 0.89 and a specificity of 0.74 (cut-off level 200 mg/l). In the interval 0-72 h, CAPAP could predict the severity of the disease in serum and urine (sensitivity 0.52/0.29, specificity 0.73/0.93, cut-off 2 nM/60 nM). The level of APC-PCI in plasma could predict the severe condition in the interval 0-24 h after the onset of symptoms (sensitivity 0.6, specificity 0.66, cut-off level 0.54 mug/l). Conclusion: Of the parameters explored, CRP is still the best biochemical marker to distinguish between severe and mild acute pancreatitis. CAPAP could be useful in combination with other tests, but the APC-PCI complex's diagnostic time interval is too short to be used in the clinical routine. and IAP.
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| 8. |
- Persson, Kristina, et al.
(författare)
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The N-terminal EGF domain of coagulation factor IX: Probing its functions in the activation of factor IX and factor X with a monoclonal antibody.
- 2002
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:38, s. 35616-35624
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Tidskriftsartikel (refereegranskat)abstract
- SUMMARY Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor (EGF)-like domains and a serine protease domain. In this study the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by FXIa, but activation by FVIIa/tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent kcat for FX both in the presence and absence of FVIIIa. Based on these results, we produced a preliminary model of the structure of the FIXa-FVIIIa-AW complex on the surface of phospholipid. The model suggests that in the Xase complex EGF1 of FIXa is not involved in direct binding to FVIIIa. Studies of the interaction of antibody AW with a mutated FIX molecule (Arg94Asp) also suggest that the Glu78-Arg94 salt-bridge is not important for maintaining the structure of FIX.
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| 9. |
- Astermark, Jan, et al.
(författare)
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Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact domains isolated from controlled proteolytic digests of bovine factor IX
- 1991
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 266:4, s. 2430-2437
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Tidskriftsartikel (refereegranskat)abstract
- Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Bjork, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42.
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| 10. |
- Brown, Mark A., et al.
(författare)
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Detection of vitamin K-dependent proteins in venoms with a monoclonal antibody specific for gamma-carboxyglutamic acid.
- 2002
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Ingår i: Toxicon. - 0041-0101. ; 40:4, s. 447-453
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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