| 1. |
- Mark, Linda, et al.
(författare)
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Kaposi's sarcoma-associated herpes virus complement control protein: KCP - complement inhibition and more.
- 2007
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 44, s. 11-22
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Tidskriftsartikel (refereegranskat)abstract
- The complement system is an important part of innate immunity providing immediate protection against pathogens without a need for previous exposure, as well as priming the adaptive immune response through opsonisation, leukocyte recruitment and enhancing humoral immune responses. Its importance is not only shown through recurring fulminant infections in individuals with complement component deficiencies, but also through the many complement evasion strategies discovered for a wide range of infectious microbes (including acquisition of endogenous host complement inhibitors and expression of own homologues). Knowledge of these mechanisms at a molecular level may aid development of vaccines and novel therapeutic strategies. Here, we review the structure-function studies of the membrane-bound complement inhibitor KCP that is expressed on the surface of Kaposi's sarcoma-associated herpesvirus (KSHV) virions and infected cells. KCP accelerates the decay of classical C3 convertase and induces the degradation of activated complement factors C4b and C3b by serine proteinase, factor I. Molecular modeling and site-directed mutagenesis have identified sites on the surface of endogenous human inhibitors. KCP additionally enhances virion binding to permissive cells through a heparin/heparan sulfate-binding site located at the N-terminus of the protein.
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| 2. |
- Mark, Linda, et al.
(författare)
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The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1-2.
- 2006
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 43:10, s. 1665-1675
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Tidskriftsartikel (refereegranskat)abstract
- The Kaposi's, sarcoma-associated herpesvirus (KSHV) complement control protein (KCP) inhibits the human complement system, and is similar in structure and function to endogenous complement inhibitors. Other inhibitors such as C4d-binding protein and factor H, as well as the viral homologue vaccinia virus complement control protein are known to bind heparin and, for the two latter, also to glycosaminoglycans at the surface of cells. We report here that KCP also binds to heparin at physiological ionic strength. With help of site directed mutagenesis, positively charged amino acids in the two N-terminal complement control protein (CCP) domains 1-2 were found to be necessary for heparin binding. In silico molecular docking of heparin to KCP confirmed the experimental data, and further explored the heparin binding site. enabling us to present a model of the KCP-heparin interaction. Furthermore, the docking analysis also yielded insights of the KCP structure, by indicating that the angle between CCP domains 1-2 during the initial binding of heparin is more extended than in the model we have previously presented. We also found that KCP binds to heparan sulfate and weakly to glycosaminoglycans at the surface of cells. This might indicate that KCP at the Surface of viral particles aids in the primary attachment to the target cells, which is known to involve binding to heparan sulfate. Therefore. the present study contributes to the knowledge of heparin-protein interactions in general its well as to the understanding of the biology of KSHV. (c) 2005 Elsevier Ltd. All rights reserved.
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| 3. |
- Okroj, Marcin, et al.
(författare)
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Characterization of the complement inhibitory function of Rhesus rhadinovirus complement control protein (RCP).
- 2009
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 2008:Nov 6., s. 505-514
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Tidskriftsartikel (refereegranskat)abstract
- Rhesus Rhadinovirus (RRV) is currently the closest known, fully sequenced homolog of human Kaposi's sarcoma-associated herpesvirus (KSHV). Both these viruses encode complement inhibitors: KSHV-complement control protein (KCP) and RRV-complement control protein (RCP). Previously we characterized in detail the functional properties of KCP as complement inhibitor. Herein, we performed comparative analyses for two variants of RCP protein, encoded by RRV strains H26-95 and 17577. Both RCP variants and KCP inhibited human and rhesus complement when tested in hemolytic assays measuring all steps of activation via the classical and the alternative pathway. RCP variants from both RRV strains supported C3b- and C4b-degradation by factor I and decay-acceleration of the classical C3 convertase, similar to KCP. Additionally, the 17577 RCP variant accelerated decay of the alternative C3 convertase, which was not seen for KCP. In contrast to KCP, RCP showed no affinity to heparin and is the first described complement inhibitor in which the binding site for C3b/C4b does not interact with heparin. Molecular modeling shows a structural disruption in the region of RCP that corresponds to the KCP-heparin binding site. This makes RRV a superior model for future in vivo investigations of complement evasion, as RCP does not play a supportive role in viral attachment as KCP does.
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| 4. |
- Blom, Anna M, et al.
(författare)
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A Novel Non-Synonymous Polymorphism (p.Arg240His) in C4b-Binding Protein Is Associated with Atypical Hemolytic Uremic Syndrome and Leads to Impaired Alternative Pathway Cofactor Activity.
- 2008
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Ingår i: Journal of Immunology. - 1550-6606. ; 180:9, s. 6385-6391
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Tidskriftsartikel (refereegranskat)abstract
- Atypical hemolytic uremic syndrome (aHUS) is a disorder characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations, polymorphisms, and copy number variation in complement factors and inhibitors are associated with aHUS. In this study, we report the first functional non-synonymous polymorphism in the complement inhibitor C4b-binding protein (C4BP) alpha-chain (c.719G>A; p.Arg240His), which is associated with aHUS. This heterozygous change was found in 6/166 aHUS patients compared with 5/542 normal (chi2 = 6.021; p = 0.014), which was replicated in a second cohort of aHUS patients in which we found 5/170 carriers. The polymorphism does not decrease expression efficiency of C4BP. p.Arg240His is equally efficient as the wild type in binding and supporting degradation of C4BP but its ability to bind C3b and act as cofactor to its degradation both in fluid phase and on surfaces is impaired. This observation supports the hypothesis that dysregulation of the alternative pathway of complement is pivotal for aHUS. Three of the patients carry also mutations in membrane cofactor protein and factor H strengthening the hypothesis that individuals may carry multiple susceptibility factors with an additive effect on the risk of developing aHUS.
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| 5. |
- Dahlbäck, Björn, et al.
(författare)
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Regulation of Blood Coagulation by the Protein C Anticoagulant Pathway. Novel Insights Into Structure-Function Relationships and Molecular Recognition.
- 2005
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1524-4636. ; 25:7, s. 1311-1320
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Forskningsöversikt (refereegranskat)abstract
- The protein C system provides important control of blood coagulation by regulating the activities of factor VIIIa (FVIIIa) and factor Va(FVa), cofactors in the activation of factor X and prothrombin, respectively. The system comprises membrane-bound and circulating proteins that assemble into multi-molecular complexes on cell surfaces. Vitamin K-dependent protein C, the key component of the system, circulates in blood as zymogen to an anticoagulant serine protease. It is efficiently activated on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The endothelial protein C receptor (EPCR) further stimulates the protein C activation. Activated protein C (APC) together with its cofactor protein S inhibits coagulation by degrading FVIIIa and FVa on the surface of negatively charged phospholipid membranes. Efficient FVIIIa degradation by APC requires not only protein S but also intact FV, which like thrombin is a Janus-faced protein with both procoagulant and anticoagulant potential. In addition to its anticoagulant properties, APC has antiinflammatory and antiapoptotic functions, which are exerted when APC binds to EPCR and proteolytic cleaves protease-activated receptor 1 (PAR-1). The protein C system is physiologically important, and genetic defects affecting the system are the most common risk factors of venous thrombosis. The proteins of the protein C system are composed of multiple domains and the 3-dimensional structures of several of the proteins are known. The molecular recognition of the protein C system is progressively being unraveled, giving us new insights into this fascinating and intricate molecular scenario at the atomic level.
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| 6. |
- Dahlbäck, Björn, et al.
(författare)
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The anticoagulant protein C pathway.
- 2005
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 579:15, s. 3310-3316
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Forskningsöversikt (refereegranskat)
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| 7. |
- Jarva, Hanna, et al.
(författare)
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Molecular Characterization of the Interaction between Porins of Neisseria gonorrhoeae and C4b-Binding Protein.
- 2007
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Ingår i: Journal of Immunology. - 1550-6606. ; 179:1, s. 540-547
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Tidskriftsartikel (refereegranskat)abstract
- Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitors C4b-binding protein (CUP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCPI) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to PorlA strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to PorlA strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to PorlA gonococci. C4BP binding to Por1-B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.
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| 8. |
- Knobe, Karin, et al.
(författare)
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Functional Analysis of the Factor IX Epidermal Growth Factor-Like Domain Mutation Ile66Thr Associated with Mild Hemophilia B.
- 2006
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Ingår i: Pathophysiology of Haemostasis and Thrombosis. - : S. Karger AG. - 1424-8832 .- 1424-8840. ; 35:5, s. 370-375
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Tidskriftsartikel (refereegranskat)abstract
- he present study focused on the functional role of the mutation Ile66Thr located in the N-terminal epidermal growth factor-like domain of coagulation factor IX (FIX). This mutation causes mild hemophilia B with approximately 25% FIX coagulant activity and FIX antigen levels of around 90% of normal. In the 3-dimensional structure of porcine FIXa and in the subsequent 3-dimensional model of human FIXa that we have previously developed, residue 66 is exposed to the solvent and can be replaced by many amino acids, including Thr, without affecting the major folding/stability of the molecule. This is consistent with the basically normal antigen levels observed. We found that the FIX Ile66Thr mutant was activated to a normal extent by FVIIa/TF and FXIa. However, the ability of FIX Ile66Thr to activate FX was impaired in both the presence and absence of FVIIIa, indicating that Ile66 is not directly involved in the binding of FIX to FVIIIa.
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| 9. |
- Nilsson, Sara, et al.
(författare)
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Genetic, molecular and functional analyses of complement factor I deficiency.
- 2009
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Ingår i: European Journal of Immunology. - : Wiley. - 1521-4141 .- 0014-2980. ; 39:1, s. 310-323
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Tidskriftsartikel (refereegranskat)abstract
- Complete deficiency of complement inhibitor factor I (FI) results in secondary complement deficiency due to uncontrolled spontaneous alternative pathway activation leading to susceptibility to infections. Current genetic examination of two patients with near complete FI deficiency and three patients with no detectable serum FI and also close family members revealed homozygous or compound heterozygous mutations in several domains of FI. These mutations were introduced into recombinant FI and the resulting proteins were purified for functional studies, while transient transfection was used to analyze expression and secretion. The G170V mutation resulted in a protein that was not expressed, whereas the mutations Q232K, C237Y, S250L, I339M and H400L affected secretion. Furthermore, the C237Y and the S250L mutants did not degrade C4b and C3b as efficiently as the WT. The truncated Q336x mutant could be expressed, in vitro, but was not functional because it lacks the serine protease domain. Furthermore, this truncated FI was not detected in serum of the patient. Structural investigations using molecular modeling were performed to predict the potential impact the mutations have on FI structure. This is the first study that investigates, at the functional level, the consequences of molecular defects identified in patients with full FI deficiency.
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| 10. |
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| 11. |
- Steen, Mårten, et al.
(författare)
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Mapping of the factor Xa-binding site on factor Va by site-directed mutagenesis.
- 2008
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 283:30, s. 20805-20812
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Tidskriftsartikel (refereegranskat)abstract
- Activated coagulation factor V functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin. Based on introduction of extra carbohydrate side chains in recombinant factor V, we recently proposed several regions in factor Va to be important for factor Xa binding. To further define which residues are important for factor Xa binding, we prepared fifteen recombinant factor V variants in which clusters of charged amino acid residues were mutated, mainly to alanines. The factor V variants were expressed in COS-1 cells and their functional properties evaluated in a prothrombinase-based assay, as well as in a direct binding test. Four of the factor V variants, 501A/510A/511D, 501A/510A/511D/513A, 513A/577A/578A, and 501A/510A/511D/513A/577A/578A exhibited markedly reduced factor Xa-cofactor activity tested in the prothrombinase assay, and reduced binding affinity as judged by the direct binding assay. These factor Va variants were normally cleaved at Arg506 by activated protein C and the interaction between the factor Xa - factor Va complex and prothrombin was unaffected by the introduced mutations. Based on the integration of all available data we propose a key factor Xa-binding surface to be centered on Arg501, Arg510, Ala511, Asp513, Asp577 and Asp578 in the factor Va A2 domain. These residues form an elongated charged factor Xa-binding cluster on the factor Va surface.
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| 12. |
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