The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform
Seppo Meri - One of the best experts on this subject based on the ideXlab platform.
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Mutations of Complement Lectin Pathway genes MBL2 and MASP2 associated with placental malaria
Malaria Journal, 2012Co-Authors: Ville Holmberg, Päivi Onkamo, Elisa Lahtela, Päivi Lahermo, George Bedu-addo, Frank P Mockenhaupt, Seppo MeriAbstract:Background Innate immunity plays a crucial role in the host defense against malaria including Plasmodium falciparum malaria in pregnancy, but the roles of the various underlying genes and mechanisms predisposing to the disease are poorly understood. Methods 98 single-nucletoide polymorphisms were genotyped in a set of 17 functionally related genes of the Complement system in 145 primiparous Ghanaian women with placental malaria, defined by placental parasitaemia or malaria pigment, and as a control, in 124 non-affected primiparae. Results Placental malaria was significantly associated with SNPs in the Lectin Pathway genes MBL2, MASP2, FCN2 and in properdin . In particular, the main African mannose-binding Lectin deficiency variant ( MBL2* G57E, rs1800451) increased the odds of placental malaria (OR 1.6; permuted p-value 0.014). In contrast, a common MASP2 mutation (R439H, rs12085877), which reduces the activity of MBL-MASP2 complexes occurred in 33% of non-affected women and in 22% primiparae with placental malaria (OR 0.55, permuted p -value 0.020). Conclusions Excessive Complement activation is of importance in the pathogenesis of placental malaria by mediating inflammation, coagulation, and endothelial dysfunction. Mutated MBL and MASP2 proteins could have direct intrinsic effects on the susceptibility to placental malaria, in addition to their roles in regulation of downstream Complement activation.
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Mutations of Complement Lectin Pathway genes MBL2 and MASP2 associated with placental malaria
Malaria journal, 2012Co-Authors: Ville Holmberg, Päivi Onkamo, Elisa Lahtela, Päivi Lahermo, George Bedu-addo, Frank P Mockenhaupt, Seppo MeriAbstract:Background Innate immunity plays a crucial role in the host defense against malaria including Plasmodium falciparum malaria in pregnancy, but the roles of the various underlying genes and mechanisms predisposing to the disease are poorly understood.
Verena Schroeder - One of the best experts on this subject based on the ideXlab platform.
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MASP-1 Induced Clotting – The First Model of Prothrombin Activation by MASP-1
PloS one, 2015Co-Authors: Lorenz Jenny, József Dobó, Péter Gál, Verena SchroederAbstract:Mannan-binding Lectin-associated serine protease-1 (MASP-1), a protein of the Complement Lectin Pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation Pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous Pathways, either cleaving at R271 or R393 first. Both Pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.
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MASP-1 of the Complement system promotes clotting via prothrombin activation
Molecular immunology, 2015Co-Authors: Lorenz Jenny, József Dobó, Péter Gál, Verena SchroederAbstract:Mannan-binding Lectin-associated serine protease-1 (MASP-1), a protein of the Complement Lectin Pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.
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Plasma levels of mannan‐binding Lectin (MBL)‐associated serine proteases (MASPs) and MBL‐associated protein in cardio‐ and cerebrovascular diseases
Clinical and experimental immunology, 2013Co-Authors: Vera Frauenknecht, Steffen Thiel, L. Storm, Niklaus Meier, Marcel Arnold, Jean-paul Schmid, Hugo Saner, Verena SchroederAbstract:Growing evidence suggests a prominent role of the Complement system in the pathogenesis of cardio- and cerebrovascular diseases (CVD). Mannan-binding Lectin-associated serine proteases (MASPs) MASP-1 and MASP-2 of the Complement Lectin Pathway contribute to clot formation and may represent an important link between inflammation and thrombosis. MBL-associated protein MAp44 has shown cardioprotective effects in murine models. However, MAp44 has never been measured in patients with CVD and data on MASP levels in CVD are scarce. Our aim was to investigate for the first time plasma levels of MAp44 and MASP-1, -2, -3 concomitantly in patients with CVD. We performed a pilot study in 50 healthy volunteers, in stable coronary artery disease (CAD) patients with one-vessel (n = 51) or three-vessel disease (n = 53) and age-matched controls with normal coronary arteries (n = 53), 49 patients after myocardial infarction (MI) and 66 patients with acute ischaemic stroke. We measured MAp44 and MASP-1 levels by in-house time-resolved immunofluorometric assays. MASP-2 and MASP-3 levels were measured using commercial enzyme-linked immunosorbent assay kits. MASP-1 levels were highest in subacute MI patients and lowest in acute stroke patients. MASP-2 levels were lower in MI and stroke patients compared with controls and CAD patients. MASP-3 and MAp44 levels did not differ between groups. MASP or MAp44 levels were not associated with severity of disease. MASP and MAp44 levels were associated with cardiovascular risk factors including dyslipidaemia, obesity and hypertension. Our results suggest that MASP levels may be altered in vascular diseases. Larger studies are needed to confirm our results and elucidate the underlying mechanisms.
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effects of masp 1 of the Complement system on activation of coagulation factors and plasma clot formation
PLOS ONE, 2012Co-Authors: Katharina Hess, József Dobó, Péter Gál, Verena Schroeder, Ramzi Ajjan, Fladia PhoenixAbstract:Background Numerous interactions between the coagulation and Complement systems have been shown. Recently, links between coagulation and mannan-binding Lectin-associated serine protease-1 (MASP-1) of the Complement Lectin Pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other Complement and coagulation proteins, may play a role in fibrin clot formation.
Misao Matsushita - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning and characterization of novel ficolins from Xenopus laevis
Immunogenetics, 2003Co-Authors: Yuji Kakinuma, Yuichi Endo, Minoru Takahashi, Misao Matsushita, Munehiro Nakata, Seiichi Takenoshita, Teizo FujitaAbstract:Ficolins are proteins characterized by the presence of collagen- and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum Lectins and are thought to play crucial roles in host defense through opsonization and Complement activation. To elucidate the evolution of ficolins and the primordial Complement Lectin Pathway, we cloned four ficolin cDNAs from Xenopus laevis, termed Xenopus ficolin (XeFCN) 1, 2, 3 and 4. The deduced amino acid sequences of the four ficolins revealed the conserved collagen- and fibrinogen-like domains. The full sequences of the four ficolins showed a 42-56% identity to human ficolins, and 60-83% between one another. Northern blots showed that XeFCN1 was expressed mainly in liver, spleen and heart, and XeFCN2 and XeFCN4 mainly in peripheral blood leukocytes, lung and spleen. We isolated ficolin proteins from Xenopus serum by affinity chromatography on N-acetylglucosamine-agarose, followed by ion-exchange chromatography. The final eluate showed polymeric bands composed of two components of 37 and 40 kDa. The N-terminal amino acid sequences and treatment with endoglycosidase F showed that the two bands are the same XeFCN1 protein with different masses of N-linked sugar. The polymeric form of the two types of XeFCN1 specifically recognized GlcNAc and GalNAc residues. These results suggest that like human L-ficolin, XeFCN1 functions in the circulation through its Lectin activity.
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an ancient Lectin dependent Complement system in an ascidian novel Lectin isolated from the plasma of the solitary ascidian halocynthia roretzi
Journal of Immunology, 2001Co-Authors: Hideharu Sekine, Minoru Takahashi, Misao Matsushita, Munehiro Nakata, Akira Kenjo, Tsuguo Mizuochi, Kaoru Azumi, Reiji Kasukawa, Narumi Ichikawa, Yuichi EndoAbstract:Mannose-binding Lectin (MBL) is a C-type Lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the Complement Lectin Pathway. To elucidate the origin and evolution of MBL, MBL-like Lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of ∼36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian Lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type Lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding Lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive Complement system, consisting of the Lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.
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an ancient Lectin dependent Complement system in an ascidian novel Lectin isolated from the plasma of the solitary ascidian halocynthia roretzi
Journal of Immunology, 2001Co-Authors: Hideharu Sekine, Minoru Takahashi, Munehiro Nakata, Akira Kenjo, Tsuguo Mizuochi, Kaoru Azumi, Reiji Kasukawa, Narumi Ichikawa, Gota Ohi, Misao MatsushitaAbstract:Mannose-binding Lectin (MBL) is a C-type Lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the Complement Lectin Pathway. To elucidate the origin and evolution of MBL, MBL-like Lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of approximately 36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian Lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type Lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding Lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive Complement system, consisting of the Lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.
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cloning and characterization of novel ficolins from the solitary ascidian halocynthia roretzi
Journal of Biological Chemistry, 2001Co-Authors: Akira Kenjo, Yuichi Endo, Minoru Takahashi, Misao Matsushita, Munehiro Nakata, Tsuguo Mizuochi, Teizo FujitaAbstract:Abstract Ficolins are animal Lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding Lectin (MBL) activates the Complement Lectin Pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated ∼40 kDa (p40) and ∼50 kDa (p50) N-acetylglucosamine-binding Lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizesN-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4–52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph Lectin activity and the correlation of different functions with binding specificity.
József Dobó - One of the best experts on this subject based on the ideXlab platform.
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key components of the Complement Lectin Pathway are not only required for the development of inflammatory arthritis but also regulate the transcription of factor d
Frontiers in Immunology, 2020Co-Authors: Michael V Holers, Annette G. Hansen, József Dobó, Anna Borodovsky, Robert I Scheinman, Nhu Ho, Joseline Ramos Ramirez, Jared J Lindenberger, Dhruv Desai, Rasmus PihlAbstract:The Complement system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Besides driving Lectin Pathway (LP) activation, the mannan-binding Lectin (MBL)-associated serine proteases (MASPs) also play a key role in regulating the alternative Pathway (AP). We evaluated the effects of N-acetylgalactosamine (GalNAc)-conjugated MASP-1 and MASP-2 duplexes in vitro and in mice with and without arthritis to examine whether knockdown of MASP-1 and MASP-2 expression affects the development of arthritis. GalNAc-siRNAs for MASP-1 and MASP-2 demonstrated robust silencing of MASP-1 or MASP-2 at pM concentrations in vitro. To evaluate the impact of silencing in arthritic mice, we used the collagen antibody-induced arthritis (CAIA) mouse model of RA. Mice were injected a 10 mg/kg dose of GalNAc-siRNAs 3x s.q. prior to the induction of CAIA. Liver gene expression was examined using qRT-PCR, and protein levels were confirmed in the circulation by sandwich immunoassays and Western blot. At day 10, CAIA mice separately treated with MASP-1 and MASP-2 duplexes had a specific reduction in expression of liver MASP-1 (70-95%, p < 0.05) and MASP-2 (90%, p < 0.05) mRNA, respectively. MASP-1-siRNA treatment resulted in a 95% reduction in levels of MASP-1 protein in circulation with no effect on MASP-2 levels and clinical disease activity (CDA). In mice injected with MASP-2 duplex, there was a significant (p < 0.05) 90% decrease in ex vivo C4b deposition on mannan, with nearly complete elimination of MASP-2 in the circulation. MASP-2 silencing initially significantly decreased CDA by 60% but subsequently changed to a 40% decrease vs. control. Unexpectedly, GalNAc-siRNA-mediated knockdown of MASP-1 and MASP-2 revealed a marked effect of these proteins on the transcription of FD under normal physiological conditions, whereas LPS-induced inflammatory conditions reversed this effect on FD levels. LPS is recognized by Toll-like receptor 4 (TLR4), we found MBL not only binds to TLR4 an interaction with a Kd of 907 nM but also upregulated FD expression in differentiated adipocytes. We show that MASP-2 knockdown impairs the development of RA and that the interrelationship between proteins of the LP and the AP may extend to the transcriptional modulation of the FD gene.
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Transcriptome analysis of inflammation-related gene expression in endothelial cells activated by Complement MASP-1
Scientific Reports, 2017Co-Authors: Endre Schwaner, Zsuzsanna Németh, Péter K. Jani, Erika Kajdácsi, Márta L. Debreczeni, Zoltán Doleschall, József Dobó, Péter Gál, János Rigó, Kinga AndrásAbstract:Mannan-binding Lectin-associated serine protease 1 (MASP-1), the most abundant enzyme of the Complement Lectin Pathway, is able to stimulate human umbilical vein endothelial cells (HUVECs) to alter the expression of several cytokines and adhesion molecules. This study has assessed to what extent MASP-1 is able to modify the transcriptional pattern of inflammation-related (IR) genes in HUVECs. We utilized Agilent microarray to analyse the effects of recombinant MASP-1 (rMASP-1) in HUVECs, on a set of 884 IR genes. Gene Set Enrichment Analysis showed an overall activation of inflammation-related genes in response to rMASP-1. rMASP-1 treatment up- and down-regulated 19 and 11 IR genes, respectively. Most of them were previously unidentified, such as genes of chemokines (CXCL1, CXCL2, CXCL3), inflammatory receptors (TLR2, BDKRB2) and other inflammatory factors (F3, LBP). Expression of IR genes changed early, during the first 2 hours of activation. Both p38-MAPK inhibitor and NFκB inhibitor efficiently suppressed the effect of rMASP-1. We delineated 12 transcriptional factors as possible regulators of rMASP-1-induced IR genes. Our microarray-based data are in line with the hypothesis that Complement Lectin Pathway activation, generating active MASP-1, directly regulates inflammatory processes by shifting the phenotype of endothelial cells towards a more pro-inflammatory type.
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masp 3 is the exclusive pro factor d activator in resting blood the Lectin and the alternative Complement Pathways are fundamentally linked
Scientific Reports, 2016Co-Authors: József Dobó, Robert Szasz, David Szakacs, Gabor Oroszlan, Elod Kortvely, Bence Kiss, Eszter Boros, Peter ZavodszkyAbstract:MASP-3 was discovered 15 years ago as the third mannan-binding Lectin (MBL)-associated serine protease of the Complement Lectin Pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key Lectin Pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative Complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in Complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or Complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or Complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the Lectin and alternative Pathways.
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Complement MASP-1 enhances adhesion between endothelial cells and neutrophils by up-regulating E-seLectin expression.
Molecular immunology, 2016Co-Authors: Péter K. Jani, Endre Schwaner, Erika Kajdácsi, Márta L. Debreczeni, Zoltán Doleschall, József Dobó, János Rigó, Rita Ungai-salánki, Miklós Geiszt, Bálint SzabóAbstract:Abstract The Complement system and neutrophil granulocytes are indispensable in the immune response against extracellular pathogens such as bacteria and fungi. Endothelial cells also participate in antimicrobial immunity largely by regulating the homing of leukocytes through their cytokine production and their pattern of cell surface adhesion molecules. We have previously shown that mannan-binding Lectin-associated serine protease-1 (MASP-1), a Complement Lectin Pathway enzyme, is able to activate endothelial cells by cleaving protease activated receptors, which leads to cytokine production and enables neutrophil chemotaxis. Therefore, we aimed to investigate how recombinant MASP-1 (rMASP-1) can modify the pattern of P‐seLectin, E‐seLectin, ICAM‐1, ICAM‐2, and VCAM‐1 adhesion molecules in human umbilical vein endothelial cells (HUVEC), and whether these changes can enhance the adherence between endothelial cells and neutrophil granulocyte model cells (differentiated PLB-985). We found that HUVECs activated by rMASP-1 decreased the expression of ICAM-2 and increased that of E-seLectin, whereas ICAM-1, VCAM-1 and P-seLectin expression remained unchanged. Furthermore, these changes resulted in increased adherence between differentiated PLB-985 cells and endothelial cells. Our finding suggests that Complement MASP-1 can increase adhesion between neutrophils and endothelial cells in a direct fashion. This is in agreement with our previous finding that MASP-1 increases the production of pro-inflammatory cytokines (such as IL-6 and IL-8) and chemotaxis, and may thereby boost neutrophil functions. This newly described cooperation between Complement Lectin Pathway and neutrophils via endothelial cells may be an effective tool to enhance the antimicrobial immune response.
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MASP-1 Induced Clotting – The First Model of Prothrombin Activation by MASP-1
PloS one, 2015Co-Authors: Lorenz Jenny, József Dobó, Péter Gál, Verena SchroederAbstract:Mannan-binding Lectin-associated serine protease-1 (MASP-1), a protein of the Complement Lectin Pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation Pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous Pathways, either cleaving at R271 or R393 first. Both Pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.
Teizo Fujita - One of the best experts on this subject based on the ideXlab platform.
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Trypanosoma cruzi calreticulin inhibits the Complement Lectin Pathway activation by direct interaction with L-Ficolin.
Molecular Immunology, 2014Co-Authors: Eduardo Sosoniuk, Teizo Fujita, Christine Gaboriaud, Gerardo Vallejos, Hany Kenawy, Nicole Thielens, Wilhelm Schwaeble, Arturo Ferreira, Carolina ValckAbstract:Trypanosoma cruzi, the agent of Chagas' disease, the sixth neglected tropical disease worldwide, infects 10-12 million people in Latin America. Differently from T. cruzi epimastigotes, trypomastigotes are Complement-resistant and infective. CRPs, T-DAF, sialic acid and lipases explain at least part of this resistance. In vitro, T. cruzi calreticulin (TcCRT), a chaperone molecule that translocates from the ER to the parasite surface: (a) Inhibits the human classical Complement activation, by interacting with C1, (b) As a consequence, an increase in infectivity is evident and, (c) It inhibits angiogenesis and tumor growth. We report here that TcCRT also binds to the L-Ficolin collagenous portion, thus inhibiting approximately between 35 and 64% of the human Complement Lectin Pathway activation, initiated by L-Ficolin, a property not shared by H-Ficolin. While L-Ficolin binds to 60% of trypomastigotes and to 24% of epimastigotes, 50% of the former and 4% of the latter display TcCRT on their surfaces. Altogether, these data indicate that TcCRT is a parasite inhibitory receptor for Ficolins. The resulting evasive activities, together with the TcCRT capacity to inhibit C1, with a concomitant increase in infectivity, may represent T. cruzi strategies to inhibit important arms of the innate immune response.
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Molecular cloning and characterization of novel ficolins from Xenopus laevis
Immunogenetics, 2003Co-Authors: Yuji Kakinuma, Yuichi Endo, Minoru Takahashi, Misao Matsushita, Munehiro Nakata, Seiichi Takenoshita, Teizo FujitaAbstract:Ficolins are proteins characterized by the presence of collagen- and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum Lectins and are thought to play crucial roles in host defense through opsonization and Complement activation. To elucidate the evolution of ficolins and the primordial Complement Lectin Pathway, we cloned four ficolin cDNAs from Xenopus laevis, termed Xenopus ficolin (XeFCN) 1, 2, 3 and 4. The deduced amino acid sequences of the four ficolins revealed the conserved collagen- and fibrinogen-like domains. The full sequences of the four ficolins showed a 42-56% identity to human ficolins, and 60-83% between one another. Northern blots showed that XeFCN1 was expressed mainly in liver, spleen and heart, and XeFCN2 and XeFCN4 mainly in peripheral blood leukocytes, lung and spleen. We isolated ficolin proteins from Xenopus serum by affinity chromatography on N-acetylglucosamine-agarose, followed by ion-exchange chromatography. The final eluate showed polymeric bands composed of two components of 37 and 40 kDa. The N-terminal amino acid sequences and treatment with endoglycosidase F showed that the two bands are the same XeFCN1 protein with different masses of N-linked sugar. The polymeric form of the two types of XeFCN1 specifically recognized GlcNAc and GalNAc residues. These results suggest that like human L-ficolin, XeFCN1 functions in the circulation through its Lectin activity.
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cloning and characterization of novel ficolins from the solitary ascidian halocynthia roretzi
Journal of Biological Chemistry, 2001Co-Authors: Akira Kenjo, Yuichi Endo, Minoru Takahashi, Misao Matsushita, Munehiro Nakata, Tsuguo Mizuochi, Teizo FujitaAbstract:Abstract Ficolins are animal Lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding Lectin (MBL) activates the Complement Lectin Pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated ∼40 kDa (p40) and ∼50 kDa (p50) N-acetylglucosamine-binding Lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizesN-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4–52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph Lectin activity and the correlation of different functions with binding specificity.