Journal articles on the topic 'C3-convertase of alternative complement pathway'
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Hourcade, Dennis E., Lynne M. Mitchell, and M. Edward Medof. "Decay acceleration of the complement alternative pathway C3 convertase." Immunopharmacology 42, no. 1-3 (May 1999): 167–73. http://dx.doi.org/10.1016/s0162-3109(99)00005-3.
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Roumenina, Lubka T., Mathieu Jablonski, Christophe Hue, Jacques Blouin, Jordan D. Dimitrov, Marie-Agnes Dragon-Durey, Mathieu Cayla, et al. "Hyperfunctional C3 convertase leads to complement deposition on endothelial cells and contributes to atypical hemolytic uremic syndrome." Blood 114, no. 13 (September 24, 2009): 2837–45. http://dx.doi.org/10.1182/blood-2009-01-197640.
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Abstract Complement is a major innate immune defense against pathogens, tightly regulated to prevent host tissue damage. Atypical hemolytic uremic syndrome (aHUS) is characterized by endothelial damage leading to renal failure and is highly associated with abnormal alternative pathway regulation. We characterized the functional consequences of 2 aHUS-associated mutations (D254G and K325N) in factor B, a key participant in the alternative C3 convertase. Mutant proteins formed high-affinity C3-binding site, leading to a hyperfunctional C3 convertase, resistant to decay by factor H. This led to enhanced complement deposition on the surface of alternative pathway activator cells. In contrast to native factor B, the 2 mutants bound to inactivated C3 and induced formation of functional C3-convertase on iC3b-coated surface. We demonstrated for the first time that factor B mutations lead to enhanced C3-fragment deposition on quiescent and adherent human glomerular cells (GEnCs) and human umbilical vein endothelial cells (HUVECs), together with the formation of sC5b-9 complexes. These results could explain the occurrence of the disease, since excessive complement deposition on endothelial cells is a central event in the pathogenesis of aHUS. Therefore, risk factors for aHUS are not only mutations leading to loss of regulation, but also mutations, resulting in hyperactive C3 convertase.
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Mullick, Jayati, John Bernet, Yogesh Panse, Sharanabasava Hallihosur, Akhilesh K. Singh, and Arvind Sahu. "Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein." Journal of Virology 79, no. 19 (October 1, 2005): 12382–93. http://dx.doi.org/10.1128/jvi.79.19.12382-12393.2005.
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ABSTRACT Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP's various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.
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Schwendinger, M. G., M. Spruth, J. Schoch, M. P. Dierich, and W. M. Prodinger. "A novel mechanism of alternative pathway complement activation accounts for the deposition of C3 fragments on CR2-expressing homologous cells." Journal of Immunology 158, no. 11 (June 1, 1997): 5455–63. http://dx.doi.org/10.4049/jimmunol.158.11.5455.
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Abstract Complement receptor type 2 (CD21, CR2), the receptor for the C3 fragment C3dg, activates complement via the alternative pathway and also serves as a preferential acceptor site for C3 fragments. The molecular basis for this phenomenon, which has recently been demonstrated for B lymphocytes in vivo, is currently not understood. Here we present a model for this CR2-dependent complement activation. The inactive C3 (iC3), which forms spontaneously in serum in low amounts by reaction of native C3 with H2O, binds noncovalently to the N-terminal part of CR2. Subsequent association of properdin and factor B, and cleavage of factor B by factor D lead to formation of a C3 convertase associated with CR2, thus focussing covalent C3 deposition to CR2 itself. This model is supported by the following experimental findings. 1) By FACS analysis and radioreceptor assays we showed that iC3, properdin, and factor B bound to CR2 on Raji B cells, MT2 T cells, and peripheral blood B cells. 2) Both binding of these proteins and complement activation by CR2-expressing cells were reduced in parallel by Abs against CR2. 3) 125I-labeled C3b was covalently deposited on CR2, when hemolytically active 125I-labeled C3 was added to Raji cells preincubated with iC3, factor B, properdin, and factor D, thus proving functionality of CR2-bound C3 convertase. This model of C3 convertase activity formed on CR2 domains inaccessible for decay-accelerating factor offers an explanation for the deposition of C3 found on CR2-expressing cells.
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Chauvet, Sophie, Romain Berthaud, Magali Devriese, Morgane Mignotet, Paula Vieira Martins, Tania Robe-Rybkine, Maria A. Miteva, et al. "Anti-Factor B Antibodies and Acute Postinfectious GN in Children." Journal of the American Society of Nephrology 31, no. 4 (February 7, 2020): 829–40. http://dx.doi.org/10.1681/asn.2019080851.
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BackgroundThe pathophysiology of the leading cause of pediatric acute nephritis, acute postinfectious GN, including mechanisms of the pathognomonic transient complement activation, remains uncertain. It shares clinicopathologic features with C3 glomerulopathy, a complement-mediated glomerulopathy that, unlike acute postinfectious GN, has a poor prognosis.MethodsThis retrospective study investigated mechanisms of complement activation in 34 children with acute postinfectious GN and low C3 level at onset. We screened a panel of anticomplement protein autoantibodies, carried out related functional characterization, and compared results with those of 60 children from the National French Registry who had C3 glomerulopathy and persistent hypocomplementemia.ResultsAll children with acute postinfectious GN had activation of the alternative pathway of the complement system. At onset, autoantibodies targeting factor B (a component of the alternative pathway C3 convertase) were found in a significantly higher proportion of children with the disorder versus children with hypocomplementemic C3 glomerulopathy (31 of 34 [91%] versus 4 of 28 [14%], respectively). In acute postinfectious GN, anti-factor B autoantibodies were transient and correlated with plasma C3 and soluble C5b-9 levels. We demonstrated that anti-factor B antibodies enhance alternative pathway convertase activity in vitro, confirming their pathogenic effect. We also identified crucial antibody binding sites on factor B, including one correlated to disease severity.ConclusionsThese findings elucidate the pathophysiologic mechanisms underlying acute postinfectious GN by identifying anti-factor B autoantibodies as contributing factors in alternative complement pathway activation. At onset of a nephritic syndrome with low C3 level, screening for anti-factor B antibodies might help guide indications for kidney biopsy to avoid misdiagnosed chronic glomerulopathy, such as C3 glomerulopathy, and to help determine therapy.
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Weiler, J. M., R. E. Edens, and G. J. Gleich. "Eosinophil granule cationic proteins regulate complement. I. Activity on the alternative pathway." Journal of Immunology 149, no. 2 (July 15, 1992): 643–48. http://dx.doi.org/10.4049/jimmunol.149.2.643.
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Abstract Eosinophil granules contain several cationic proteins that mediate tissue damage in allergic disease. The present study examined the capacity and mechanisms by which these cationic proteins regulate activity of the alternative pathway of C. Eosinophil peroxidase and eosinophil cationic protein inhibited formation of cell-bound alternative pathway C3 convertase, causing 50% inhibition of lysis at about 0.19 and 0.75 microgram/10(7) cellular intermediates, respectively. Major basic protein inhibited alternative pathway C3 activity by only 19% at 1.5 micrograms/10(7) cellular intermediates. Eosinophil-derived neurotoxin had no activity on the alternative pathway. The eosinophil granule proteins were examined for the mechanism by which they inhibited alternative pathway activity. Eosinophil peroxidase and major basic protein inhibited fluid phase factor B consumption in a reaction mixture that also contained factors D and C3b, eosinophil-derived neurotoxin had no activity on factor B consumption, and eosinophil cationic protein consumed factor B in the absence of C3b and factor D. Both eosinophil cationic protein and eosinophil peroxidase enhanced the decay of preformed alternative pathway convertase. Lysis of EAC4b,3b cellular intermediates formed to contain a low surface amount of C3b was more inhibited than was lysis of cells formed with a standard amount of C3b on the surface. This suggests that these eosinophil proteins acted predominantly on C3b to regulate alternative pathway activity. We also found that none of the eosinophil granule cationic proteins had any effect on later events after the formation of the C3 convertase. We conclude that although eosinophil-derived neurotoxin (isoelectric pH value (pI) = 8.9) does not regulate alternative pathway activity, the more highly charged eosinophil granule cationic proteins--major basic protein (pI = 10.9), eosinophil cationic protein (pI = 10.8), and eosinophil peroxidase (pI = 10.8)--do share the capacity to regulate C activity and may exert this activity in vivo.
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Irmscher, Sarah, Nadia Döring, Luke D. Halder, Emeraldo A. H. Jo, Isabell Kopka, Christine Dunker, Ilse D. Jacobsen, et al. "Kallikrein Cleaves C3 and Activates Complement." Journal of Innate Immunity 10, no. 2 (December 14, 2017): 94–105. http://dx.doi.org/10.1159/000484257.
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The human plasma contact system is an immune surveillance system activated by the negatively charged surfaces of bacteria and fungi and includes the kallikrein-kinin, the coagulation, and the fibrinolytic systems. Previous work shows that the contact system also activates complement, and that plasma enzymes like kallikrein, plasmin, thrombin, and FXII are involved in the activation process. Here, we show for the first time that kallikrein cleaves the central complement component C3 directly to yield active components C3b and C3a. The cleavage site within C3 is identical to that recognized by the C3 convertase. Also, kallikrein-generated C3b forms C3 convertases, which trigger the C3 amplification loop. Since kallikrein also cleaves factor B to yield Bb and Ba, kallikrein alone can trigger complement activation. Kallikrein-generated C3 convertases are inhibited by factor H; thus, the kallikrein activation pathway merges with the amplification loop of the alternative pathway. Taken together, these data suggest that activation of the contact system locally enhances complement activation on cell surfaces. The human pathogenic microbe Candida albicans activates the contact system in normal human serum. However, C. albicans immediately recruits factor H to the surface, thereby evading the alternative and likely kallikrein-mediated complement pathways.
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Pangburn, M. K., and H. J. Müller-Eberhard. "The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase." Biochemical Journal 235, no. 3 (May 1, 1986): 723–30. http://dx.doi.org/10.1042/bj2350723.
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The association of Factor B with C3b (the major fragment of complement component C3) in the presence of Mg2+ results in the formation of a bimolecular zymogen, C3b,B, which is activated by the serine proteinase Factor D, generating the C3 convertase, C3b,Bb (EC 3.4.21.47). Cleavage of native C3 by the C3 convertase was monitored by recording the increase in fluorescence associated with C3b formation in the presence of the fluorescent probe 8-anilinonaphthalene-1-sulphonate. Measurements of initial rates of C3b formation at various C3 concentrations were analysed in accordance with the Michaelis-Menten equation, yielding kcat. = 1.78 +/- 0.08 s-1, Km = 5.86 × 10(-6) M and turnover number = 107 min-1. The assay was used to measure the Ki values of a variety of proteinase inhibitors. The C3 convertase has a short half-life, owing to spontaneous dissociation of the complex. The t1/2 and kcat./Km of the enzyme were determined by fitting an equation modelling both the kinetic reaction and enzyme decay to the fluorimetrically measured progress curve. The enzyme, C3b,Bb, exhibited a t1/2 of 90 +/- 2 s and a kcat./Km of 31.1 × 10(4) +/- 0.8 × 10(4) M-1 × s-1 at physiological pH, ionic strength and temperature. The enzyme that initiates activation of the alternative pathway, C3(H2O),Bb, was also examined. It was slightly less stable (t1/2 = 77 +/- 3 s) and exhibited only half the activity of C3b,Bb (kcat./Km = 16.3 × 10(4) +/- 1.0 × 10(4) M-1 × s-1).
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Sahu, Arvind, Stuart N. Isaacs, Athena M. Soulika, and John D. Lambris. "Interaction of Vaccinia Virus Complement Control Protein with Human Complement Proteins: Factor I-Mediated Degradation of C3b to iC3b1 Inactivates the Alternative Complement Pathway." Journal of Immunology 160, no. 11 (June 1, 1998): 5596–604. http://dx.doi.org/10.4049/jimmunol.160.11.5596.
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Abstract Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.
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Jiang, Haixiang, Eric Wagner, Huamei Zhang, and Michael M. Frank. "Complement 1 Inhibitor Is a Regulator of the Alternative Complement Pathway." Journal of Experimental Medicine 194, no. 11 (December 3, 2001): 1609–16. http://dx.doi.org/10.1084/jem.194.11.1609.
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We studied complement 1 inhibitor (C1-INH) as an inhibitor of the alternative complement pathway. C1-INH prevented lysis, induced by the alternative complement pathway, of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes in human serum. It inhibited the binding of both factors B and C3 to PNH and rabbit erythrocytes and blocked the ability of factor B to restore alternative-pathway function in factor B–depleted serum. C1-INH did not bind to factors B or D but did bind to immobilized C3b and cobra venom factor (CVF), a C3b analogue. C1-INH prevented factor B from binding to CVF-coated beads and dissociated bound factor B from such beads. Factor B and C1-INH showed cross competition in binding to CVF-coated beads. Factor D cleaved factor B into Bb and Ba in the presence of C3b. Cleavage was markedly inhibited when C3b was preincubated with C1-INH. C1-INH inhibited the formation of CVFBb and decreased the C3 cleavage. Removal of C1-INH from serum, in the presence of Mg-EGTA with an anti–C1-INH immunoabsorbant, markedly increased alternative-pathway lysis. C1-INH interacts with C3b to inhibit binding of factor B to C3b. At physiologic concentrations, it is a downregulator of the alternative pathway convertase.
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Jongerius, Ilse, Jörg Köhl, Manoj K. Pandey, Maartje Ruyken, Kok P. M. van Kessel, Jos A. G. van Strijp, and Suzan H. M. Rooijakkers. "Staphylococcal complement evasion by various convertase-blocking molecules." Journal of Experimental Medicine 204, no. 10 (September 24, 2007): 2461–71. http://dx.doi.org/10.1084/jem.20070818.
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To combat the human immune response, bacteria should be able to divert the effectiveness of the complement system. We identify four potent complement inhibitors in Staphylococcus aureus that are part of a new immune evasion cluster. Two are homologues of the C3 convertase modulator staphylococcal complement inhibitor (SCIN) and function in a similar way as SCIN. Extracellular fibrinogen-binding protein (Efb) and its homologue extracellular complement-binding protein (Ecb) are identified as potent complement evasion molecules, and their inhibitory mechanism was pinpointed to blocking C3b-containing convertases: the alternative pathway C3 convertase C3bBb and the C5 convertases C4b2aC3b and C3b2Bb. The potency of Efb and Ecb to block C5 convertase activity was demonstrated by their ability to block C5a generation and C5a-mediated neutrophil activation in vitro. Further, Ecb blocks C5a-dependent neutrophil recruitment into the peritoneal cavity in a mouse model of immune complex peritonitis. The strong antiinflammatory properties of these novel S. aureus–derived convertase inhibitors make these compounds interesting drug candidates for complement-mediated diseases.
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Agostoni, A., M. Cicardi, M. Gardinali, and L. Bergamaschini. "The Complement System." International Journal of Immunopathology and Pharmacology 5, no. 2 (May 1992): 123–30. http://dx.doi.org/10.1177/039463209200500207.
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The human complement system (C) acts to lyse susceptible cells, to promote phagocytosis of target particles, and to solubilize immune-complexes, its activation generates peptides that mediate features of the inflammatory response. It is comprised of a series of plasma zymogens, activated by proteolytic cleavage in a cascade manner, and of plasma and cell membrane control proteins. Activation is achieved by two independent routes: the classical pathway, started by immunoglobulins, and the alternative pathway, started by cell membrane components. Both of them promote the generation of an enzyme-complex (C3 convertase) able to cleave the pivotal protein of the complement system, C3, thus initiating the common pathway with the formation of the lytic complex (Figure 1). In this paper we will briefly review the physiologic phenomena related to the complement activation and its role in pathogenesis of illness particularly focusing on the studies carried out in our laboratory.
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Michelfelder, Stefan, Friedericke Fischer, Astrid Wäldin, Kim V. Hörle, Martin Pohl, Juliana Parsons, Ralf Reski, et al. "The MFHR1 Fusion Protein Is a Novel Synthetic Multitarget Complement Inhibitor with Therapeutic Potential." Journal of the American Society of Nephrology 29, no. 4 (January 15, 2018): 1141–53. http://dx.doi.org/10.1681/asn.2017070738.
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The complement system is essential for host defense, but uncontrolled complement system activation leads to severe, mostly renal pathologies, such as atypical hemolytic uremic syndrome or C3 glomerulopathy. Here, we investigated a novel combinational approach to modulate complement activation by targeting C3 and the terminal pathway simultaneously. The synthetic fusion protein MFHR1 links the regulatory domains of complement factor H (FH) with the C5 convertase/C5b-9 inhibitory fragment of the FH-related protein 1. In vitro, MFHR1 showed cofactor and decay acceleration activity and inhibited C5 convertase activation and C5b-9 assembly, which prevented C3b deposition and reduced C3a/C5a and C5b-9 generation. Furthermore, this fusion protein showed the ability to escape deregulation by FH-related proteins and form multimeric complexes with increased inhibitory activity. In addition to substantially inhibiting alternative and classic pathway activation, MFHR1 blocked hemolysis mediated by serum from a patient with aHUS expressing truncated FH. In FH−/− mice, MFHR1 administration augmented serum C3 levels, reduced abnormal glomerular C3 deposition, and ameliorated C3 glomerulopathy. Taking the unique design of MFHR1 into account, we suggest that the combination of proximal and terminal cascade inhibition together with the ability to form multimeric complexes explain the strong inhibitory capacity of MFHR1, which offers a novel basis for complement therapeutics.
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Inal, Jameel Malhador, Andreas Laich, and Sylvie Miot. "Complement C2 bypass mechanism involving the C3-convertase C4bBb (53.16)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S106. http://dx.doi.org/10.4049/jimmunol.178.supp.53.16.
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Abstract The majority of patients with a homozygous complement C2 (C2) deficiency show no or only minor disease, at most one third showing mild symptoms of systemic lupus erythematosus. This suggests that classical pathway activation is not solely dependent on C2. We show that factor B (FB) the alternative pathway homologue of C2 can substitute for C2. In ELISA, FB was found to complex with immobilised C4b. This was confirmed by surface plasmon resonance, giving an estimated binding constant of 2.0 x10−5 M. Furthermore Factor D was able to cleave factor B in the presence of C4b. The hybrid C3 convertase formed, C4bBb, also cleaved 125I-C3 at a low level, further supporting previous evidence of a C2-bypass mechanism.
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Wilcox, LA, JL Ezzell, NJ Bernshaw, and CJ Parker. "Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum." Blood 78, no. 3 (August 1, 1991): 820–29. http://dx.doi.org/10.1182/blood.v78.3.820.bloodjournal783820.
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When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay- accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane- associated activation of the APC.
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Nolasco, Jennifer, Leticia Nolasco, Qi Da, Sonya Cirlos, Zaverio Ruggeri, Joel Moake, and Miguel Cruz. "Complement Component C3 Binds to the A3 Domain of von Willebrand Factor." TH Open 02, no. 03 (July 2018): e338-e345. http://dx.doi.org/10.1055/s-0038-1672189.
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Abstractvon Willebrand factor (VWF) is a multimeric protein composed of monomeric subunits (∼280 kD) linked by disulfide bonds. During hemostasis and thrombosis, ultralarge (UL) VWF (ULVWF) multimers initiate platelet adhesion. In vitro, human C3 binds to ULVWF multimeric strings secreted by and anchored to human endothelial cell to promote the assembly and activation of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b) of the alternative complement pathway (AP). The purified and soluble C3 avidly binds to recombinant human VWF A1A2A3, as well as the recombinant isolated human VWF A3 domain. Notably, the binding of soluble human ULVWF multimers to purified human C3 was blocked by addition of a monovalent Fab fragment antibody to the VWF A3 domain. We conclude that the A3 domain in VWF/ULVWF contains a docking site for C3. In contrast, purified human C4, an essential component of the classical and lectin complement pathways, binds to soluble, isolated A1, but not to ULVWF strings secreted by and anchored to endothelial cells. Our findings should facilitate the design of new therapeutic agents to suppress the initiation of the AP on ULVWF multimeric strings during thrombotic and inflammatory disorders.
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Kinoshita, T., A. W. Dodds, S. K. A. Law, and K. Inoue. "The low C5 convertase activity of the C4A6 allotype of human complement component C4." Biochemical Journal 261, no. 3 (August 1, 1989): 743–48. http://dx.doi.org/10.1042/bj2610743.
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We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.
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Hindmarsh, Elizabeth J., and Rory M. Marks. "Complement Activation Occurs on Subendothelial Extracellular Matrix In Vitro and Is Initiated by Retraction or Removal of Overlying Endothelial Cells." Journal of Immunology 160, no. 12 (June 15, 1998): 6128–36. http://dx.doi.org/10.4049/jimmunol.160.12.6128.
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Abstract Vascular endothelium is continuously exposed to plasma complement, which could generate a potent proinflammatory signal if activated on the vascular wall. Normal endothelium, however, expresses an anti-inflammatory phenotype, which includes resistance to complement fixation. As activated endothelium converts to a proinflammatory phenotype, we investigated the effect of cytokines on endothelial susceptibility to complement fixation. Cytokine-treated HUVEC were exposed to human serum as a source of complement, and C3 deposition was quantified. IL-1β and TNF-α in combination with IFN-γ markedly increased endothelial C3 deposition; however, immunofluorescence microscopy revealed that the endothelial cells had retracted, and that bound C3 was concentrated not on cells but in areas of exposed subendothelial extracellular matrix (ECM). Studies with cell-free ECM indicated that complement activation required only ECM exposure and was independent of cellular activation. C3 deposition on ECM was reproduced by reconstituting the alternative pathway, which generated a stable C3 convertase on ECM, but not on endothelial cells. C3b and iC3b were identified on ECM exposed to purified alternative pathway components and serum, respectively. In conditions associated with endothelial disruption, exposure of subendothelial ECM could induce complement fixation and contribute to inflammation and vascular damage.
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Kinoshita, T., Y. Takata, H. Kozono, J. Takeda, K. S. Hong, and K. Inoue. "C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme." Journal of Immunology 141, no. 11 (December 1, 1988): 3895–901. http://dx.doi.org/10.4049/jimmunol.141.11.3895.
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Abstract C5 convertase of the alternative C pathway is a complex enzyme consisting of three C fragments--one molecule of a major fragment of factor B (Bb) and two molecules of a major fragment of C3 (C3b). Within this C3bBbC3b complex, the first C3b binds covalently to the target surface, and Bb, which bears a catalytic site, binds noncovalently to the first C3b. In the present investigation, we studied the nature of the convertase that is assembled on E surfaces and obtained evidence that the second C3b binds directly to the alpha'-chain of the first through an ester bond rather than to the target surface. Thus, the alternative pathway C5 convertase could be described as a trimolecular complex in which Bb binds noncovalently to a covalently linked C3b dimer. We also obtained evidence that not only the second C3b but also the first C3b participates in binding C5, that is, covalently-linked C3b dimer acts as a substrate-binding site. Because of this two-site binding, the convertase has a much higher affinity for C5 than the surrounding monomeric C3b molecules. Based on this evidence, a new model of the alternative pathway C5 convertase is proposed. Covalent association of two subunits and the bivalent binding of the substrate are then common properties of the alternative and classical pathway C5 convertases.
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Iatropoulos, Paraskevas, Erica Daina, Manuela Curreri, Rossella Piras, Elisabetta Valoti, Caterina Mele, Elena Bresin, et al. "Cluster Analysis Identifies Distinct Pathogenetic Patterns in C3 Glomerulopathies/Immune Complex–Mediated Membranoproliferative GN." Journal of the American Society of Nephrology 29, no. 1 (October 13, 2017): 283–94. http://dx.doi.org/10.1681/asn.2017030258.
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Membranoproliferative GN (MPGN) was recently reclassified as alternative pathway complement–mediated C3 glomerulopathy (C3G) and immune complex–mediated membranoproliferative GN (IC-MPGN). However, genetic and acquired alternative pathway abnormalities are also observed in IC-MPGN. Here, we explored the presence of distinct disease entities characterized by specific pathophysiologic mechanisms. We performed unsupervised hierarchical clustering, a data-driven statistical approach, on histologic, genetic, and clinical data and data regarding serum/plasma complement parameters from 173 patients with C3G/IC-MPGN. This approach divided patients into four clusters, indicating the existence of four different pathogenetic patterns. Specifically, this analysis separated patients with fluid-phase complement activation (clusters 1–3) who had low serum C3 levels and a high prevalence of genetic and acquired alternative pathway abnormalities from patients with solid-phase complement activation (cluster 4) who had normal or mildly altered serum C3, late disease onset, and poor renal survival. In patients with fluid-phase complement activation, those in clusters 1 and 2 had massive activation of the alternative pathway, including activation of the terminal pathway, and the highest prevalence of subendothelial deposits, but those in cluster 2 had additional activation of the classic pathway and the highest prevalence of nephrotic syndrome at disease onset. Patients in cluster 3 had prevalent activation of C3 convertase and highly electron-dense intramembranous deposits. In addition, we provide a simple algorithm to assign patients with C3G/IC-MPGN to specific clusters. These distinct clusters may facilitate clarification of disease etiology, improve risk assessment for ESRD, and pave the way for personalized treatment.
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JELEZAROVA, Emiliana, Anna VOGT, and Hans U. LUTZ. "Interaction of C3b2–IgG complexes with complement proteins properdin, factor B and factor H: implications for amplification." Biochemical Journal 349, no. 1 (June 26, 2000): 217–23. http://dx.doi.org/10.1042/bj3490217.
Full textAbstract:
Nascent C3b can form ester bonds with various target molecules on the cell surface and in the fluid phase. Previously, we showed that C3b2-IgG complexes represent the major covalent product of C3 activation in serum [Lutz, Stammler, Jelezarova, Nater and Späth (1996) Blood 88, 184-193]. In the present report, binding of alternative pathway proteins to purified C3b2-IgG complexes was studied in the fluid phase by using biotinylated IgG for C3b2-IgG generation and avidin-coated plates to capture complexes. Up to seven moles of properdin ‘monomer’ bound per mole of C3b2-IgG at physiological conditions in the absence of any other complement protein. At low properdin/C3b2-IgG ratios bivalent binding was preferred. Neither factor H nor factor B affected properdin binding. On the other hand, properdin strongly stimulated factor B binding. Interactions of all three proteins with C3b2-IgG exhibited pH optima. An ionic strength optimum was most pronounced for properdin, while factor B binding was largely independent of the salt concentration. C3b2-IgG complexes were powerful precursors of the alternative pathway C3 convertase. In the presence of properdin, C3 convertase generated from C3b2-IgG cleaved about sevenfold more C3 than the enzyme generated on C3b. C3b2-IgG complexes could therefore maintain the amplification loop of complement longer than free C3b.
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Mold, C., B. M. Bradt, G. R. Nemerow, and N. R. Cooper. "Epstein-Barr virus regulates activation and processing of the third component of complement." Journal of Experimental Medicine 168, no. 3 (September 1, 1988): 949–69. http://dx.doi.org/10.1084/jem.168.3.949.
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Serum incubated with purified EBV was found to contain C3 cleavage fragments characteristic of C3c. Since the cofactors necessary for such cleavage of C3b by factor I are not normally present in serum, EBV was tested for factor I cofactor activity. Purified EBV from both human and marmoset EBV-producing cell lines was found to act as a cofactor for the factor I-mediated breakdown C3b to iC3b and iC3b to C3c and C3dg. EBV also acted as a cofactor for the factor I-mediated cleavage of C4b to iC4b and iC4b to C4c and C4d. EBV from both the human and marmoset cell lines accelerated the decay of the alternative pathway C3 convertase. The classical pathway C3 convertase was unaffected. Multiple lines of evidence eliminated the possibility that marmoset or human CR1 was responsible for the functional activities of EBV preparations. The spectrum of activities was different from CR1 in that EBV and EBV-expressing cell lines failed to rosette with C3b or particles bearing C3b, the primary functional assay for CR1, and EBV did not accelerate classical pathway C3 convertase decay, another property of CR1. In addition, CR1 could not be detected immunologically on marmoset or human EBV-expressing cells and mAbs to CR1 failed to alter EBV-produced decay acceleration and factor I cofactor activities, although the antibodies blocked the same CR1-dependent functional activities. The multiple complement regulatory activities exhibited by purified EBV derived from human and marmoset cells differ from those of any of the known C3 or C4 regulatory proteins. These various activities would be anticipated to provide survival value for the virus by subverting complement- and cell-dependent host defense mechanisms.
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Wilcox, LA, JL Ezzell, NJ Bernshaw, and CJ Parker. "Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum." Blood 78, no. 3 (August 1, 1991): 820–29. http://dx.doi.org/10.1182/blood.v78.3.820.820.
Full textAbstract:
Abstract When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay- accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane- associated activation of the APC.
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Moulton, Elizabeth A., Paula Bertram, Nanhai Chen, R. Mark L. Buller, and John P. Atkinson. "Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement." Journal of Virology 84, no. 18 (July 7, 2010): 9128–39. http://dx.doi.org/10.1128/jvi.02677-09.
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ABSTRACT Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.
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Kim, Y. U., T. Kinoshita, H. Molina, D. Hourcade, T. Seya, L. M. Wagner, and V. M. Holers. "Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein." Journal of Experimental Medicine 181, no. 1 (January 1, 1995): 151–59. http://dx.doi.org/10.1084/jem.181.1.151.
Full textAbstract:
Normal host cells are protected from the destructive action of complement by cell surface complement regulatory proteins. In humans, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) play such a biologic role by inhibiting C3 and C5 convertases. DAF and MCP accomplish this task by specific mechanisms designated decay-accelerating activity and factor I cofactor activity, respectively. In other species, including mice, structural and/or functional homologues of these proteins are not yet well characterized. Previous studies have shown that the mouse protein Crry/p65 has certain characteristics of self-protecting complement regulatory proteins. For example, Crry/p65 is expressed on a wide variety of murine cells, and when expressed on human K562 erythroleukemic cells, it prevents deposition of mouse C3 fragments on the cell surface during activation of either the classical or alternative complement pathway. We have now studied factor I cofactor and decay-accelerating activities of Crry/p65. Recombinant Crry/p65 demonstrates cofactor activity for factor I-mediated cleavage of both mouse C3b and C4b. Surprisingly, Crry/p65 also exhibits decay-accelerating activity for the classical pathway C3 convertase strongly and for the alternative pathway C3 convertase weakly. Therefore, mouse Crry/p65 uses the specific mechanisms of both human MCP and DAF. Although Crry/p65, like MCP and DAF, contains tandem short consensus repeats (SCR) characteristic of C3/C4 binding proteins, Crry/p65 is not considered to be a genetic homologue of either MCP or DAF. Thus, Crry/p65 is an example of evolutionary conservation of two specific activities in a single unique protein in one species that are dispersed to individual proteins in another. We propose that the repeating SCR motif in this family has allowed this unusual process of evolution to occur, perhaps driven by the use of MCP and DAF as receptors by human pathogens such as the measles virus.
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Matsushita, M., and H. Okada. "Alternative complement pathway activation by C4b deposited during classical pathway activation." Journal of Immunology 136, no. 8 (April 15, 1986): 2994–98. http://dx.doi.org/10.4049/jimmunol.136.8.2994.
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Abstract Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.
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Murray, Hannah, Beiying Qiu, Sze Yuan Ho, and Xiaomeng Wang. "Complement Factor B Mediates Ocular Angiogenesis through Regulating the VEGF Signaling Pathway." International Journal of Molecular Sciences 22, no. 17 (September 3, 2021): 9580. http://dx.doi.org/10.3390/ijms22179580.
Full textAbstract:
Complement factor B (CFB), a 95-kDa protein, is a crucial catalytic element of the alternative pathway (AP) of complement. After binding of CFB to C3b, activation of the AP depends on the proteolytic cleavage of CFB by factor D to generate the C3 convertase (C3bBb). The C3 convertase contains the catalytic subunit of CFB (Bb), the enzymatic site for the cleavage of a new molecule of C3 into C3b. In addition to its role in activating the AP, CFB has been implicated in pathological ocular neovascularization, a common feature of several blinding eye diseases, however, with somewhat conflicting results. The focus of this study was to investigate the direct impact of CFB on ocular neovascularization in a tightly controlled environment. Using mouse models of laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), our study demonstrated an increase in CFB expression during pathological angiogenesis. Results from several in vitro and ex vivo functionality assays indicated a promoting effect of CFB in angiogenesis. Mechanistically, CFB exerts this pro-angiogenic effect by mediating the vascular endothelial growth factor (VEGF) signaling pathway. In summary, we demonstrate compelling evidence for the role of CFB in driving ocular angiogenesis in a VEGF-dependent manner. This work provides a framework for a more in-depth exploration of CFB-mediated effects in ocular angiogenesis in the future.
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Alcorlo, M., A. Tortajada, S. Rodriguez de Cordoba, and O. Llorca. "Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin." Proceedings of the National Academy of Sciences 110, no. 33 (July 30, 2013): 13504–9. http://dx.doi.org/10.1073/pnas.1309618110.
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Holguin, M. H., C. B. Martin, N. J. Bernshaw, and C. J. Parker. "Analysis of the effects of activation of the alternative pathway of complement on erythrocytes with an isolated deficiency of decay accelerating factor." Journal of Immunology 148, no. 2 (January 15, 1992): 498–502. http://dx.doi.org/10.4049/jimmunol.148.2.498.
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Abstract E from individuals with the Inab blood group phenotype have an isolated deficiency of decay accelerating factor (DAF, CD55). DAF is a glycosyl phosphatidylinositol anchored membrane protein that inhibits activation of both the classical and alternative pathways of complement. Deficiency of DAF from the E of paroxysmal nocturnal hemoglobinuria (PNH) is thought to contribute to their greater sensitivity to complement-mediated lysis. Unlike PNH E, however, Inab cells are not susceptible to acidified serum lysis, a process that is mediated through activation of the alternative pathway. This observation led us to hypothesize that membrane constituents other than DAF control susceptibility to acidified serum lysis. To investigate this hypothesis, Inab E were incubated in acidified serum, and hemolysis and C3 deposition (as a measure of alternative pathway activation) were quantitated. C3 deposition of Inab cells was approximately 20 times greater than normal, however, hemolysis was not observed. Inab E expressed a normal amount of membrane inhibitor of reactive lysis (MIRL, CD59), a glycosyl phosphatidylinositol anchored protein that is also deficient in PNH. When MIRL function was blocked with antibody, C3 deposition markedly increased, and 100% of the Inab cells hemolyzed in acidified serum. These studies demonstrate that susceptibility to acidified serum lysis is controlled primarily by MIRL, and that in addition to its regulatory affect on the membrane attack complex, MIRL also modulates the activity of the C3 convertase of the alternative pathway by a mechanism that remains to be determined.
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Manderson, Anthony P., Matthew C. Pickering, Marina Botto, Mark J. Walport, and Christopher R. Parish. "Continual Low-Level Activation of the Classical Complement Pathway." Journal of Experimental Medicine 194, no. 6 (September 10, 2001): 747–56. http://dx.doi.org/10.1084/jem.194.6.747.
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There is evidence that the classical complement pathway may be activated via a “C1-tickover” mechanism, analogous to the C3-tickover of the alternative pathway. We have quantitated and characterized this pathway of complement activation. Analysis of freshly collected mouse and human plasma revealed that spontaneous C3 activation rapidly occurred with the generation of C3 fragments in the plasma. By the use of complement- and Ig-deficient mice it was found that C1q, C4, C2, and plasma Ig were all required for this spontaneous C3 activation, with the alternative complement pathway further amplifying C3 fragment generation. Study of plasma from a human with C1q deficiency before and after therapeutic C1q infusion confirmed the existence of a similar pathway for complement activation in humans. Elevated levels of plasma C3 were detected in mice deficient in complement components required for activation of either the classical or alternative complement pathways, supporting the hypothesis that there is continuous complement activation and C3 consumption through both these pathways in vivo. Blood stasis was found to stimulate C3 activation by classical pathway tick-over. This antigen-independent mechanism for classical pathway activation may augment activation of the complement system at sites of inflammation and infarction.
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Meri, S., V. Koistinen, A. Miettinen, T. Törnroth, and I. J. Seppälä. "Activation of the alternative pathway of complement by monoclonal lambda light chains in membranoproliferative glomerulonephritis." Journal of Experimental Medicine 175, no. 4 (April 1, 1992): 939–50. http://dx.doi.org/10.1084/jem.175.4.939.
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Immunopathological evidence suggests that activation of the alternative pathway of complement (AP) is involved in membranoproliferative glomerulonephritis (MPGN) and in immunoglobulin A nephropathy. In this report we describe an AP dysfunction-associated factor that was isolated from the serum and urine of a patient with hypocomplementemic MPGN. Extensive glomerular deposits of C3, properdin, and of the terminal complement components were observed in the kidney of the patient. In her serum the AP hemolytic activity was virtually absent. When mixed with fresh normal serum, the patient's serum induced a 96% C3 conversion during a 30-min incubation at +37 degrees C. This activity was found to be due to a circulating factor that by immunochemical characterization proved to be a 46-kD monoclonal immunoglobulin lambda light (L) chain dimer (lambda L). Purified lambda L, but not control lambda or kappa L chains from patients with L chain disease, activated the AP in a dose- and ionic strength-dependent manner. Functionally, lambda L was differentiated from C3 nephritic factor (an autoantibody against the AP C3 convertase, C3bBb) by its inability to bind to and stabilize the C3bBb enzyme. Instead, lambda L was observed to interact directly with the AP control factor H. Thus, lambda L represents a novel type of immunoglobulin-related AP-activating factor with the capacity to initiate alternative complement pathway activation in the fluid phase.
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Pedersen, Henrik, Rasmus K. Jensen, Annette G. Hansen, Trine A. F. Gadeberg, Steffen Thiel, Nick S. Laursen, and Gregers R. Andersen. "A C3-specific nanobody that blocks all three activation pathways in the human and murine complement system." Journal of Biological Chemistry 295, no. 26 (May 6, 2020): 8746–58. http://dx.doi.org/10.1074/jbc.ra119.012339.
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The complement system is a tightly controlled proteolytic cascade in the innate immune system, which tags intruding pathogens and dying host cells for clearance. An essential protein in this process is complement component C3. Uncontrolled complement activation has been implicated in several human diseases and disorders and has spurred the development of therapeutic approaches that modulate the complement system. Here, using purified proteins and several biochemical assays and surface plasmon resonance, we report that our nanobody, hC3Nb2, inhibits C3 deposition by all complement pathways. We observe that the hC3Nb2 nanobody binds human native C3 and its degradation products with low nanomolar affinity and does not interfere with the endogenous regulation of C3b deposition mediated by Factors H and I. Using negative stain EM analysis and functional assays, we demonstrate that hC3Nb2 inhibits the substrate–convertase interaction by binding to the MG3 and MG4 domains of C3 and C3b. Furthermore, we notice that hC3Nb2 is cross-reactive and inhibits the lectin and alternative pathway in murine serum. We conclude that hC3Nb2 is a potent, general, and versatile inhibitor of the human and murine complement cascades. Its cross-reactivity suggests that this nanobody may be valuable for analysis of complement activation within animal models of both acute and chronic diseases.
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Risitano, Antonio M., Patrizia Ricci, Caterina Pascariello, Maddalena Raia, Christoph Q. Schmidt, Yingxue Li, Edimara S. Reis, et al. "Novel Complement Modulators for Paroxysmal Nocturnal Hemoglobinuria: Peptide and Protein Inhibitors of C3 Convertase Prevent Both Surface C3 Deposition and Subsequent Hemolysis of Affected Erythrocytes in Vitro." Blood 120, no. 21 (November 16, 2012): 370. http://dx.doi.org/10.1182/blood.v120.21.370.370.
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Abstract Abstract 370 Paroxysmal nocturnal hemoglobinuria (PNH) is a complex hematological disorder characterized by the expansion of blood cells deficient in the surface complement inhibitors CD55 and CD59; affected erythrocytes suffer from uncontrolled complement activation on their surface, and subsequent membrane attack complex (MAC)-mediated intravascular hemolysis. The anti-C5 antibody eculizumab has proven effective in controlling intravascular hemolysis in vivo, leading to remarkable clinical benefit in almost all PNH patients. Yet, we have demonstrated that persistent C3 activation occurring during eculizumab treatment may lead to progressive C3 deposition on affected erythrocyte and subsequent C3-mediated extravascular hemolysis, possibly limiting the hematological benefit of anti-C5 treatment (Risitano et al, Blood 2009). Thus, upstream inhibition of the complement cascade seems an appropriate strategy to improve the results of current anti-complement treatment; indeed, we have recently documented that the CD21/factor H (FH) fusion protein TT30 efficiently prevents both hemolysis and C3 deposition of PNH erythrocytes (Risitano et al, Blood 2012). Here we used the same in vitro model to evaluate two novel complement inhibitors that both act at the level of C3 convertases. Cp30 is an analog of the peptidic inhibitor compstatin, which is a 13-residue disulphide-bridged peptide that selectively binds to C3 and its activate fragment C3b. Compstatin and its analogues thereby prevent the initiation, amplification and terminal damage of the complement cascade via all its major pathways (classical, alternative, and mannose/lectin). Cp30 is one of the analogues developed to increase potency and stability of compstatin. Mini-FH, on the other hand, is an engineered 43kDa protein that combines the regulatory and surface-recognition activities of FH while showing increased affinity for the opsonins C3b, iC3b and C3d. Indeed, mini-FH retained both convertase decay acceleration and cofactor activities typical of endogenous human FH, resulting in a potent and selective inhibition of activation and amplification of the complement alternative pathway, without affecting the classical and the mannose/lectin pathway. Erythrocytes from PNH patients were washed and incubated in ABO-matched sera and exposed to pH-lowering to activate the alternative pathway, both in absence and presence of Cp30, mini-FH, and appropriate controls. Assessment of hemolysis and of C3 activation and deposition on PNH erythrocytes was performed by flow cytometry analyses of erythrocytes using anti-C3 and anti-CD59 antibodies, as previously described (Risitano et al Blood 2012). In absence of inhibitors, >90% of PNH erythrocytes lysed within 24 hours of incubation. Cp30 demonstrated a dose-dependent inhibition of hemolysis, with an IC50 of 4 μM and full inhibition at 8 μM. Cp30 also prevented deposition of any C3 fragment on the surface of surviving PNH erythrocyte. Similarly, mini-FH also showed dose-dependent inhibition of hemolysis, with an IC50 of 0.05 μM and full inhibition at 0.1 μM. Notably, both full-length fH and fH SCR1-4 were much less efficient in preventing hemolysis and C3 deposition (IC50 ∼ 0.5 μM; full inhibition >1 μM), supporting the higher potency of the engineered protein mini-FH. As expected, mini-FH also prevented surface deposition of C3 fragments on PNH erythrocytes. In conclusion, we confirm that inhibition of early phases of complement activation efficiently prevents hemolysis of PNH erythrocytes and their opsonization with C3 fragments in vitro. This effect may be obtained using either broad or pathway-specific inhibitors of C3 convertase, namely Cp30 and mini-FH, respectively. Thus, both strategies promise to prevent in vivo both MAC-mediated intravascular and C3-mediated extravascular hemolysis; however, according to their effect on specific complement pathways, they likely entail distinct patterns of potential risks. Our study provides the rationale for future translational plans to investigate the risk-to-benefit of these novel complement modulators in PNH. Disclosures: Risitano: Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Hiramatsu, M., J. E. Balow, and G. C. Tsokos. "Production of nephritic factor of the alternative complement pathway by Epstein Barr virus-transformed B cell lines derived from a patient with membranoproliferative glomerulonephritis." Journal of Immunology 136, no. 12 (June 15, 1986): 4451–55. http://dx.doi.org/10.4049/jimmunol.136.12.4451.
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Abstract Nephritic factor of the alternative complement pathway (C3NeF) is an IgG autoantibody which binds to and stabilizes the C3 convertase (C3bBb) enzyme, and which has been detected mainly in sera from patients with membranoproliferative glomerulonephritis (MPGN) and partial lipodystrophy. To study the production of C3NeF, mononuclear cells isolated from the peripheral blood of patients with MPGN and C3NeF activity in their sera were infected with Epstein Barr virus (EBV) to establish active B lymphocyte cell lines. By using a modified C3NeF screening assay, we detected C3NeF activity in the supernatant of a B cell line derived from a patient with MPGN Type II, but in none of the supernatants of B cell lines derived from normal individuals. C3NeF-positive supernatants were investigated for their ability to conserve classical or alternative pathway C3 convertase activity by using EAC3bBb and EAC4b2a stabilization assays. C3NeF-positive supernatants stabilized the C3bBb convertase activity, but not the C4b2a convertase activity. Studies of the supernatants, using anti-human IgG affinity columns, showed that the C3NeF activity was in the IgG fraction; furthermore, C3NeF antibody agglutinated sheep erythrocytes coated with C3bBb, but not with C3b alone. On gel electrophoresis, both heavy and light chains of the C3NeF were comparable in size to that of normal human IgG molecules. We conclude that C3NeF, produced in vitro by EBV-transformed B cell lines derived from a patient with MPGN Type II, is functionally identical to the conventional C3NeF in serum. In vitro preparation of homogeneous NeF(s) should greatly facilitate the studies of the role of these autoantibodies in complement dysmetabolism.
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Sahu, A., B. K. Kay, and J. D. Lambris. "Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library." Journal of Immunology 157, no. 2 (July 15, 1996): 884–91. http://dx.doi.org/10.4049/jimmunol.157.2.884.
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Abstract We have screened a phage-displayed random peptide library for binding to C3b, the proteolytically activated form of complement component C3, and have identified a novel peptide that suppresses complement activation. This phage-displayed peptide bound to C3, C3b, and C3c, but not to C3d, indicating that it binds to the C3c region of C3. A synthetic 27-amino acid peptide corresponding to the phage-displayed peptide also bound to C3 and C3 fragments and inhibited both the classical and alternative pathways of complement activation. The inhibition of complement activation was reversible. Studies with overlapping peptides indicated that the functional activity was located in the cyclic 13-amino acid N-terminal region (ICVVQDWGHHRCT) of the parent peptide. Reduction and alkylation of this 13-residue synthetic peptide destroyed its inhibitory activity. Analysis of the mechanism of inhibition revealed that the peptide inhibited C3 cleavage in normal human serum as well as when the alternative pathway was reconstituted with purified complement components, and the observed inhibition was not due to sterically hindered access to the C3a/C3b cleavage site. Further, the peptide did not inhibit the cleavage of factor B, indicating that it did not affect the interaction of CA with factor B or the formation of C3b,Bb. The peptide also had no effect on the binding of properdin to C3, demonstrating that the observed inhibition of C3 cleavage in normal human serum was not due in part to its effect on the properdin-stabilized C3 convertase, C3b,Bb,P. These results indicate that the peptide we have identified interacts with C3 to inhibit its activation.
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Fremeaux-Bacchi, Veronique, and Christoph Licht. "Hereditary and acquired complement dysregulation in membranoproliferative glomerulonephritis." Thrombosis and Haemostasis 101, no. 02 (2009): 271–78. http://dx.doi.org/10.1160/th08-09-0575.
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SummaryMembranoproliferative glomerulonephritis (MPGN) is a chronic progressive renal disease that is diagnosed on the basis of renal histological features. Several MPGN subtypes have been defined by the localization and composition of glomerular deposits (electron dense, Ig and C3). MPGN II or dense deposit disease (DDD) which is defined by the occurrence of electron dense deposits within the lamina densa of the glomerular basement membrane (GBM) is strongly associated with dysregulation of the alternative complement pathway (AP). However, C3 Nephritic Factor (C3NeF), an autoantibody against the alternative C3 convertase C3bBb, and mutations in regulatory proteins of the AP have also been identified in other subtypes of MPGN and even in glomerulonephritis with mesangial C3 deposits. Clinically, MPGN is characterized by proteinuria (up to nephrotic range) and hypertension, frequent progression to end-stage kidney disease and disease recurrence after renal transplantation. The age of onset varies from childhood to adulthood. In the following we will review our current knowledge of pathogenesis of MPGN and will present a novel classification system of the disease based on pathogenesis rather than on morphology. A better understanding of the pathogenesis of MPGN is crucial for the development of novel, specific treatment strategies.
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Lindorfer, Margaret A., Andrew W. Pawluczkowycz, Elizabeth M. Peek, Kimberly Hickman, Ronald P. Taylor, and Charles J. Parker. "A novel approach to preventing the hemolysis of paroxysmal nocturnal hemoglobinuria: both complement-mediated cytolysis and C3 deposition are blocked by a monoclonal antibody specific for the alternative pathway of complement." Blood 115, no. 11 (March 18, 2010): 2283–91. http://dx.doi.org/10.1182/blood-2009-09-244285.
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Abstract The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is chronic intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement (APC). Intravascular hemolysis can be inhibited in patients by treatment with eculizumab, a monoclonal antibody that binds complement C5 thereby preventing formation of the cytolytic membrane attack complex of complement. However, in essentially all patients treated with eculizumab, persistent anemia, reticulocytosis, and biochemical evidence of hemolysis are observed; and in a significant proportion, their PNH erythrocytes become opsonized with complement C3. These observations suggest that PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anemia because the antibody does not block APC activation. With a goal of improving PNH therapy, we characterized the activity of anti-C3b/iC3b monoclonal antibody 3E7 in an in vitro model of APC-mediated hemolysis. We show that 3E7 and its chimeric-deimmunized derivative H17 block both hemolysis and C3 deposition on PNH erythrocytes. The antibody is specific for the APC C3/C5 convertase because classical pathway–mediated hemolysis is unaffected by 3E7/H17. These findings suggest an approach to PNH treatment in which both intravascular and extravascular hemolysis can be inhibited while preserving important immune functions of the classical pathway of complement.
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Cheung, A. K., C. J. Parker, J. Janatova, and E. Brynda. "Modulation of complement activation on hemodialysis membranes by immobilized heparin." Journal of the American Society of Nephrology 2, no. 8 (February 1992): 1328–37. http://dx.doi.org/10.1681/asn.v281328.
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To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.
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Figueroa, J. E., and P. Densen. "Infectious diseases associated with complement deficiencies." Clinical Microbiology Reviews 4, no. 3 (July 1991): 359–95. http://dx.doi.org/10.1128/cmr.4.3.359.
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The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies.
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Pangburn, M. K., and N. Rawal. "Structure and function of complement C5 convertase enzymes." Biochemical Society Transactions 30, no. 6 (November 1, 2002): 1006–10. http://dx.doi.org/10.1042/bst0301006.
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The multisubunit enzymes of the complement system that cleave C5 have many unusual properties, the most striking of which is that they acquire their specificity for C5 following cleavage of another substrate C3. C5 convertases are assemblies of two proteins C4b and C2a (classical or lectin pathways) or C3b and Bb (alternative pathway) and additional C3b molecules. The catalytic complexes (C4b, C2a or C3b, Bb) are intrinsically unstable (t1,2 = 1–3 min) and the enzymes are controlled by numerous regulatory proteins that accelerate this natural decay rate. Immediately after assembly, the bi-molecular enzymes preferentially cleave the protein C3 and exhibit poor activity toward C5 (a Km of approx. 25 μM and a C5 cleavage rate of 0.3-1 C5/min at Vmax). Efficient C3 activation results in the covalent attachment of C3b to the cell surface and to the enzyme itself, resulting in formation of C3b-C3b and C4b-C3b complexes. Our studies have shown that deposition of C3b alters the specificity of the enzymes of both pathways by changing the Km for C5 more than 1000-fold from far above the physiological C5 concentration to far below it. Thus, after processing sufficient C3 at the surface of a microorganism, the enzymes switch to processing C5, which initiates the formation of the cytolytic membrane attack complex of complement.
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Taylor, Ronald P., Margaret A. Lindorfer, Andrew W. Pawluczkowycz, and Charles J. Parker. "A Novel Approach to Treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH): Both Hemolysis and C3 Deposition Are Blocked by a Monoclonal Antibody (mAb) Specific for the Alternative Pathway of Complement (APC) C3/C5 Convertase." Blood 114, no. 22 (November 20, 2009): 157. http://dx.doi.org/10.1182/blood.v114.22.157.157.
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Abstract Abstract 157 In PNH, red blood cells (RBCs) lack key complement control proteins, CD55 and CD59 and are therefore sensitive to complement activation and intravascular hemolysis. These regulatory proteins function at two different steps in the complement cascade; CD55 (decay accelerating factor, DAF) controls the formation and stability of the APC C3 and C5 convertases, while CD59 (membrane inhibitor of reactive lysis, MIRL) blocks formation of the cytolytic membrane attack complex (MAC). The intravascular hemolysis of PNH can be inhibited in vivo by eculizumab, a humanized mAb that binds complement C5, thereby preventing formation of the MAC. However, PNH patients treated with eculizumab continue to manifest evidence of ongoing hemolysis as they remain anemic with an elevated reticulocyte count and low serum haptoglobin concentration, and approximately 50% of eculizumab-treated patients require transfusion. This observation is consistent with the hypothesis that, in patients treated with eculizumab, PNH RBCs undergo extravascular hemolysis as a consequence of C3 opsonization because eculizumab does not compensate for deficiency of DAF. Recent studies (Risitano et al., Blood, 2009) support this hypothesis as patients undergoing treatment with eculizumab were found to have a positive Coombs test for C3 but not IgG, and flow cytometry demonstrated C3 activation and degradation products bound to the PNH RBCs. This process appeared clinically relevant as transfusion requirement correlated with the percentage of C3 opsonized PNH RBCs. These observations suggest that blocking the APC C3/C5 convertase would be a better way to treat the hemolysis of PNH because this approach has the advantage of blocking both extravascular hemolysis by inhibiting C3 opsonization and preventing intravascular hemolysis by inhibiting MAC generation. We have developed a mAb 3E7 and its deimmunized chimeric humanized derivative H17 that specifically block the APC C3/C5 convertase by binding to a neoepitope expressed when complement C3 is activated. In vitro, 3E7/H17 prevents APC-mediated lysis of rabbit RBCs in human serum and blocks deposition of human C3 activation fragments on APC activator substrates such as zymosan (Mol Immunol, 2006; J Immunol, 2007). We now report that mAb H17/3E7 blocks lysis in acidified normal human serum (aNHS) (a process mediated by the APC) of RBCs from patients with PNH (n=5). Representative results for patients 1 and 2 are as follows: 60% and 40% of RBCs were lysed after a one hour incubation at 37°C; lysis was reduced to 10% and 6%, respectively, at 80 ug/ml of mAb H17, and to 1% lysis (both patients) at 170 ug/ml of mAb H17. We also showed that mAb H17/3E7 blocks deposition of C3 activation fragments on PNH RBCs. After lysis in aNHS, blood samples from PNH patients were probed with Al488 mAb 1H8, specific for C3b/iC3b/C3dg. Flow cytometry experiments revealed C3 fragment deposition on lysed cells corresponding to 30,500 molecules of equivalent soluble fluorochrome (MESF) compared to a background signal of 225 MESF on unlysed RBCs in the same sample. Addition of mAb H17 blocked C3 fragment deposition not only on the unlysed cells but also on the small number of recovered ghosts . Importantly, mAb H17/3E7 inhibits the APC specifically. PNH RBCs, opsonized with IgM in serum from a patient with chronic cold agglutinin disease, were lysed in NHS by the classical complement pathway, and this lysis was not inhibited by mAbH17/3E7. Together, these experiments demonstrate that both hemolysis and C3 opsonization of PNH RBCs can be inhibited by a novel mAb that specifically blocks the APC C3/C5 convertase while leaving intact the classical pathway of complement. These findings suggest an approach to therapy of PNH in which both intravascular and extravascular hemolysis can be inhibited while preserving the important immune functions of the classical pathway of complement. Disclosures: No relevant conflicts of interest to declare.
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Rawal, Nenoo, and Michael K. Pangburn. "C5 Convertase of the Alternative Pathway of Complement." Journal of Biological Chemistry 273, no. 27 (July 3, 1998): 16828–35. http://dx.doi.org/10.1074/jbc.273.27.16828.
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Iwata, K., T. Seya, H. Ariga, and S. Nagasawa. "Expression of a hybrid complement regulatory protein, membrane cofactor protein decay accelerating factor on Chinese hamster ovary. Comparison of its regulatory effect with those of decay accelerating factor and membrane cofactor protein." Journal of Immunology 152, no. 7 (April 1, 1994): 3436–44. http://dx.doi.org/10.4049/jimmunol.152.7.3436.
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Abstract C activation on the cell surface is supposedly regulated by membrane cofactor protein (MCP) and decay accelerating factor (DAF). These are complementary in function: MCP acts as a cofactor in factor I-mediated C3b and C4b inactivation, thus preventing the assembly of C3 convertases, whereas DAF accelerates spontaneous decay of the assembled C3 convertase. In this report, a hybrid MCP-DAF was expressed on Chinese hamster ovary cells by transfecting cDNA, and its regulatory activity was compared with those of MCP and DAF transfectants and with a transfectant expressing both MCP and DAF (MCP + DAF). The C3 deposition on sensitized CHO cells through activation of the classical pathway was blocked to a different degree with these transfectants, the order being MCP + DAF > DAF > hybrid MCP-DAF > MCP. Likewise, the C3 deposition via the alternative pathway was blocked efficiently in the order hybrid > MCP + DAF > MCP. The C-mediated cytolysis of CHO cells virtually reflected the degree of C3 fragment deposition. The MCP-DAF transfectant acquired additive protective activity against alternative pathway-mediated C3 deposition and cytolysis but was less potent in circumventing classical pathway attack than cells that expressed DAF alone or DAF + MCP. Hybrid MCP-DAF may be useful for alleviating C-mediated cell damage, especially via the alternative pathway.
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Tu, Anh-Hue T., Robert L. Fulgham, Mark A. McCrory, David E. Briles, and Alexander J. Szalai. "Pneumococcal Surface Protein A Inhibits Complement Activation by Streptococcus pneumoniae." Infection and Immunity 67, no. 9 (September 1, 1999): 4720–24. http://dx.doi.org/10.1128/iai.67.9.4720-4724.1999.
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ABSTRACT Pneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor for Streptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the α chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.
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ISAAC, Lourdes, Dikran AIVAZIAN, Aiko TANIGUCHI-SIDLE, O. Roger EBANKS, S. Chuck FARAH, Marlene P. C. FLORIDO, K. Michael PANGBURN, and E. David ISENMAN. "Native conformations of human complement components C3 and C4 show different dependencies on thioester formation." Biochemical Journal 329, no. 3 (February 1, 1998): 705–12. http://dx.doi.org/10.1042/bj3290705.
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The thioester bond in complement components C3 and C4 and the protease inhibitor α2-macroglobulin have traditionally been thought of as fulfilling the dual roles of mediating covalent attachment and maintaining the native conformational states of these molecules. We previously reported that several human C3 thioester-region mutants, including variants E1012Q and C1010A, in the latter of which thioester-bond formation is precluded, display an unexpected phenotype. Despite the lack of a thioester bond in these mutants, they appear to adopt a native-like conformation as suggested by the finding that they are cleavable by the classical pathway C3 convertase, C4b2a, whereas the C3b-like C3(H2O) species is not. Subsequently, a species referred to as C3(NH3)* was described which potentially could account for the observations with the above mutants. C3(NH3)* is a transient species formed on aminolysis of native C3 that can spontaneously re-form the thioester bond. Importantly, it has a mobility on cation-exchange HPLC that is distinct from both native C3 and C3(H2O), but like the native molecule, it is cleavable by an alternative-pathway C3 convertase. In this study we showed by using cation-exchange HPLC as an additional conformational probe that C3 C1010A and E1012Q mutant proteins did not resemble C3(NH3)*. Instead they displayed a chromatographic behaviour that was indistinguishable from that of native C3. To assess the general applicability of these observations, we engineered the equivalent mutations into human C4, specifically C4 C1010A and C4 E1012Q. As expected, thioester-bond formation did not occur in either of these C4 mutants, but in contrast with the results with C3 we found no evidence for the formation of a stable native-like conformation in either C4 mutant, as assessed using cleavability by C1s as the conformational probe. A possible interpretation of our data is that the adoption of the native conformational state during biosynthesis of C3 and C4 is an energetically permissible process, even if it is not locked in via thioester-bond formation. Whereas this conformational state is stable in mature C3, it is unstable in mature C4, perhaps reflecting the additional post-translational cleavage of C4 before its secretion.
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Kimura, Yuko, Takashi Miwa, Lin Zhou, and Wen-Chao Song. "Activator-specific requirement of properdin in the initiation and amplification of the alternative pathway complement." Blood 111, no. 2 (January 15, 2008): 732–40. http://dx.doi.org/10.1182/blood-2007-05-089821.
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Properdin is a positive regulator of alternative pathway (AP) complement. The current understanding of properdin function is that it facilitates AP complement activation by stabilizing the C3 convertase C3bBb. Properdin-deficient patients are susceptible to lethal meningococcal infection, but the mechanism of this selective predisposition is not fully understood. By gene targeting in the mouse, we show here that properdin is essential for AP complement activation induced by bacterial lipopolysacharride (LPS) and lipooligosacharride (LOS) and other, but not all, AP complement activators. LPS- and LOS-induced AP complement activation was abolished in properdin−/− mouse serum, and properdin−/− mice were unable to clear Crry-deficient erythrocytes, which are known to be susceptible to AP complement–mediated extravascular hemolysis. In contrast, zymosan- and cobra venom factor–induced AP complement activation, and classical pathwaytriggered AP complement amplification were only partially or minimally affected in properdin−/− mice. We further show that the ability of human properdin to restore LPS-dependent AP complement activity in properdin−/− mouse serum correlated with the human properdin-binding affinity of the LPS. These results reveal a novel role of properdin in AP complement initiation and have implications for understanding the selective predisposition of properdin-deficient patients to meningococcal infection.
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Koistinen, V. "Effect of complement-protein-C3b density on the binding of complement factor H to surface-bound C3b." Biochemical Journal 280, no. 1 (November 15, 1991): 255–59. http://dx.doi.org/10.1042/bj2800255.
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Various amounts of the activation fragment C3b of the complement (C) protein C3 were coupled to Sepharose 4B by catalysis with the C3 convertase of the alternative pathway of C. The binding of radioactively labelled C proteins B and H (= factor H) to the C3b-carrying particles was assayed. It was found that the relative binding of H, but not of B, fell rapidly with decreasing densities of solid-phase C3b, suggesting a sigmoidal relationship between C3b density and binding of H. To study the phenomenon in more detail, preformed C3b was coupled to activated thiopropyl-Sepharose 6B at various densities. By using this model system, it was shown that the binding of H/unit amount of C3b was positively correlated to C3b density up to a C3b concentration of about 0.5 mg/ml of gel, whereas binding of B was independent of C3b density. The results show that accumulation of high densities of C3b on a surface creates high-affinity binding sites for H. Because H has recently been shown to form dimers in solution, the interaction of dimeric H with neighbouring C3b molecules is a likely explanation for the phenomenon. The C3b density effect may be a regulatory mechanism keeping the activation of the alternative pathway of C on activating surface within reasonable limits.
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Rooijakkers, Suzan H. M., Jin Wu, Maartje Ruyken, Robert van Domselaar, Karel L. Planken, Apostolia Tzekou, Daniel Ricklin, et al. "Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor." Nature Immunology 10, no. 7 (June 7, 2009): 721–27. http://dx.doi.org/10.1038/ni.1756.
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Jelezarova, Emiliana, and Hans U. Lutz. "IgG naturally occurring antibodies stabilize and promote the generation of the alternative complement pathway C3 convertase." Molecular Immunology 42, no. 11 (July 2005): 1393–403. http://dx.doi.org/10.1016/j.molimm.2004.12.014.
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Ricklin, Daniel. "Manipulating the mediator: Modulation of the alternative complement pathway C3 convertase in health, disease and therapy." Immunobiology 217, no. 11 (November 2012): 1057–66. http://dx.doi.org/10.1016/j.imbio.2012.07.016.
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