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1.
Pediatr Nephrol ; 34(8): 1349-1367, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30141176

RESUMO

Properdin is known as the only positive regulator of the complement system. Properdin promotes the activity of this defense system by stabilizing its key enzymatic complexes: the complement alternative pathway (AP) convertases. Besides, some studies have indicated a role for properdin as an initiator of complement activity. Though the AP is a powerful activation route of the complement system, it is also involved in a wide variety of autoimmune and inflammatory diseases, many of which affect the kidneys. The role of properdin in regulating complement in health and disease has not received as much appraisal as the many negative AP regulators, such as factor H. Historically, properdin deficiency has been strongly associated with an increased risk for meningococcal disease. Yet only recently had studies begun to link properdin to other complement-related diseases, including renal diseases. In the light of the upcoming complement-inhibiting therapies, it is interesting whether properdin can be a therapeutic target to attenuate AP-mediated injury. A full understanding of the basic concepts of properdin biology is therefore needed. Here, we first provide an overview of the function of properdin in health and disease. Then, we explore its potential as a therapeutic target for the AP-associated renal diseases C3 glomerulopathy, atypical hemolytic uremic syndrome, and proteinuria-induced tubulointerstitial injury. Considering current knowledge, properdin-inhibiting therapy seems promising in certain cases. However, knowing the complexity of properdin's role in renal pathologies in vivo, further research is required to clarify the exact potential of properdin-targeted therapy in complement-mediated renal diseases.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/imunologia , Via Alternativa do Complemento , Glomerulonefrite Membranoproliferativa/imunologia , Nefrite Intersticial/imunologia , Properdina/metabolismo , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Complemento C3/imunologia , Complemento C3/metabolismo , C3 Convertase da Via Alternativa do Complemento/metabolismo , Glomerulonefrite Membranoproliferativa/tratamento farmacológico , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Nefrite Intersticial/complicações , Nefrite Intersticial/tratamento farmacológico , Nefrite Intersticial/urina , Properdina/antagonistas & inibidores , Estabilidade Proteica/efeitos dos fármacos , Proteinúria/imunologia
2.
PLoS One ; 13(3): e0194662, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29579105

RESUMO

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and constitutes one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, cleave NHBA protein upstream or downstream of a conserved Arg-rich region, respectively. The cleavage results in the release of the C-terminal portion of the protein. The C-terminal fragment originating from the processing of meningococcal proteases, referred to as C2 fragment, exerts a toxic effect on endothelial cells altering the endothelial permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistent with previous findings showing that Neisseria meningitidis traverses the epithelial barrier without disrupting the junctional structures. We showed that epithelial cells constantly secrete proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich region, a putative docking domain reported to be essential for C2-mediated toxic effect. Moreover, we found that the C3-convertase of the alternative complement pathway is one of the proteases responsible for this processing. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. NHBA cleavage may occur at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , C3 Convertase da Via Alternativa do Complemento/metabolismo , Neisseria/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Transporte/química , Linhagem Celular , Ácido Edético/farmacologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Humanos , Magnésio/química , Magnésio/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise/efeitos dos fármacos , Proteômica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Zinco/química , Zinco/metabolismo
3.
J Biol Chem ; 292(32): 13345-13360, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28637873

RESUMO

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of ES PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on ES Conversely, PspCN boosted the CA, on ES, of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Ativação do Complemento , Degeneração Macular/genética , Mutação , Substituição de Aminoácidos , Animais , Síndrome Hemolítico-Urêmica Atípica/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , C3 Convertase da Via Alternativa do Complemento/química , C3 Convertase da Via Alternativa do Complemento/genética , C3 Convertase da Via Alternativa do Complemento/metabolismo , Complemento C3d/química , Complemento C3d/genética , Complemento C3d/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Fator I do Complemento/química , Fator I do Complemento/genética , Fator I do Complemento/metabolismo , Eritrócitos/química , Hemólise , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Degeneração Macular/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Carneiro Doméstico , Solubilidade , Streptococcus pneumoniae/metabolismo , Propriedades de Superfície
4.
J Immunol ; 193(11): 5567-75, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25355917

RESUMO

The alternative pathway (AP) is critical for the efficient activation of complement regardless of the trigger. It is also a major player in pathogenesis, as illustrated by the long list of diseases in which AP activation contributes to pathology. Its relevance to human disease is further emphasized by the high prevalence of pathogenic inherited defects and acquired autoantibodies disrupting components and regulators of the AP C3-convertase. Because pharmacological downmodulation of the AP emerges as a broad-spectrum treatment alternative, there is a powerful interest in developing new molecules to block formation and/or activity of the AP C3-convertase. In this paper, we describe the generation of a novel mAb targeting human factor B (FB). mAb FB48.4.2, recognizing with high affinity an evolutionary-conserved epitope in the Ba fragment of FB, very efficiently inhibited formation of the AP C3-proconvertase by blocking the interaction between FB and C3b. In vitro assays using rabbit and sheep erythrocytes demonstrated that FB28.4.2 was a potent AP inhibitor that blocked complement-mediated hemolysis in several species. Using ex vivo models of disease we demonstrated that FB28.4.2 protected paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and inhibited both C3 fragment and C5b-9 deposition on ADP-activated HMEC-1 cells, an experimental model for atypical hemolytic uremic syndrome. Moreover, i.v. injection of FB28.4.2 in rats blocked complement activation in rat serum and prevented the passive induction of experimental autoimmune Myasthenia gravis. As a whole, these data demonstrate the potential value of FB28.4.2 for the treatment of disorders associated with AP complement dysregulation in man and animal models.


Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Síndrome Hemolítico-Urêmica Atípica/terapia , Complemento C3b/metabolismo , Fator B do Complemento/metabolismo , Hemoglobinúria Paroxística/terapia , Miastenia Gravis Autoimune Experimental/terapia , Animais , Anticorpos Bloqueadores/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Síndrome Hemolítico-Urêmica Atípica/imunologia , Bovinos , Linhagem Celular , C3 Convertase da Via Alternativa do Complemento/metabolismo , Fator B do Complemento/genética , Fator B do Complemento/imunologia , Via Alternativa do Complemento/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Hemoglobinúria Paroxística/imunologia , Humanos , Camundongos , Camundongos Knockout , Miastenia Gravis Autoimune Experimental/imunologia , Ligação Proteica/efeitos dos fármacos , Coelhos , Ratos , Ratos Endogâmicos Lew , Ovinos
5.
J Am Soc Nephrol ; 25(9): 2053-65, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24652797

RESUMO

Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Síndrome Hemolítico-Urêmica Atípica/imunologia , Fator B do Complemento/genética , Mutação , Substituição de Aminoácidos , Sítios de Ligação/genética , C3 Convertase da Via Alternativa do Complemento/química , C3 Convertase da Via Alternativa do Complemento/genética , C3 Convertase da Via Alternativa do Complemento/metabolismo , Complemento C3b/metabolismo , C5 Convertase da Via Alternativa do Complemento/química , C5 Convertase da Via Alternativa do Complemento/genética , C5 Convertase da Via Alternativa do Complemento/metabolismo , Fator B do Complemento/química , Fator B do Complemento/metabolismo , Via Alternativa do Complemento/genética , Simulação por Computador , Frequência do Gene , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Modelos Moleculares , Complexos Multiproteicos/química , Polimorfismo Genético , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Immunobiology ; 217(11): 1057-66, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22964231

RESUMO

The complement network is increasingly recognized as an important triage system that is able to differentiate between healthy host cells, microbial intruders, cellular debris and immune complexes, and tailor its actions accordingly. At the center of this triage mechanism is the alternative pathway C3 convertase (C3bBb), a potent enzymatic protein complex capable of rapidly converting the inert yet abundant component C3 into powerful effector fragments (C3a and C3b), thereby amplifying the initial response on unprotected surfaces and inducing a variety of effector functions. A fascinating molecular mechanism of convertase assembly and intrinsic regulation, as well as the interplay with a panel of cell surface-bound and soluble inhibitors are essential for directing complement attack to intruders and protecting healthy host cells. While efficiently keeping immune surveillance and homeostasis on track, the reliance on an intricate cascade of interaction and conversion steps also renders the C3 convertase vulnerable to derail. On the one hand, tissue damage, accumulation of debris, or polymorphisms in complement genes may unfavorably shift the balance between activation and regulation, thereby contributing to a variety of clinical conditions. On the other hand, pathogens developed powerful evasion strategies to avoid complement attack by targeting the convertase. Finally, we increasingly challenge our bodies with foreign materials such as biomaterial implants or drug delivery vehicles that may induce adverse effects that are at least partially caused by complement activation and amplification via the alternative pathway. The involvement of the C3 convertase in a range of pathological conditions put this complex into the spotlight of complement-targeted drug discovery efforts. Fortunately, the physiological regulation and microbial evasion approaches provide a rich source of inspiration for the development of powerful treatment options. This review provides insight into the current knowledge about the molecular mechanisms that drive C3 convertase activity, reveals common and divergent strategies of convertase inhibition employed by host and pathogens, and how this inhibitory arsenal can be tapped for developing therapeutic options to treat complement-related diseases.


Assuntos
C3 Convertase da Via Alternativa do Complemento/metabolismo , Via Alternativa do Complemento , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Complemento C3/imunologia , Complemento C3/metabolismo , C3 Convertase da Via Alternativa do Complemento/antagonistas & inibidores , Descoberta de Drogas , Humanos , Evasão da Resposta Imune , Infecções/tratamento farmacológico , Infecções/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Terapia de Alvo Molecular
7.
Biochim Biophys Acta ; 1812(1): 12-22, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20837143

RESUMO

Complement is an essential component of innate immunity and a major trigger of inflammatory responses. A critical step in complement activation is the formation of the C3 convertase of the alternative pathway (AP), a labile bimolecular complex formed by activated fragments of the C3 and factor B components that is fundamental to provide exponential amplification of the initial complement trigger. Regulation of the AP C3 convertase is essential to maintain complement homeostasis in plasma and to protect host cells and tissues from damage by complement. During the last decade, several studies have associated genetic variations in components and regulators of the AP C3 convertase with a number of chronic inflammatory diseases and susceptibility to infection. The functional characterization of these protein variants has helped to decipher the critical pathogenic mechanisms involved in some of these complement related disorders. In addition, these functional data together with recent 3D structures of the AP C3 convertase have provided fundamental insights into the assembly, activation and regulation of the AP C3 convertase.


Assuntos
Complemento C3/genética , Fator B do Complemento/genética , Via Alternativa do Complemento/genética , Mutação , Complemento C3/química , Complemento C3/metabolismo , C3 Convertase da Via Alternativa do Complemento/química , C3 Convertase da Via Alternativa do Complemento/genética , C3 Convertase da Via Alternativa do Complemento/metabolismo , Fator B do Complemento/química , Fator B do Complemento/metabolismo , Via Alternativa do Complemento/fisiologia , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/fisiopatologia , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica
8.
J Immunol ; 185(1): 507-16, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20530262

RESUMO

Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological "native" (dimeric, trimeric, and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, preincubation of bacteria with unfractionated properdin, followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of 10 Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the "conventional" mechanism of stabilizing AP C3 convertases.


Assuntos
Via Alternativa do Complemento/imunologia , Neisseria gonorrhoeae/imunologia , Neisseria meningitidis Sorogrupo A/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Neisseria meningitidis Sorogrupo C/imunologia , Neisseria meningitidis Sorogrupo W-135/imunologia , Neisseria meningitidis Sorogrupo Y/imunologia , Properdina/fisiologia , Aderência Bacteriana/imunologia , Complemento C3/metabolismo , C3 Convertase da Via Alternativa do Complemento/metabolismo , Via Alternativa do Complemento/genética , Estabilidade Enzimática/imunologia , Humanos , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Neisseria meningitidis Sorogrupo A/genética , Neisseria meningitidis Sorogrupo A/metabolismo , Neisseria meningitidis Sorogrupo B/genética , Neisseria meningitidis Sorogrupo B/metabolismo , Neisseria meningitidis Sorogrupo C/genética , Neisseria meningitidis Sorogrupo C/metabolismo , Neisseria meningitidis Sorogrupo W-135/genética , Neisseria meningitidis Sorogrupo W-135/metabolismo , Neisseria meningitidis Sorogrupo Y/genética , Neisseria meningitidis Sorogrupo Y/metabolismo , Properdina/isolamento & purificação , Properdina/metabolismo , Ligação Proteica/imunologia
9.
J Am Soc Nephrol ; 21(5): 859-67, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20203157

RESUMO

In contrast to pregnancy-associated thrombotic thrombocytopenic purpura, the pathogenesis and presentation of pregnancy-associated atypical hemolytic uremic syndrome (P-aHUS) remain ill-defined. We conducted a retrospective study to assess the presentation and outcomes of patients presenting with P-aHUS and the prevalence of alternative C3 convertase dysregulation. P-aHUS occurred in 21 of the 100 adult female patients with atypical HUS, with 79% presenting postpartum. We detected complement abnormalities in 18 of the 21 patients. The outcomes were poor: 62% reached ESRD by 1 month and 76% by last follow-up. The risk for P-aHUS was highest during a second pregnancy. Thirty-five women, 26 (74%) of whom had complement abnormalities, had at least one pregnancy before the onset of a non-pregnancy-related aHUS. Outcomes did not differ between patients with pregnancy-related and non-pregnancy-related aHUS. Mutations in the SCR19-20 domains of factor H were less frequent in P-aHUS patients compared with non-pregnancy-related aHUS. Pregnancies in female patients with complement abnormalities (n = 44) were complicated by fetal loss and preeclampsia in 4.8% and 7.7%, respectively. Better understanding of complement dysregulation in pregnancy complications is essential, especially to guide development of pharmacologic agents to modulate this system.


Assuntos
C3 Convertase da Via Alternativa do Complemento/metabolismo , Síndrome Hemolítico-Urêmica/enzimologia , Complicações Hematológicas na Gravidez/enzimologia , Adulto , C3 Convertase da Via Alternativa do Complemento/genética , Feminino , Síndrome Hemolítico-Urêmica/genética , Humanos , Gravidez , Complicações Hematológicas na Gravidez/genética , Resultado da Gravidez , Estudos Retrospectivos , Adulto Jovem
10.
Science ; 330(6012): 1816-20, 2010 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-21205667

RESUMO

Activation of the complement cascade induces inflammatory responses and marks cells for immune clearance. In the central complement-amplification step, a complex consisting of surface-bound C3b and factor B is cleaved by factor D to generate active convertases on targeted surfaces. We present crystal structures of the pro-convertase C3bB at 4 angstrom resolution and its complex with factor D at 3.5 angstrom resolution. Our data show how factor B binding to C3b forms an open "activation" state of C3bB. Factor D specifically binds the open conformation of factor B through a site distant from the catalytic center and is activated by the substrate, which displaces factor D's self-inhibitory loop. This concerted proteolytic mechanism, which is cofactor-dependent and substrate-induced, restricts complement amplification to C3b-tagged target cells.


Assuntos
C3 Convertase da Via Alternativa do Complemento/química , Complemento C3b/química , Fator B do Complemento/química , Fator D do Complemento/química , Sítios de Ligação , Domínio Catalítico , C3 Convertase da Via Alternativa do Complemento/metabolismo , Complemento C3b/metabolismo , Fator B do Complemento/metabolismo , Fator D do Complemento/metabolismo , Via Alternativa do Complemento , Cristalografia por Raios X , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína
11.
J Immunol ; 183(4): 2565-74, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19625656

RESUMO

Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its beta-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies.


Assuntos
Complemento C3b/metabolismo , Proteínas Inativadoras do Complemento/fisiologia , Família Multigênica/imunologia , Staphylococcus aureus/imunologia , C3 Convertase da Via Alternativa do Complemento/metabolismo , C3 Convertase da Via Alternativa do Complemento/fisiologia , Complemento C3b/química , Fator B do Complemento/metabolismo , Fator H do Complemento/metabolismo , Proteínas Inativadoras do Complemento/metabolismo , Humanos , Ligação Proteica/imunologia , Staphylococcus aureus/patogenicidade , Virulência
12.
Nat Immunol ; 10(7): 721-7, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19503103

RESUMO

Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg(2+)-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.


Assuntos
Proteínas de Bactérias/química , C3 Convertase da Via Alternativa do Complemento/química , Proteínas Inativadoras do Complemento/química , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Complemento C3/química , Complemento C3/metabolismo , C3 Convertase da Via Alternativa do Complemento/metabolismo , Convertases de Complemento C3-C5/química , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Proteínas Inativadoras do Complemento/imunologia , Proteínas Inativadoras do Complemento/metabolismo , Via Alternativa do Complemento/imunologia , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Staphylococcus aureus/química , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Especificidade por Substrato , Ressonância de Plasmônio de Superfície
13.
Structure ; 17(4): 611-9, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19368894

RESUMO

Cobra venom factor (CVF) is a functional analog of human complement component C3b, the active fragment of C3. Similar to C3b, in human and mammalian serum, CVF binds factor B, which is then cleaved by factor D, giving rise to the CVFBb complex that targets the same scissile bond in C3 as the authentic complement convertases C4bC2a and C3bBb. Unlike the latter, CVFBb is a stable complex and an efficient C5 convertase. We solved the crystal structure of CVF, isolated from Naja naja kouthia venom, at 2.6 A resolution. The CVF crystal structure, an intermediate between C3b and C3c, lacks the TED domain and has the CUB domain in an identical position to that seen in C3b. The similarly positioned CUB and slightly displaced C345c domains of CVF could play a vital role in the formation of C3 convertases by providing important primary binding sites for factor B.


Assuntos
C3 Convertase da Via Alternativa do Complemento/metabolismo , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Venenos Elapídicos/química , Animais , Sítios de Ligação/genética , C3 Convertase da Via Alternativa do Complemento/genética , Convertases de Complemento C3-C5/genética , Complemento C3b/genética , Cristalografia por Raios X , Venenos Elapídicos/genética , Venenos Elapídicos/isolamento & purificação , Venenos Elapídicos/metabolismo , Modelos Químicos , Modelos Moleculares , Ligação Proteica/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
J Biol Chem ; 284(16): 10473-9, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19196712

RESUMO

Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in complex with a Fab fragment of this antibody (S77) illustrates the structural basis for this selectivity. Cleavage of C3 to C3b results in a plethora of structural changes within C3, including the rearrangement of macroglobulin domain 6 enabling binding of S77 to the adjacent macroglobulin domain 7 domain. S77 blocks binding of factor B to C3b inhibiting the first step in the formation of the alternative pathway C3 convertase. In addition, S77 inhibits C5 binding to C3b. This results in significantly reduced formations of anaphylatoxins and membrane-attack complexes. This study for the first time demonstrates the structural basis for complement inhibition by a C3b-selective antibody and provides insights into the molecular mechanisms of alternative pathway complement activation.


Assuntos
Anticorpos , Complemento C3b/imunologia , Via Alternativa do Complemento/fisiologia , Fragmentos Fab das Imunoglobulinas , Conformação Proteica , Animais , Anticorpos/química , Anticorpos/metabolismo , C3 Convertase da Via Alternativa do Complemento/metabolismo , Complemento C3b/química , Complemento C3b/genética , Complemento C3b/metabolismo , C5 Convertase da Via Alternativa do Complemento/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Macaca mulatta , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Receptores de Complemento 3b/química , Receptores de Complemento 3b/metabolismo
15.
Proc Natl Acad Sci U S A ; 106(3): 882-7, 2009 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-19136636

RESUMO

Generation of the alternative pathway C3-convertase, the central amplification enzyme of the complement cascade, initiates by the binding of factor B (fB) to C3b to form the proconvertase, C3bB. C3bB is subsequently cleaved by factor D (fD) at a single site in fB, producing Ba and Bb fragments. Ba dissociates from the complex, while Bb remains bound to C3b, forming the active alternative pathway convertase, C3bBb. Using single-particle electron microscopy we have determined the 3-dimensional structures of the C3bB and the C3bBb complexes at approximately 27A resolution. The C3bB structure shows that fB undergoes a dramatic conformational change upon binding to C3b. However, the C3b-bound fB structure was easily interpreted after independently fitting the atomic structures of the isolated Bb and Ba fragments. Interestingly, the divalent cation-binding site in the von Willebrand type A domain in Bb faces the C345C domain of C3b, whereas the serine-protease domain of Bb points outwards. The structure also shows that the Ba fragment interacts with C3b separately from Bb at the level of the alpha'NT and CUB domains. Within this conformation, the long and flexible linker between Bb and Ba is likely exposed and accessible for cleavage by fD to form the active convertase, C3bBb. The architecture of the C3bB and C3bBb complexes reveals that C3b could promote cleavage and activation of fB by actively displacing the Ba domain from the von Willebrand type A domain in free fB. These structures provide a structural basis to understand fundamental aspects of the activation and regulation of the alternative pathway C3-convertase.


Assuntos
C3 Convertase da Via Alternativa do Complemento/metabolismo , Complemento C3b/química , Fator B do Complemento/química , Antígenos CD55/fisiologia , C3 Convertase da Via Alternativa do Complemento/química , Fator H do Complemento/fisiologia , Precursores Enzimáticos/química , Humanos , Imageamento Tridimensional , Microscopia Eletrônica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Complemento 3b/fisiologia
16.
Blood ; 111(2): 732-40, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17916747

RESUMO

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 pathway-triggered 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.


Assuntos
Via Alternativa do Complemento , Properdina/metabolismo , Animais , C3 Convertase da Via Alternativa do Complemento/genética , C3 Convertase da Via Alternativa do Complemento/metabolismo , Via Alternativa do Complemento/genética , Marcação de Genes , Predisposição Genética para Doença , Humanos , Lipopolissacarídeos/farmacologia , Infecções Meningocócicas/genética , Infecções Meningocócicas/metabolismo , Camundongos , Camundongos Knockout , Properdina/genética , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Receptores de Complemento 3b
17.
J Immunol ; 179(4): 2600-8, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17675523

RESUMO

Complement promotes the rapid recognition and elimination of pathogens, infected cells, and immune complexes. The biochemical basis for its target specificity is incompletely understood. In this report, we demonstrate that properdin can directly bind to microbial targets and provide a platform for the in situ assembly and function of the alternative pathway C3 convertases. This mechanism differs from the standard model wherein nascent C3b generated in the fluid phase attaches nonspecifically to its targets. Properdin-directed complement activation occurred on yeast cell walls (zymosan) and Neisseria gonorrhoeae. Properdin did not bind wild-type Escherichia coli, but it readily bound E. coli LPS mutants, and the properdin-binding capacity of each strain correlated with its respective serum-dependent AP activation rate. Moreover, properdin:single-chain Ab constructs were used to direct serum-dependent complement activation to novel targets. We conclude properdin participates in two distinct complement activation pathways: one that occurs by the standard model and one that proceeds by the properdin-directed model. The properdin-directed model is consistent with a proposal made by Pillemer and his colleagues >50 years ago.


Assuntos
C3 Convertase da Via Alternativa do Complemento/química , Via Alternativa do Complemento , Escherichia coli K12/química , Lipopolissacarídeos/química , Neisseria gonorrhoeae/química , Properdina/química , Zimosan/química , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , C3 Convertase da Via Alternativa do Complemento/metabolismo , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Gonorreia/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Mutação , Neisseria gonorrhoeae/metabolismo , Properdina/genética , Properdina/metabolismo , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ovinos , Células U937 , Zimosan/metabolismo
18.
J Immunol ; 178(1): 352-9, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17182573

RESUMO

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplification. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bBb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 does not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR2-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF.


Assuntos
Antígenos CD55/metabolismo , C3 Convertase da Via Alternativa do Complemento/metabolismo , Complemento C3b/metabolismo , Fator B do Complemento/metabolismo , Bioensaio , Antígenos CD55/genética , Ensaio de Imunoadsorção Enzimática , Hemólise , Humanos , Properdina/metabolismo , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Deleção de Sequência , Solubilidade
19.
Mol Immunol ; 43(1-2): 97-106, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16026839

RESUMO

The complement system is a central part of innate immunity and in its normal setting aimed to recognize and eliminate microbes. For elimination toxic activation products are generated locally and are reported directly of the surface of the invading microbe. A deregulation of the alternative pathway results in defective recognition and toxic activation products can be formed on the surface of host tissues and structures. Recent studies have shown that mutated or defective regulators of the alternative pathway of complement are associated with auto immune diseases of the kidney, including the atypical form of hemolytic uremic syndrome (HUS), membranoproliferative glomerulonephritis (MPGN) and also of the eye, such as age-related macular degeneration (ARMD). Current research provides clues how mutations occurring in genes coding for single complement components or the inactivation of single regulators lead to defective alternative pathway amplification, via the convertase C3bBb. These scenarios explain how defects of a single regulator lead to local, organ specific damage.


Assuntos
C3 Convertase da Via Alternativa do Complemento/deficiência , Via Alternativa do Complemento/genética , Glomerulonefrite Membranoproliferativa/metabolismo , Síndrome Hemolítico-Urêmica/metabolismo , Degeneração Macular/metabolismo , Mutação , Animais , C3 Convertase da Via Alternativa do Complemento/genética , C3 Convertase da Via Alternativa do Complemento/metabolismo , Fator H do Complemento/deficiência , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Modelos Animais de Doenças , Glomerulonefrite Membranoproliferativa/genética , Glomerulonefrite Membranoproliferativa/patologia , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/patologia , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Camundongos Knockout , Suínos
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