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1.
Nat Commun ; 11(1): 1326, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165615

RESUMO

Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface. Receptor expression is highest in developmental stages transmitted to the tsetse fly vector and those exposed to blood meals in the tsetse gut. Receptor gene deletion reduced tsetse infection, identifying this receptor as a virulence factor for transmission. This demonstrates how a pathogen evolved a molecular mechanism to increase transmission to an insect vector by exploitation of a mammalian complement regulator.


Assuntos
Fator H do Complemento/metabolismo , Trypanosoma/fisiologia , Moscas Tsé-Tsé/parasitologia , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Bovinos , Membrana Celular/metabolismo , Complemento C3b/metabolismo , Fator H do Complemento/química , Cricetinae , Cricetulus , Camundongos Endogâmicos BALB C , Parasitemia/sangue , Ligação Proteica , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Regulação para Cima
2.
J Am Soc Nephrol ; 31(2): 241-256, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31980588

RESUMO

Sequence and copy number variations in the human CFHR-Factor H gene cluster comprising the complement genes CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, and Factor H are linked to the human kidney diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy. Distinct genetic and chromosomal alterations, deletions, or duplications generate hybrid or mutant CFHR genes, as well as hybrid CFHR-Factor H genes, and alter the FHR and Factor H plasma repertoire. A clear association between the genetic modifications and the pathologic outcome is emerging: CFHR1, CFHR3, and Factor H gene alterations combined with intact CFHR2, CFHR4, and CFHR5 genes are reported in atypical hemolytic uremic syndrome. But alterations in each of the five CFHR genes in the context of an intact Factor H gene are described in C3 glomerulopathy. These genetic modifications influence complement function and the interplay of the five FHR proteins with each other and with Factor H. Understanding how mutant or hybrid FHR proteins, Factor H::FHR hybrid proteins, and altered Factor H, FHR plasma profiles cause pathology is of high interest for diagnosis and therapy.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Complemento C3/análise , Glomerulonefrite Membranoproliferativa/genética , Síndrome Hemolítico-Urêmica Atípica/etiologia , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/fisiologia , Variações do Número de Cópias de DNA , Predisposição Genética para Doença , Variação Genética , Glomerulonefrite Membranoproliferativa/etiologia , Humanos , Rim/patologia , Família Multigênica
3.
J Biol Chem ; 294(52): 20148-20163, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31719147

RESUMO

Activation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·µm-2 AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, KD values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical.


Assuntos
Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Microscopia de Força Atômica , Ressonância de Plasmônio de Superfície , Ativação do Complemento , Complemento C3b/química , Complemento C3d/química , Complemento C3d/metabolismo , Fator H do Complemento/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Ligação Proteica
4.
Semin Immunol ; 45: 101341, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31757608

RESUMO

The complement system, while being an essential and very efficient effector component of innate immunity, may cause damage to the host and result in various inflammatory, autoimmune and infectious diseases or cancer, when it is improperly activated or regulated. Factor H is a serum glycoprotein and the main regulator of the activity of the alternative complement pathway. Factor H, together with its splice variant factor H-like protein 1 (FHL-1), inhibits complement activation at the level of the central complement component C3 and beyond. In humans, there are also five factor H-related (FHR) proteins, whose function is poorly characterized. While data indicate complement inhibiting activity for some of the FHRs, there is increasing evidence that FHRs have an opposite role compared with factor H and FHL-1, namely, they enhance complement activation directly and also by competing with the regulators FH and FHL-1. This review summarizes the current stand and recent data on the roles of factor H family proteins in health and disease, with focus on the function of FHR proteins.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Ativação do Complemento/imunologia , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Fator H do Complemento/química , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Suscetibilidade a Doenças , Humanos , Imunomodulação , Ligantes , Família Multigênica , Ligação Proteica , Relação Estrutura-Atividade
5.
Front Immunol ; 10: 1230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214187

RESUMO

Streptococcus pyogenes infects over 700 million people worldwide annually. Immune evasion strategies employed by the bacteria include binding of the complement inhibitors, C4b-binding protein (C4BP) and Factor H in a human-specific manner. We recently showed that human IgG increased C4BP binding to the bacterial surface, which promoted streptococcal immune evasion and increased mortality in mice. We sought to identify how IgG promotes C4BP binding to Protein H, a member of the M protein family. Dimerization of Protein H is pivotal for enhanced binding to human C4BP. First, we illustrated that Protein H, IgG, and C4BP formed a tripartite complex. Second, surface plasmon resonance revealed that Protein H binds IgG solely through Fc, but not Fab domains, and with high affinity (IgG-Protein H: KD = 0.4 nM; IgG-Fc-Protein H: KD ≤ 1.6 nM). Each IgG binds two Protein H molecules, while up to six molecules of Protein H bind one C4BP molecule. Third, interrupting Protein H dimerization either by raising temperature to 41°C or with a synthetic peptide prevented IgG-Protein H interactions. IgG-Fc fragments or monoclonal human IgG permitted maximal C4BP binding when used at concentrations from 0.1 to 10 mg/ml. In contrast, pooled human IgG enhanced C4BP binding at concentrations up to 1 mg/ml; decreased C4BP binding at 10 mg/ml occurred probably because of Fab-streptococcal interactions at these high IgG concentrations. Taken together, our data show how S. pyogenes exploits human IgG to evade complement and enhance its virulence. Elucidation of this mechanism could aid design of new therapeutics against S. pyogenes.


Assuntos
Proteína de Ligação ao Complemento C4b/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunoglobulina G/imunologia , Imunomodulação , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Proteína de Ligação ao Complemento C4b/metabolismo , Fator H do Complemento/química , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/metabolismo , Humanos , Cinética , Ligantes , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Multimerização Proteica , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia
6.
Int J Mol Sci ; 20(10)2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31130605

RESUMO

An understanding of the interaction between the antibody and its targeted antigen and knowing of the epitopes are critical for the development of monoclonal antibody drugs. Complement factor H (CFH) is implied to play a role in tumor growth and metastasis. An autoantibody to CHF is associated with anti-tumor cell activity. The interaction of a human monoclonal antibody Ab42 that was isolated from a cancer patient with CFH polypeptide (pCFH) antigen was analyzed by molecular docking, molecular dynamics (MD) simulation, free energy calculation, and computational alanine scanning (CAS). Experimental alanine scanning (EAS) was then carried out to verify the results of the theoretical calculation. Our results demonstrated that the Ab42 antibody interacts with pCFH by hydrogen bonds through the Tyr315, Ser100, Gly33, and Tyr53 residues on the complementarity-determining regions (CDRs), respectively, with the amino acid residues of Pro441, Ile442, Asp443, Asn444, Ile447, and Thr448 on the pCFH antigen. In conclusion, this study has explored the mechanism of interaction between Ab42 antibody and its targeted antigen by both theoretical and experimental analysis. Our results have important theoretical significance for the design and development of relevant antibody drugs.


Assuntos
Anticorpos Monoclonais/imunologia , Peptídeos/imunologia , Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Autoanticorpos/química , Autoanticorpos/imunologia , Fator H do Complemento/química , Fator H do Complemento/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Epitopos/química , Epitopos/imunologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neoplasias/imunologia , Peptídeos/química , Conformação Proteica
7.
J Thromb Thrombolysis ; 48(1): 95-102, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30915671

RESUMO

Coagulation factor XIII (FXIII) covalently crosslinks pre-formed fibrin clots preventing their premature fibrinolysis. In plasma, FXIII circulates as a zymogenic heterotetramer composed of catalytic FXIII-A subunits, and carrier/regulatory FXIII-B subunits. FXIII-A is a well characterized component of this complex, and has been associated with several pleiotropic roles outside coagulation as well. In comparison only protective/regulatory roles towards the FXIII-A subunit have been identified for FXIII-B. Strong homology between FXIII-B and complement regulator Complement factor H suggests a putative role of FXIII-B in complement activation. In the current study we have analyzed the similarities and yet functional divergence of these two proteins using in silico sequence alignment and structural analysis. We have evaluated complement activation post reconstitution of FXIII components into FXIII deficient and CFH deficient plasma. We have also transiently expressed FXIII-B in SH-SY5Y cell lines and evaluated its effect on the endogenous complement activation. Our investigations show no effect of FXIII-B subunit on the rate of complement activation. Therefore we conclude that at a physiological level, FXIII-B subunit plays no role in the complement system, although a vestigial function in altered pathological states might still exist.


Assuntos
Ativação do Complemento , Fator XIII/química , Coleta de Amostras Sanguíneas , Linhagem Celular , Fator H do Complemento/química , Fator H do Complemento/fisiologia , Simulação por Computador , Fator XIII/fisiologia , Humanos , Estrutura Molecular , Domínios Proteicos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Transfecção
8.
Biophys J ; 116(2): 215-226, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30616835

RESUMO

A single nucleotide polymorphism, tyrosine at position 402 to histidine (Y402H), within the gene encoding complement factor H (FH) predisposes individuals to acquiring age-related macular degeneration (AMD) after aging. This polymorphism occurs in short consensus repeat (SCR) 7 of FH and results in decreased binding affinity of SCR6-8 for heparin. As FH is responsible for regulating the complement system, decreased affinity for heparin results in decreased regulation on surfaces of self. To understand the involvement of the Y402H polymorphism in AMD, we leverage methods from bioinformatics and computational biophysics to quantify structural and dynamical differences between SCR7 isoforms that contribute to decreased pattern recognition in SCR7H402. Our data from molecular and Brownian dynamics simulations suggest a revised mechanism for decreased heparin binding. In this model, transient contacts not observed in structures for SCR7 are predicted to occur in molecular dynamics simulations between coevolved residues Y402 and I412, stabilizing SCR7Y402 in a conformation that promotes association with heparin. H402 in the risk isoform is less likely to form a contact with I412 and samples a larger conformational space than Y402. We observe energy minima for sidechains of Y402 and R404 from SCR7Y402 that are predicted to associate with heparin at a rate constant faster than energy minima for sidechains of H402 and R404 from SCR7H402. As both carbohydrate density and degree of sulfation decrease with age in Bruch's membrane of the macula, the decreased heparin recognition of SCR7H402 may contribute to the pathogenesis of AMD.


Assuntos
Fator H do Complemento/química , Degeneração Macular/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Sítios de Ligação , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Heparina/química , Humanos , Ligação Proteica
9.
J Phys Chem B ; 122(47): 10653-10658, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30351116

RESUMO

Activation of proteins often involves conformational transitions, and these switches are often difficult to characterize in multidomain proteins. Full-length factor H (FH), consisting of 20 small consensus repeat domains (150 kD), is a complement control protein that regulates the activity of the alternative complement pathway. Different preparations of FH can also reduce the disulfide bonds linking large Von Willebrand factor (VWF) multimers into smaller, less adhesive forms. In contrast, commercially available purified FH (pFH) has little or no VWF reductase activity unless the pFH is chemically modified by either ethylenediaminetetraacetic acid (EDTA) or urea. We used atomic force microscopy single molecule force measurements to investigate different forms of FH, including recombinant FH and pFH, in the presence or absence of EDTA and urea, and to correlate the conformational changes to its activities. We found that the FH conformation depends on the method used for sample preparation, which affects the VWF reductase activity of FH.


Assuntos
Oxirredutases/química , Fator de von Willebrand/química , Catálise , Fator H do Complemento/química , Detergentes/química , Células HEK293 , Humanos , Microscopia de Força Atômica/métodos , Octoxinol/química , Organofosfatos/química , Oxirredução , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Termodinâmica
10.
J Biol Chem ; 293(44): 17166-17187, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30217822

RESUMO

Factor H (FH) is the major regulator of C3b in the alternative pathway of the complement system in immunity. FH comprises 20 short complement regulator (SCR) domains, including eight glycans, and its Y402H polymorphism predisposes those who carry it to age-related macular degeneration. To better understand FH complement binding and self-association, we have studied the solution structures of both the His-402 and Tyr-402 FH allotypes. Analytical ultracentrifugation revealed that up to 12% of both FH allotypes self-associate, and this was confirmed by small-angle X-ray scattering (SAXS), MS, and surface plasmon resonance analyses. SAXS showed that monomeric FH has a radius of gyration (Rg ) of 7.2-7.8 nm and a length of 25 nm. Starting from known structures for the SCR domains and glycans, the SAXS data were fitted using Monte Carlo methods to determine atomistic structures of monomeric FH. The analysis of 29,715 physically realistic but randomized FH conformations resulted in 100 similar best-fit FH structures for each allotype. Two distinct molecular structures resulted that showed either an extended N-terminal domain arrangement with a folded-back C terminus or an extended C terminus and a folded-back N terminus. These two structures are the most accurate to date for glycosylated full-length FH. To clarify FH functional roles in host protection, crystal structures for the FH complexes with C3b and C3dg revealed that the extended N-terminal conformation accounted for C3b fluid-phase regulation, the extended C-terminal conformation accounted for C3d binding, and both conformations accounted for bivalent FH binding to glycosaminoglycans on the target cell surface.


Assuntos
Complemento C3b , Fator H do Complemento , Fragmentos de Peptídeos , Complemento C3b/química , Complemento C3b/genética , Complemento C3b/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Cristalografia por Raios X , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Ressonância de Plasmônio de Superfície , Difração de Raios X
11.
Glycobiology ; 28(10): 765-773, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29982679

RESUMO

Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains several glycan binding sites which mediate recognition of α2-3-linked sialic acid (FH domain 20) and glycosaminoglycans (domains 6-8 and 19-20). FH also binds the complement C3-activation product C3b, a powerful opsonin and focal point for the formation of C3-convertases of the AP feedback loop. In freely circulating FH the C3b binding site in domains 19-20 is occluded, a phenomenon that is not fully understood and could be mediated by an intramolecular interaction between FH's intrinsic sialylated glycosylation and its own sialic acid binding site. In order to assess this possibility, we characterized FH's sialylation with respect to glycosidic linkage type and searched for further potential, not yet characterized sialic acid binding sites in FH and its seven-domain spanning splice variant and fellow complement regulator FH like-1 (FHL-1). We also probed FH binding to the sialic acid variant Neu5Gc which is not expressed in humans but on heterologous erythrocytes that restrict the human AP and in FH transgenic mice. We find that FH contains mostly α2-6-linked sialic acid, making an intramolecular interaction with its α2-3-sialic acid specific binding site and an associated self-lock mechanism unlikely, substantiate that there is only a single sialic acid binding site in FH and none in FHL-1, and demonstrate direct binding of FH to the nonhuman sialic acid Neu5Gc, supporting the use of FH transgenic mouse models for studies of complement-related diseases.


Assuntos
Ácido N-Acetilneuramínico/análise , Animais , Sítios de Ligação , Configuração de Carboidratos , Fator H do Complemento/química , Fator H do Complemento/isolamento & purificação , Fator H do Complemento/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo
12.
J Korean Med Sci ; 33(1): e4, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29215813

RESUMO

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a rare disease that is often associated with genetic defects. Mutations of complement factor H (CFH) are the most common genetic defects that cause aHUS and often result in end-stage renal disease. Since CFH is mainly produced in the liver, liver transplantation (LT) has been performed in patients with defective CFH. METHODS: The clinical courses of four kidney allograft recipients who lost their native kidney functions due to aHUS associated with a CFH mutation were reviewed. RESULTS: Subject A underwent kidney transplantation (KT) twice, aHUS recurred and the allograft kidney failed within a few years. Subject B received a KT and soon experienced a recurrence of aHUS coinciding with infection. Her allograft kidney function has worsened, and she remains on plasma infusion therapy. Subject C underwent LT followed by KT. She is doing well without plasma infusion therapy after combined LT-KT for 3 years. Subject D received KT following LT and is now recurrence-free from aHUS. CONCLUSION: In patients with aHUS associated with a CFH mutation, KT without LT was complicated with a recurrence of aHUS, which might lead to allograft loss. Conversely, LT was successful in preventing the recurrence of aHUS and thus might be another option for a recurrence-free life for aHUS patients associated with CFH mutation.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/etiologia , Transplante de Rim/efeitos adversos , Adolescente , Criança , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Creatinina/sangue , Análise Mutacional de DNA , Feminino , Humanos , Falência Renal Crônica/terapia , Transplante de Fígado , Masculino , Plasma Rico em Plaquetas , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Transplante Homólogo , Adulto Jovem
13.
Sci Rep ; 7(1): 6004, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729648

RESUMO

Despite distinct renal lesions, a series of rare glomerular nephropathies are reportedly mediated by complement overactivation. Genetic variations in complement genes contribute to disease risk, but the relationship of genotype to phenotype has not been straightforward. Here, we screened 11 complement genes from 91 patients with atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G) and membranoproliferative glomerulonephritis type I (MPGN I), and identified the concomitant presence of three missense variations located within the human complement Factor H (CFH) gene cluster. The three variations, rs55807605, rs61737525 and rs57960694, have strong linkage disequilibrium; subsequent haplotype analysis indicated that ATA increased the susceptibility of these renal diseases. In silico analysis, the CFHR3 rs61737525-T risk allele altered the physical and structural properties and generated a reduction in binding affinity of the CFHR3/C3b complex. Surface plasmon resonance (SPR) binding analysis further demonstrated the substitution induced a decrease of two orders of magnitude in C3b-binding properties, with a declined cofactor activity in fluid phase. These data suggest that the haplotype carrying the causative allele behaves as a partial C3 convertase deficiency, predisposing individuals to diverse pathologic lesions underlying complement overactivation. Such genotype-phenotype discrepancies allow better understanding about these nephropathies mediated by genetic complement disorders.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Predisposição Genética para Doença , Variação Genética , Glomerulonefrite Membranoproliferativa/genética , Haplótipos , Família Multigênica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/imunologia , Síndrome Hemolítico-Urêmica Atípica/metabolismo , Ativação do Complemento , Complemento C3/imunologia , Complemento C3/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Feminino , Estudos de Associação Genética , Ligação Genética , Glomerulonefrite Membranoproliferativa/diagnóstico , Glomerulonefrite Membranoproliferativa/imunologia , Glomerulonefrite Membranoproliferativa/metabolismo , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Adulto Jovem
14.
Nat Struct Mol Biol ; 24(8): 643-651, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28671664

RESUMO

The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains 1-4 with 19-20). FI binds C3b-FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.


Assuntos
Complemento C3b/química , Complemento C3b/metabolismo , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Fator I do Complemento/química , Fator I do Complemento/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteólise
15.
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
16.
Biochem J ; 474(11): 1803-1806, 2017 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-28490660

RESUMO

The human immune system is responsible for identification and destruction of invader cells, such as the bacterial pathogen Staphylococcus aureus In response, S. aureus brings to the fight a large number of virulence factors, including several that allow it to evade the host immune response. The staphylococcal surface protein SdrE was recently reported to bind to complement Factor H, an important regulator of complement activation. Factor H attaches to the surface of host cells to inhibit complement activation and amplification, preventing the destruction of the host cell. SdrE binding to Factor H allows S. aureus to mimic a host cell and reduces bacterial killing by granulocytes. In a new study published in Biochemical Journal, Zhang et al. describe crystal structures of SdrE and its complex with the C-terminal portion of Factor H. The structure of SdrE and its interaction with the Factor H peptide closely resemble a family of surface proteins that recognize extracellular matrix components such as fibrinogen. However, unbound SdrE forms a novel 'Closed' conformation with an occluded peptide-binding groove. These structures reveal a fascinating mechanism for immune evasion and provide a potential avenue for the development of novel antimicrobial agents to target SdrE.


Assuntos
Proteínas de Bactérias/metabolismo , Evasão da Resposta Imune , Modelos Moleculares , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Ativação do Complemento , Fator H do Complemento/antagonistas & inibidores , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Humanos , Conformação Proteica , Dobramento de Proteína , Desdobramento de Proteína , Staphylococcus aureus/imunologia
17.
Biochem J ; 474(10): 1619-1631, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28258151

RESUMO

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine-aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE-CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH1206-1226), which binds SdrE N2 and N3 domains (SdrEN2N3) with high affinity, and determined the crystal structures of apo-SdrEN2N3 and the SdrEN2N3-CFH1206-1226 complex. Comparison of the structure of the CFH-SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrEN2N3 adopts a 'close' state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel 'close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a 'clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion.


Assuntos
Proteínas de Bactérias/metabolismo , Modelos Moleculares , Staphylococcus aureus , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Evasão da Resposta Imune , Cinética , Ligantes , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Desdobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/imunologia
18.
Microbiol Res ; 196: 17-25, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28164787

RESUMO

Factor H (FH), a regulatory protein of the complement system, can bind specifically to factor H-binding proteins (FHBPs) of Streptococcus suis serotype 2 (SS2), which contribute to evasion of host innate immune defenses. In the present study, we aimed to identify novel FHBPs and characterize the biological functions of FH in SS2 pathogenesis. Here, a method that combined proteomics and Far-western blotting was developed to identify the surface FHBPs of SS2. With this method, fourteen potential novel FHBPs were identified among SS2 surface proteins. We selected eight newly identified proteins and further confirmed their binding activity to FH. The binding of SS2 to immobilized FH decreased dramatically after pre-incubation with anti-FHBPs polyclonal antibodies. We showed for the first time that SS2 also interact specifically with mouse FH. Furthermore, we found that FH play an important role in adherence and invasion of SS2 to HEp-2 cells. Additionally, using a mouse model of intraperitoneal challenge, we confirmed that SS2 pre-incubated with FH enhanced bacteremia and brain invasion, compared with SS2 not pretreated with FH. Taken together, this study provides a useful method to characterize the host-bacteria interactions. These results first indicated that binding of FH to the cell surface improved the adherence and invasion of SS2 to HEp-2 cells, promoting SS2 to resist killing and leading to enhance virulence.


Assuntos
Antígenos de Bactérias/imunologia , Fator H do Complemento/imunologia , Streptococcus suis/imunologia , Streptococcus suis/patogenicidade , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Aderência Bacteriana/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Fator H do Complemento/farmacologia , Escherichia coli/genética , Feminino , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/imunologia , Proteínas Imobilizadas/metabolismo , Evasão da Resposta Imune , Imunidade Inata , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Streptococcus suis/metabolismo , Virulência
19.
Oncotarget ; 8(30): 49016-49032, 2017 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-28159936

RESUMO

Nitrosative stress has been implicated in the pathogenesis of age related macular degeneration (AMD). Tyrosine nitration is a unique type of post translational modification that occurs in the setting of inflammation and nitrosative stress. To date, the significance and functional implications of tyrosine nitration of complement factor H (CFH), a key complement regulator in the eye has not been explored, and is examined in this study in the context of AMD pathogenesis.Sections of eyes from deceased individuals with AMD (n = 5) demonstrated the presence of immunoreactive nitrotyrosine CFH. We purified nitrated CFH from retinae from 2 AMD patients. Mass spectrometry of CFH isolated from AMD eyes revealed nitrated residues in domains critical for binding to heparan sulphate glycosaminoglycans (GAGs), lipid peroxidation by-products and complement (C) 3b.Functional studies revealed that nitrated CFH did not bind to lipid peroxidation products, nor to the GAG of perlecan nor to C3b. There was loss of cofactor activity for Factor I mediated cleavage of C3b with nitrated CFH compared to non-nitrated CFH. CFH inhibits, but nitrated CFH significantly potentiates, the secretion of the pro-inflammatory and angiogenic cytokine IL-8 from monocytes that have been stimulated with lipid peroxidation by-products. AMD patients (n = 30) and controls (n = 30) were used to measure plasma nitrated CFH using a novel ELISA. AMD patients had significantly elevated nitrated CFH levels compared to controls (p = 0.0117). These findings strongly suggest that nitrated CFH contributes to AMD progression, and is a target for therapeutic intervention.


Assuntos
Fator H do Complemento/metabolismo , Suscetibilidade a Doenças , Imunomodulação , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Tirosina/genética , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Biomarcadores , Estudos de Casos e Controles , Corioide/imunologia , Corioide/metabolismo , Corioide/patologia , Complemento C3b/imunologia , Complemento C3b/metabolismo , Fator H do Complemento/química , Ensaio de Imunoadsorção Enzimática , Feminino , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Degeneração Macular/diagnóstico , Masculino , Monócitos/imunologia , Monócitos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico , Proteólise , Espécies Reativas de Nitrogênio/metabolismo , Retina/imunologia , Retina/metabolismo , Retina/patologia , Índice de Gravidade de Doença , Espectrometria de Massas em Tandem
20.
PLoS One ; 12(1): e0168814, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28125581

RESUMO

The surface protein SdrE, a microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family protein expressed on the surface of Staphylococcus aureus (S. aureus), can recognize human complement regulator Factor H and C4BP, thus making it a potentially promising vaccine candidate. In this study, SdrE278-591 was found to directly affect S. aureus host cell invasion. Additionally, the crystal structure of SdrE278-591 at a resolution of 1.25 Å was established, with the three-dimensional structure revealing N2-N3 domains which fold in a manner similar to an IgG fold. Furthermore, a putative ligand binding site located at a conserved charged groove formed by the interface between N2 and N3 domains was identified, with ß2 suspected to occupy the ligand recognizing site and undergo a structural rearrangement to allow ligand binding. Overall, these findings have further contributed to the understanding of SdrE as a key factor for S. aureus invasivity and will enable a better understanding of bacterial infection processes.


Assuntos
Proteínas de Bactérias/química , Proteína de Ligação ao Complemento C4b/química , Fator H do Complemento/química , Mutação , Staphylococcus aureus/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação , Linhagem Celular Tumoral , Clonagem Molecular , Proteína de Ligação ao Complemento C4b/genética , Proteína de Ligação ao Complemento C4b/imunologia , Fator H do Complemento/genética , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Ligantes , Modelos Moleculares , Osteoblastos/imunologia , Osteoblastos/microbiologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Staphylococcus aureus/patogenicidade
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