RESUMO
The use of biomaterials in modern medicine has enabled advanced drug delivery strategies and led to reduced morbidity and mortality in a variety of interventions such as transplantation or hemodialysis. However, immune-mediated reactions still present a serious complication of these applications. One of the drivers of such reactions is the complement system, a central part of humoral innate immunity that acts as a first-in-line defense system in its own right but also coordinates other host defense responses. A major regulator of the complement system is the abundant plasma protein factor H (FH), which impairs the amplification of complement responses. Previously, we could show that it is possible to recruit FH to biomedical surfaces using the phage display-derived cyclic peptide 5C6 and, consequently, reduce deposition of C3b, an activation product of the complement system. However, the optimal orientation of 5C6 on surfaces, structural determinants within the peptide for the binding, and the exact binding region on FH remained unknown. Here, we show that the cyclic core and C-terminal region of 5C6 are essential for its interaction with FH and that coating through its N-terminus strongly increases FH recruitment and reduces C3-mediated opsonization in a microparticle-based assay. Furthermore, we could demonstrate that 5C6 selectively binds to FH but not to related proteins. The observation that 5C6 also binds murine FH raises the potential for translational evaluation in animal models. This work provides important insight for the future development of 5C6 as a probe or therapeutic entity to reduce complement activation on biomaterials. STATEMENT OF SIGNIFICANCE: Biomaterials have evolved into core technologies critical to biomedical and drug delivery applications alike, yet their safe and efficient use may be adversely impacted by immune responses to the foreign materials. Taking inspiration from microbial immune evasion strategies, our group developed a peptide-based surface coating that recruits factor H (FH), a host regulator of the complement system, from plasma to the material surface and prevents unwanted activation of this innate immunity pathway. In this study, we identified the molecular determinants that define the interaction between FH and the coated peptide, developed tethering strategies with largely enhanced binding capacity and provided important insight into the target selectivity and species specificity of the FH-binding peptide, thereby paving the way for preclinical development steps.
Assuntos
Complemento C3b , Fator H do Complemento , Animais , Camundongos , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Materiais Biocompatíveis/farmacologia , Ligação Proteica , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
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-AtividadeRESUMO
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 ProteicaRESUMO
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/patogenicidadeRESUMO
Complement is the major humoral component of the innate immune system. It recognizes pathogen- and damage-associated molecular patterns, and initiates the immune response in coordination with innate and adaptive immunity. When activated, the complement system unleashes powerful cytotoxic and inflammatory mechanisms, and thus its tight control is crucial to prevent damage to host tissues and allow restoration of immune homeostasis. Factor H is the major soluble inhibitor of complement, where its binding to self markers (i.e., particular glycan structures) prevents complement activation and amplification on host surfaces. Not surprisingly, mutations and polymorphisms that affect recognition of self by factor H are associated with diseases of complement dysregulation, such as age-related macular degeneration and atypical haemolytic uremic syndrome. In addition, pathogens (i.e., non-self) and cancer cells (i.e., altered-self) can hijack factor H to evade the immune response. Here we review recent (and not so recent) literature on the structure and function of factor H, including the emerging roles of this protein in the pathophysiology of infectious diseases and cancer.
Assuntos
Fator H do Complemento/metabolismo , Evasão da Resposta Imune , Imunidade Inata , Animais , Fator H do Complemento/química , Doença , Humanos , Polissacarídeos/metabolismo , Ligação ProteicaRESUMO
Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Alanina/genética , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Autoanticorpos/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Clonagem Molecular , Fator H do Complemento/química , Fator H do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Cristalografia por Raios X , Citotoxicidade Imunológica , Modelos Animais de Doenças , Epitopos/imunologia , Rearranjo Gênico , Humanos , Neoplasias Pulmonares/patologia , Camundongos Nus , Modelos Moleculares , Mutagênese/genética , Peptídeos/química , Peptídeos/imunologiaRESUMO
Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19-20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19-20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19-20 bound dose-dependently to mGEnCs and TNF-α treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19-20 binding to mGEnCs. Compared with wild type FH19-20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19-20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19-20 mutants.
Assuntos
Fator H do Complemento/metabolismo , Células Endoteliais/metabolismo , Mutação , Animais , Linhagem Celular , Ativação do Complemento , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/imunologia , Células Endoteliais/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Heparina/farmacologia , Humanos , Glomérulos Renais/citologia , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 µs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures.
Assuntos
Capsídeo/química , Fator H do Complemento/química , Gangliosídeo G(M1)/análogos & derivados , Simulação de Dinâmica Molecular , Vírus 40 dos Símios/química , Capsídeo/metabolismo , Fator H do Complemento/metabolismo , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/metabolismo , Humanos , Vírus 40 dos Símios/metabolismoRESUMO
Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a ß-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.
Assuntos
Enzimas Ativadoras do Complemento/química , Ativação do Complemento , Proteínas Inativadoras do Complemento/química , Desenho de Fármacos , Modelos Moleculares , Engenharia de Proteínas , Animais , Sítios de Ligação , Enzimas Ativadoras do Complemento/genética , Enzimas Ativadoras do Complemento/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Inativadores do Complemento/química , Inativadores do Complemento/metabolismo , Inativadores do Complemento/farmacologia , Proteínas Inativadoras do Complemento/genética , Proteínas Inativadoras do Complemento/metabolismo , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Enhanced fucosylation has been suggested as a marker for serologic monitoring of liver disease and hepatocellular carcinoma (HCC). We present a workflow for quantitative site-specific analysis of fucosylation and apply it to a comparison of hemopexin (HPX) and complement factor H (CFH), two liver-secreted glycoproteins, in healthy individuals and patients with liver cirrhosis and HCC. Label-free LC-MS quantification of glycopeptides derived from these purified glycoproteins was performed on pooled samples (2 pools/group, 5 samples/pool) and complemented by glycosidase assisted analysis using sialidase and endoglycosidase F2/F3, respectively, to improve resolution of glycoforms. Our analysis, presented as relative abundance of individual fucosylated glycoforms normalized to the level of their nonfucosylated counterparts, revealed a consistent increase in fucosylation in liver disease with significant site- and protein-specific differences. We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples. Glycosidase assisted LC-MS-MRM analysis of individual patient samples prepared by a simplified protocol confirmed the quantitative differences. Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX. Further verification is needed to confirm that enhanced fucosylation of HPX and CFH may serve as an indicator of premalignant liver disease. The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.
Assuntos
Fator H do Complemento/metabolismo , Fucose/química , Hemopexina/metabolismo , Hepatopatias/sangue , Sequência de Aminoácidos , Fator H do Complemento/química , Feminino , Hemopexina/química , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.
Assuntos
Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Transporte/química , Cromatografia de Afinidade , Peptídeos/química , Substituição de Aminoácidos , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/isolamento & purificação , Fator H do Complemento/metabolismo , Variação Genética , Humanos , Peptídeos/metabolismo , Ligação ProteicaRESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membrane-targeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membrane-targeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients.
Assuntos
Fator H do Complemento/química , Eritrócitos/efeitos dos fármacos , Hemoglobinúria Paroxística/sangue , Hemólise/efeitos dos fármacos , Proteínas de Fusão Oncogênica/farmacologia , Receptores de Complemento 3d/química , Proteínas Recombinantes de Fusão/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Complemento C3/efeitos adversos , Complemento C3/antagonistas & inibidores , Complemento C3/farmacologia , Fator H do Complemento/metabolismo , Fator H do Complemento/farmacologia , Proteínas do Sistema Complemento/efeitos adversos , Proteínas do Sistema Complemento/fisiologia , Citoproteção/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Eritrócitos/fisiologia , Hemoglobinúria Paroxística/patologia , Humanos , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Receptores de Complemento 3d/metabolismo , Receptores de Complemento 3d/fisiologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Structural knowledge of interactions amongst the ~ 40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X-ray diffraction of co-crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co-crystal structures is valuable since intermolecular affinities are generally weak-to-medium and inter-domain mobility may be important. In this current work, Förster resonance energy transfer (FRET) was used to investigate the 10 µM K(D) (210 kD) complex between the N-terminal region of the soluble complement regulator, factor H (FH1-4), and the key activation-specific complement fragment, C3b. Using site-directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1-4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1-4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time-resolved FRET measurements were undertaken to explore interactions between FH1-4 and C3b in fluid phase and under near-physiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1-4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex.
Assuntos
Complemento C3b/química , Fator H do Complemento/química , Transferência Ressonante de Energia de Fluorescência/métodos , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8-11 and SCR19-20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19-20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.
Assuntos
Fator H do Complemento/metabolismo , Streptococcus pneumoniae/metabolismo , Actinas/metabolismo , Animais , Aderência Bacteriana , Transporte Biológico , Células CHO , Linhagem Celular , Fator H do Complemento/química , Sequência Consenso , Cricetinae , Cricetulus , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/química , Heparina/metabolismo , Humanos , Antígeno de Macrófago 1/metabolismo , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequências Repetitivas de Aminoácidos , Transdução de Sinais , Solubilidade , Streptococcus pneumoniae/fisiologiaRESUMO
We recently discovered that the vascular responsiveness to adrenomedullin (AM), a potent vasoactive peptide, decreased during sepsis and hemorrhage in the rat and was markedly improved by its novel binding protein (AMBP-1). Moreover, AM/AMBP-1 appears to be one of the leading candidates for further development to treat sepsis and hemorrhage. However, the extremely high cost of commercial AMBP-1 limits the development of human AM and AMBP-1 as therapeutic agents. The purpose of this study was to isolate and purify AMBP-1 from normal human serum and test its stability and biological activity under in vitro and in vivo conditions. AMBP-1 was isolated and purified from normal human serum with a yield of about 3.0 mg per 100 mL and purity of >99%. The purified AMBP-1 has a AM-binding capacity similar to that of the commercial AMBP-1. Human AM and human AMBP-1 in combination significantly inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production from macrophages. The biological activity of the purified human AMBP-1 was well preserved when stored at 45 degrees C for 5 d in solution or at 100 degrees C for 1 h in powder. Moreover, administration of AM and purified AMBP-1 to hemorrhaged rats attenuated tissue injury and neutrophil accumulation. Purified AMBP-1 in combination with AM also suppressed the hemorrhage-induced rise in serum cytokines TNF-alpha and IL-6. Thus, we have successfully purified biologically active AMBP-1 from human normal serum and demonstrated the stability of purified human AMBP-1. This technique will enable us to further develop human AM/AMBP-1 as a novel treatment for safe and effective therapy of patients with hemorrhagic shock, sepsis, and ischemic injury.
Assuntos
Fator H do Complemento/química , Fator H do Complemento/isolamento & purificação , Animais , Células Cultivadas , Quimiocina CCL2/metabolismo , Fator H do Complemento/metabolismo , Fator H do Complemento/farmacologia , Avaliação Pré-Clínica de Medicamentos , Hemorragia/complicações , Hemorragia/patologia , Hemorragia/prevenção & controle , Humanos , Interleucina-6/metabolismo , Isquemia/patologia , Lipopolissacarídeos/farmacologia , Hepatopatias/etiologia , Hepatopatias/patologia , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Soro/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Age-related macular degeneration (AMD) and membranoproliferative glomerulonephritis type II (MPGN2) are dense deposit diseases that share a genetic association with complement genes and have complement proteins as important components of the dense deposits. Here, we present the case of a 64-year-old smoker male who developed both AMD and MPGN2 in his late 50s. The patient presented persistent low plasma levels of C3, factor H levels in the lower part of the normal range and C3NeF traces. Genetic analyses of the CFH, CFB, C3, CFHR1-CFHR3 and LOC387715/HTRA1 genes revealed that the patient was heterozygote for a novel missense mutation in exon 9 of CFH (c.1292 G>A) that results in a Cys431Tyr substitution in SCR7 of the factor H protein. In addition, he was homozygote for the His402 CFH allele, heterozygote for the Ser69 LOC387715 allele, homozygote for the Arg32 (BFS) CFB allele, heterozygote for the Gly102 (C3F) C3 allele and carried no deletion of the CFHR1/CFHR3 genes. Proteomic and functional analyses indicate absence in plasma of the factor H allele carrying the Cys431Tyr mutation. As a whole, these data recapitulate a prototypical complement genetic profile, including a partial factor H deficiency and the presence of major risk factors for AMD and MPGN2, which support the hypothesis that these dense deposit diseases have a common pathogenic mechanism involving dysregulation of the alternative pathway of complement activation.
Assuntos
Fator H do Complemento/genética , Glomerulonefrite Membranoproliferativa/complicações , Glomerulonefrite Membranoproliferativa/genética , Degeneração Macular/complicações , Degeneração Macular/genética , Substituição de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Células COS , Chlorocebus aethiops , Fator H do Complemento/química , Cisteína , Olho/patologia , Predisposição Genética para Doença , Humanos , Rim/patologia , Espectrometria de Massas , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação/genética , Proteínas Recombinantes , TirosinaRESUMO
Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.
Assuntos
Envelhecimento/fisiologia , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Sítios de Ligação , Fator H do Complemento/genética , Cristalografia por Raios X , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Ligantes , Modelos Moleculares , Mutação/genética , Polissacarídeos/química , Polissacarídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sacarose/análogos & derivados , Sacarose/química , Sacarose/metabolismo , Propriedades de SuperfícieRESUMO
Age-related macular degeneration (AMD), the leading cause of severe vision loss in the elderly, is a complex disease that results from genetic modifications that increase susceptibility to environmental exposures. Smoking, a major source of oxidative stress, increases the incidence and severity of AMD, and antioxidants slow progression, suggesting that oxidative stress plays a major role. Polymorphisms in the complement factor H (CFH) gene that reduce activity of CFH increase the risk of AMD. In this study we demonstrate an interaction between these two risk factors, because oxidative stress reduces the ability of an inflammatory cytokine, interferon-gamma, to increase CFH expression in retinal pigmented epithelial cells. The interferon-gamma-induced increase in CFH is mediated by transcriptional activation by STAT1, and its suppression by oxidative stress is mediated by acetylation of FOXO3, which enhances FOXO3 binding to the CFH promoter, reduces its binding to STAT1, inhibits STAT1 interaction with the CFH promoter, and reduces expression of CFH. Expression of SIRT1, a mammalian homolog of NAD-dependent protein deacetylase sir2, attenuated FOXO3 recruitment to the CFH regulatory region and reversed the H(2)O(2)-induced repression of CFH gene expression. These data suggest an important interaction between environmental exposure and genetic susceptibility in the pathogenesis of AMD and, by elucidating molecular signaling involved in the interaction, provide potential targets for therapeutic intervention.
Assuntos
Fator H do Complemento/biossíntese , Fator H do Complemento/química , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Estresse Oxidativo , Epitélio Pigmentado Ocular/metabolismo , Acetilação , Citocinas/metabolismo , Meio Ambiente , Proteína Forkhead Box O3 , Predisposição Genética para Doença , Humanos , Inflamação , Interferon gama/metabolismo , Polimorfismo Genético , Ligação Proteica , Transdução de SinaisRESUMO
Tick-borne relapsing fever in North America is primarily caused by the spirochete Borrelia hermsii. The pathogen employs multiple strategies, including the acquisition of complement regulators and antigenic variation, to escape innate and humoral immunity. In this study we identified in B. hermsii a novel member of the complement regulator-acquiring surface protein (CRASP) family, designated BhCRASP-1, that binds the complement regulators factor H (FH) and FH-related protein 1 (FHR-1) but not FH-like protein 1 (FHL-1). BhCRASP-1 specifically interacts with the short consensus repeat 20 of FH, thereby maintaining FH-associated cofactor activity for factor I-mediated C3b inactivation. Furthermore, ectopic expression of BhCRASP- 1 converted the serum-sensitive Borrelia burgdorferi B313 strain into an intermediate complement-resistant strain. Finally, we report for the first time that BhCRASP-1 binds plasminogen/plasmin in addition to FH via, however, distinct nonoverlapping domains. The fact that surface-bound plasmin retains its proteolytic activity suggest that the dual binding specificity of BhCRASP-1 for FH and plasminogen/plasmin contributes to both the dissemination/invasion of B. hermsii and its resistance to innate immunity.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Borrelia/imunologia , Fator H do Complemento/metabolismo , Plasminogênio/metabolismo , Febre Recorrente/imunologia , Febre Recorrente/microbiologia , Fatores de Virulência/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Atividade Bactericida do Sangue/imunologia , Proteínas Sanguíneas/metabolismo , Borrelia/genética , Borrelia/patogenicidade , Clonagem Molecular , Fator H do Complemento/química , Fator H do Complemento/fisiologia , Sequência Consenso , Citotoxicidade Imunológica , Fibrinolisina/química , Fibrinolisina/metabolismo , Humanos , Lipoproteínas/genética , Lipoproteínas/isolamento & purificação , Lipoproteínas/metabolismo , Dados de Sequência Molecular , Plasminogênio/química , Ativadores de Plasminogênio/metabolismo , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Febre Recorrente/metabolismo , Sequências Repetitivas de Aminoácidos , Fatores de Virulência/genética , Fatores de Virulência/isolamento & purificaçãoRESUMO
Pneumococcal surface protein C (PspC) of Streptococcus pneumoniae is a key virulence factor that mediates adhesion to host cells and immune evasion of the host complement. PspC binds the host immune and complement regulator factor H, which is composed of 20 short consensus repeats (SCR). This interaction contributes to pneumococcal virulence. In this study, we identified within the factor H protein two separate PspC binding regions, which were localized to SCR8-11 and SCR19-20, by using recombinant factor H deletion constructs for Western blotting assays and surface plasmon resonance studies. A detailed analysis of binding epitopes in these SCR by peptide spot arrays identified several linear binding regions within the sequences of SCR8-11 and SCR19-20. In addition, the factor H binding site was mapped within the pneumococcal PspC protein to a 121-aa-long stretch positioned in the N terminus (residues 38-158). Factor H attached to the surface of pneumococci via PspC significantly enhanced pneumococcal adherence to host epithelial and endothelial cells. This adhesion was specific and was blocked with a truncated N-terminal factor H-binding fragment of PspC. In conclusion, the acquisition of factor H by pneumococci via PspC occurs via two contact sites located in SCR8-11 and SCR19-20, and factor H attached to the surface of the pneumococcus promotes adhesion to both host epithelial and endothelial cells.