RESUMEN
The complement is a conserved cascade that plays a central role in the innate immune system. To maintain a delicate equilibrium preventing excessive complement activation, complement inhibitors are essential. One of the major fluid-phase complement inhibitors is C4b-binding protein (C4BP). Human C4BP is a macromolecular glycoprotein composed of two distinct subunits, C4BPα and C4BPß. These associate with vitamin K-dependent protein S (ProS) forming an ensemble of co-occurring higher-order structures. Here, we characterize these C4BP assemblies. We resolve and quantify isoforms of purified human serum C4BP using distinct single-particle detection techniques: charge detection mass spectrometry, and mass photometry accompanied by high-speed atomic force microscopy. Combining cross-linking mass spectrometry, glycoproteomics, and structural modeling, we report comprehensive glycoproteoform profiles and full-length structural models of the endogenous C4BP assemblies, expanding knowledge of this key complement inhibitor's structure and composition. Finally, we reveal that an increased C4BPα to C4BPß ratio coincides with elevated C-reactive protein levels in patient plasma samples. This observation highlights C4BP isoform variation and affirms a distinct role of co-occurring C4BP assemblies upon acute phase inflammation.
Asunto(s)
Proteína de Unión al Complemento C4b , Humanos , Proteína C-Reactiva/metabolismo , Proteína C-Reactiva/química , Proteína de Unión al Complemento C4b/metabolismo , Espectrometría de Masas , Microscopía de Fuerza Atómica , Modelos Moleculares , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/sangreRESUMEN
Complement offers a first line of defence against infection through the opsonization of microbial pathogens, recruitment of professional phagocytes to the infection site and the coordination of inflammatory responses required for the resolution of infection. Staphylococcus aureus is a successful pathogen that has developed multiple mechanisms to thwart host immune responses. Understanding the precise strategies employed by S. aureus to bypass host immunity will be paramount for the development of vaccines and or immunotherapies designed to prevent or limit infection. To gain a better insight into the specific immune evasion mechanisms used by S. aureus we examined the pathogen's interaction with the soluble complement inhibitor, C4b-binding protein (C4BP). Previous studies indicated that S. aureus recruits C4BP using a specific cell-wall-anchored surface protein and that bound C4BP limits complement deposition on the staphylococcal surface. Using flow-cytometric-based bacterial-protein binding assays we observed no interaction between S. aureus and C4BP. Moreover, we offer a precautionary warning that C4BP isolated from plasma can be co-purified with minute quantities of human IgG, which can distort binding analysis between S. aureus and human-derived proteins. Combined our data indicates that recruitment of C4BP is not a complement evasion strategy employed by S. aureus.
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Proteína de Unión al Complemento C4b , Infecciones Estafilocócicas , Humanos , Staphylococcus aureus/genética , Proteínas del Sistema Complemento , Staphylococcus , Proteínas de la MembranaRESUMEN
The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.
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Parásitos , Vacunas , Animales , Humanos , Proteína de Unión al Complemento C4b , Inactivadores del Complemento , Péptidos , Plasmodium falciparum , EsporozoítosRESUMEN
Coronavirus Disease 2019 (COVID-19) has been widely associated with increased thrombotic risk, with many different proposed mechanisms. One such mechanism is acquired deficiency of protein S (PS), a plasma protein that regulates coagulation and inflammatory processes, including complement activation and efferocytosis. Acquired PS deficiency is common in patients with severe viral infections and has been reported in multiple studies of COVID-19. This deficiency may be caused by consumption, degradation, or clearance of the protein, by decreased synthesis, or by binding of PS to other plasma proteins, which block its anticoagulant activity. Here, we review the functions of PS, the evidence of acquired PS deficiency in COVID-19 patients, the potential mechanisms of PS deficiency, and the evidence that those mechanisms may be occurring in COVID-19.
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COVID-19 , Deficiencia de Proteína S , Proteína S , Trombosis , Humanos , COVID-19/complicaciones , COVID-19/genética , COVID-19/metabolismo , Proteína S/genética , Proteína S/metabolismo , Deficiencia de Proteína S/complicaciones , Deficiencia de Proteína S/metabolismo , Trombosis/complicacionesRESUMEN
Classical pediatric Hodgkin Lymphoma (HL) is a rare malignancy. Therapeutic regimens for its management may be optimized by establishing treatment response early on. The aim of this study was to identify plasma protein biomarkers enabling the prediction of relapse in pediatric/adolescent HL patients treated under the pediatric EuroNet-PHL-C2 trial. We used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics at the time of diagnosisbefore any therapyas semiquantitative method to profile plasma proteins specifically associated with relapse in 42 children with nodular sclerosing HL. In both the exploratory and the validation cohorts, six proteins (apolipoprotein E, C4b-binding protein α chain, clusterin, fibrinogen γ chain, prothrombin, and vitronectin) were more abundant in the plasma of patients whose HL relapsed (|fold change| ≥ 1.2, p < 0.05, Student's t-test). Predicting protein function with the Gene Ontology classification model, the proteins were included in four biological processes (p < 0.01). Using immunoblotting and Luminex assays, we validated two of these candidate biomarkersC4b-binding protein α chain and clusterinlinked to innate immune response function (GO:0045087). This study identified C4b-binding protein α chain and clusterin as candidate early plasma biomarkers of HL relapse, and important for the purpose of shedding light on the molecular scenario associated with immune response in patients treated under the EuroNet-PHL-C2 trial.
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Enfermedad de Hodgkin , Proteómica , Adolescente , Biomarcadores , Niño , Cromatografía Liquida , Clusterina , Proteína de Unión al Complemento C4b , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/genética , Humanos , Recurrencia Local de Neoplasia , Proteómica/métodos , Espectrometría de Masas en TándemRESUMEN
OBJECTIVE: Narcolepsy is a chronic sleep disorder long hypothesised to be an autoimmune disease. Complement-mediated immune mechanisms have not been investigated in detail in narcolepsy. Our aim was to establish the significance of classical pathway activation in narcolepsy. METHODS: Sera of 42 narcolepsy patients and 26 healthy controls were screened with ELISA to determine the levels of C1q, C3a, C4d and complement component 4 binding protein (C4BP). A home-made ELISA method was developed to detect antibodies to C4BP-alpha (anti-C4BPA). The correlation between complement levels and clinical findings was examined. RESULTS: C1q levels were significantly higher in narcolepsy patients while C4d and C4BP levels were significantly lower compared to healthy controls. C3a levels were comparable among patients and controls. Eleven narcolepsy patients showed serum anti-C4BPA levels. Total rapid eye movements (REM) time, sleep onset latency, REM sleep latency, sleep activity, percentage of wakefulness after sleep onset and Epworth sleepiness scale scores were correlated with levels of different complement factors. CONCLUSION: Complement-mediated immune mechanisms might partake in narcolepsy pathogenesis. The precise role of autoantibodies on complement level alterations needs to be investigated. Levels of complement factors and degradation products may potentially be utilised as biomarkers to predict the clinical severity of narcolepsy.
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Vía Clásica del Complemento , Narcolepsia , Complemento C1q , Humanos , Narcolepsia/diagnóstico , Sueño REM/fisiología , Vigilia/fisiologíaRESUMEN
Neisseria gonorrhoeae is the etiological agent of gonorrhea, the second most common bacterial sexually transmitted infection worldwide. Reproductive sequelae of gonorrhea include infertility, ectopic pregnancy and chronic pelvic pain. Most antibiotics currently in clinical use have been rendered ineffective due to the rapid spread of antimicrobial resistance among gonococci. The developmental pipeline of new antibiotics is sparse and novel therapeutic approaches are urgently needed. Previously, we utilized the ability of N. gonorrhoeae to bind the complement inhibitor C4b-binding protein (C4BP) to evade killing by human complement to design a chimeric protein that linked the two N-terminal gonococcal binding domains of C4BP with the Fc domain of IgM. The resulting molecule, C4BP-IgM, enhanced complement-mediated killing of gonococci. Here we show that C4BP-IgM induced membrane perturbation through complement deposition and membrane attack complex pore insertion facilitates the access of antibiotics to their intracellular targets. Consequently, bacteria become more susceptible to killing by antibiotics. Remarkably, C4BP-IgM restored susceptibility to azithromycin of two azithromycin-resistant clinical gonococcal strains because of overexpression of the MtrC-MtrD-MtrE efflux pump. Our data show that complement activation can potentiate activity of antibiotics and suggest a role for C4BP-IgM as an adjuvant for antibiotic treatment of drug-resistant gonorrhea.
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Antibacterianos/farmacología , Activación de Complemento , Proteína de Unión al Complemento C4b/administración & dosificación , Farmacorresistencia Bacteriana/efectos de los fármacos , Inmunoglobulina M/administración & dosificación , Neisseria gonorrhoeae/efectos de los fármacos , Azitromicina/farmacología , Ciprofloxacina/farmacología , Proteína de Unión al Complemento C4b/genética , Humanos , Inmunoglobulina M/genética , Neisseria gonorrhoeae/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/administración & dosificación , Espectinomicina/farmacologíaRESUMEN
BACKGROUND: HIV is a chronic inflammatory state with the production of many acute-phase-reactant proteins. Some of these proteins have procoagulant activities that predispose HIV-infected patients to thrombosis. OBJECTIVES: The aim of the study was to evaluate the effects of HIV infection on the serum levels of C4b-binding protein (C4BP) and protein S as markers of predisposition to thrombosis in HIV-infected adults. METHODS: The study population comprised of 61 HIV-infected adults on antiretroviral treatment (ART) who had achieved virological suppression, 58 HIV-infected adults not yet on ART and 59 HIV-negative healthy controls. The serum levels of free protein S, C4BP and the euglobulin clot lysis time (ECLT) were determined. RESULTS: The mean plasma-free protein S level of HIV-infected patients on ART (86.9% ± 25.8%) was significantly higher than that of treatment-naïve HIV-infected patients (75.7% ± 27.3%) (p = 0.005). Conversely, there was no statistically significant difference between the protein S levels of the HIV-infected subjects on ART (86.9% ± 25.8%) and those of the controls (94.9% ± 7.9%) (p = 0.119). The mean C4BP was significantly higher in the treatment-naïve HIV-infected subjects (36.7 ± 1.7â ng/dL) than that in those on ART (30.7 ± 2.6â ng/dL) and that in the controls (22.4 ± 2.4â ng/dL) (p < 0.0001). Protein S deficiency was more prevalent among the subjects with elevated C4BP (p = 0.023). The mean ECLT was significantly more prolonged in the treatment-naïve HIV-infected subjects (241.9 ± 34.7â s) than controls (189.5 ± 40.7â s) (p < 0.0001). CONCLUSION: HIV infection causes elevated levels of C4BP and diminishes the serum levels of free protein S. We infer that the risk of thrombosis (as measured by these biomarkers) decreases with the use of antiretroviral drugs.
RESUMEN
Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, while Neisseria meningitidis is an important cause of bacterial meningitis and sepsis. Complement is a central arm of innate immune defenses and plays an important role in combating Neisserial infections. Persons with congenital and acquired defects in complement are at a significantly higher risk for invasive Neisserial infections such as invasive meningococcal disease and disseminated gonococcal infection compared to the general population. Of note, Neisseria gonorrhoeae and Neisseria meningitidis can only infect humans, which in part may be related to their ability to evade only human complement. This review summarizes the epidemiologic and clinical aspects of Neisserial infections in persons with defects in the complement system. Mechanisms used by these pathogens to subvert killing by complement and preclinical studies showing how these complement evasion strategies may be used to counteract the global threat of meningococcal and gonococcal infections are discussed.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Gonorrea/inmunología , Evasión Inmune , Infecciones Meningocócicas/inmunología , Neisseria gonorrhoeae , Neisseria meningitidis , Animales , Gonorrea/patología , Humanos , Infecciones Meningocócicas/patología , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/patogenicidad , Neisseria meningitidis/inmunología , Neisseria meningitidis/patogenicidadRESUMEN
Streptococcus pyogenes is a major human pathogen that causes a variety of diseases ranging from mild skin and throat infections to fatal septicemia. In severe invasive infections, S. pyogenes encounters and interacts with components of the extracellular matrix (ECM), including small leucine rich-proteoglycans (SLRPs). In this study, we report a novel antimicrobial role played by SLRPs biglycan, decorin, fibromodulin and osteoadherin, specifically in promoting the eradication of S. pyogenes in a human sepsis model of infection. SLRPs can be released from the ECM and de novo synthesized by a number of cell types. We reveal that infection of human monocytes by S. pyogenes induces the expression of decorin. Furthermore, we show that the majority of genetically distinct and clinically relevant S. pyogenes isolates interact with SLRPs resulting in decreased survival in blood killing assays. Biglycan and decorin induce TLR2 and TLR4 signaling cascades resulting in secretion of proinflammatory and chemotactic molecules and recruitment of professional phagocytes. Surprisingly, SLRP-mediated elimination of S. pyogenes occurs independently of TLR activation. Our results indicate that SLRPs act in concert with human serum, enhancing deposition of complement activation fragments and the classical activator C1q on the bacterial surface, facilitating efficient microbial eradication. Addition of the complement C3 inhibitor compstatin significantly reverses SLRP-induced blood killing, confirming active complement as a key mediator in SLRP-mediated bacterial destruction. Taken together our results add to the functional repertoire of SLRPs, expanding to encompass their role in controlling bacterial infection.
RESUMEN
Complement is a critical component of antimicrobial immunity. Various complement regulatory proteins prevent host cells from being attacked. Many pathogens have acquired the ability to sequester complement regulators from host plasma to evade complement attack. We describe here how Streptococcus pneumoniae adopts a strategy to prevent the formation of the C3 convertase C4bC2a by the rapid conversion of surface bound C4b and iC4b into C4dg, which remains bound to the bacterial surface but no longer forms a convertase complex. Noncapsular virulence factors on the pneumococcus are thought to facilitate this process by sequestering C4b-binding protein (C4BP) from host plasma. When S. pneumoniae D39 was opsonized with human serum, the larger C4 activation products C4b and iC4b were undetectable, but the bacteria were liberally decorated with C4dg and C4BP. With targeted deletions of either PspA or PspC, C4BP deposition was markedly reduced, and there was a corresponding reduction in C4dg and an increase in the deposition of C4b and iC4b. The effect was greatest when PspA and PspC were both knocked out. Infection experiments in mice indicated that the deletion of PspA and/or PspC resulted in the loss of bacterial pathogenicity. Recombinant PspA and PspC both bound serum C4BP, and both led to increased C4b and reduced C4dg deposition on S. pneumoniae D39. We conclude that PspA and PspC help the pneumococcus to evade complement attack by binding C4BP and so inactivating C4b.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteína de Unión al Complemento C4b/metabolismo , Complemento C4b/antagonistas & inhibidores , Evasión Inmune , Streptococcus pneumoniae/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Infecciones Neumocócicas/microbiología , Unión Proteica , Streptococcus pneumoniae/patogenicidadRESUMEN
Protein S (PS) is a Vitamin K-dependent protein that functions as a cofactor for the regulation of the coagulation system. PS works in conjunction with Activated Protein C to inactivate factors V and VIII. PS circulates in plasma either complexed to the complement protein, C4b Binding Protein or unbound. The unbound (or free) component is the functional form for the regulation of the coagulation system. PS can be measured in plasma by functional activity, the free (or unbound form) or both free and bound fractions (Total PS). The test most widely used for clinical evaluations is the Free PS Antigen assay (which is the surrogate of PS anticoagulant activity) and represents the protocol described in this chapter. The Free PS Antigen assay is an immunologic assay which specifically measures the unbound fraction of PS in test plasma. Other methods for assessing PS are also available, including PS activity and total PS Antigen assays, but protocols for these assays are not provided.
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Inmunoturbidimetría/métodos , Proteína S/análisis , Trombofilia/sangre , Trombofilia/diagnóstico , Proteína de Unión al Complemento C4b/metabolismo , Humanos , Unión Proteica , Proteína S/metabolismo , Trombofilia/metabolismoRESUMEN
Complement is a crucial arm of the innate immune response against invading bacterial pathogens, and one of its main functions is to recognize and destroy target cells. Similar to other pathogens, Escherichia coli has evolved mechanisms to overcome complement activation. It is well known that capsular polysaccharide may confer resistance to complement-mediated killing and phagocytosis, being one of the strategies adopted by this bacterium to survive in serum. In addition, proteases produced by E. coli have been shown to downregulate the complement system. Pic, an autotransporter secreted by different pathogens in the Enterobacteriaceae family, is able to cleave C2, C3/C3b, and C4/C4b and works synergistically with human Factor I and Factor H (FH), thereby promoting inactivation of C3b. Extracellular serine protease P, a serine protease of enterohemorrhagic E. coli (EHEC), downregulates complement activation by cleaving C3/C3b and C5. StcE, a metalloprotease secreted by EHEC, inhibits the classical complement-mediated cell lysis by potentiating the action of C1 inhibitor, and the periplasmic protease Prc contributes to E. coli complement evasion by interfering with the classical pathway activation and by preventing membrane attack complex deposition. Finally, it has been described that E. coli proteins interact with negative complement regulators to modulate complement activation. The functional consequences resulting from the interaction of outer membrane protein A, new lipoprotein I, outer membrane protein W, and Stx2 with proteins of the FH family and C4b-binding protein (C4BP) are discussed in detail. In brief, in this review, we focused on the different mechanisms used by pathogenic E. coli to circumvent complement attack, allowing these bacteria to promote a successful infection.
RESUMEN
Recently, we reported a novel function for C4b-binding protein (C4BP) in inhibiting the toll-like receptor (TLR)1/2 response by interacting with TLR2. TLRs share a common structure; hence, we examined the effect of C4BP on activation of other TLRs-TLR4 and TLR3. The results of immunoprecipitation assays suggest that C4BP interacts with TLR4/MD-2 but not TLR3. C4BP inhibits TLR4/MD-2-mediated, but not TLR3-mediated, proinflammatory cytokine production and nuclear factor (NF)-κB signaling. C4BP-deficient mice show increased interleukin (IL)-6 production in response to the TLR4/MD-2 ligand. A competition assay revealed that C4BP prevents an interaction between TLR4/MD-2 and its ligand. These findings indicate that C4BP binds to cell surface TLRs and inhibits the TLR-TLR ligand interaction, thereby inhibiting TLR activation.
Asunto(s)
Regulación hacia Abajo , Antígenos de Histocompatibilidad/metabolismo , Antígeno 96 de los Linfocitos/antagonistas & inhibidores , Activación de Macrófagos , Macrófagos/metabolismo , Transducción de Señal , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Citocinas/agonistas , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Femenino , Células HEK293 , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/genética , Humanos , Ligandos , Lípido A/toxicidad , Lipopolisacáridos/toxicidad , Antígeno 96 de los Linfocitos/agonistas , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Noqueados , FN-kappa B/agonistas , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Células RAW 264.7 , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 3/química , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
TLR2 associates with TLR1 and recognizes microbial lipoproteins. Pam3CSK4, a triacylated lipoprotein, is anchored to the extracellular domain of TLR1 and TLR2 and induces pro-inflammatory signals. Here we show that C4b binding protein (C4BP), which is a complement pathway inhibitor, is a TLR2-associated molecule. Immunoprecipitation assay using anti-TLR2 mAb shows that C4BP binds to TLR2. In C4BP-deficient mice, Pam3CSK4-induced IL-6 levels were increased compared with wild type mice. In C4BP-expressing cells, Pam3CSK4-induced IL-8 production was reduced depending on the C4BP expression levels. These results reveal the important role of C4BP in negative regulation of TLR1/2-dependent pro-inflammatory cytokine production. Furthermore, using a fluorescent conjugated Pam3CSK4, we show that C4BP blocks the binding of Pam3CSK4 to TLR1/2. Finally, we show that exogenous C4BP also inhibits Pam3CSK4-induced signaling leading to IL-8 production. Our results indicate C4BP binding to TLR2 and consequent neutralization of its activity otherwise inducing pro-inflammatory cytokine production. C4BP is a negative regulator of TLR1/2 activity.
Asunto(s)
Proteína de Unión al Complemento C4b/metabolismo , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Sitios de Unión , Activación de Complemento , Células HEK293 , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopéptidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Transducción de SeñalRESUMEN
A previous study had demonstrated that Leptospira enolase is secreted extracellularly by a yet unknown mechanism and reassociates with the bacterial membrane. Surface-anchored leptospiral enolase displays plasminogen binding activity. In this work, we explored the consequences of this interaction and also assessed whether Leptospira enolase might display additional moonlighting functions by interacting with other host effector proteins. We first demonstrated that enolase-bound plasminogen is converted to its active form, plasmin. The protease plasmin targets human fibrinogen and vitronectin, but not the complement proteins C3b and C5. Leptospira enolase also acts as an immune evasion protein by interacting with the negative complement regulators C4b binding protein and factor H. Once bound to enolase, both regulators remain functional as cofactors of factor I, mediating cleavage of C4b and C3b. In conclusion, enolase may facilitate leptospiral survival and dissemination, thus contributing to bacterial virulence. The identification and characterization of moonlighting proteins is a growing field of bacterial pathogenesis, as these multifaceted proteins may represent potential future therapeutic targets to fight bacterial infections.
Asunto(s)
Leptospira/enzimología , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Proteína de Unión al Complemento C4b , Complemento C5/química , Complemento C5/metabolismo , Factor H de Complemento/química , Factor H de Complemento/metabolismo , Fibrinolisina/metabolismo , Humanos , Evasión Inmune , Leptospira/patogenicidad , Fosfopiruvato Hidratasa/genética , Plasminógeno/metabolismo , Unión ProteicaRESUMEN
C4b-binding protein (C4BP) is best known as a potent soluble inhibitor of the classical and lectin pathways of the complement system. This large 500 kDa multimeric plasma glycoprotein is expressed mainly in the liver but also in lung and pancreas. It consists of several identical 75 kDa α-chains and often also one 40 kDa ß-chain, both of which are mainly composed of complement control protein (CCP) domains. Structure-function studies revealed that one crucial binding site responsible for inhibition of complement is located to CCP1-3 of the α-chain. Binding of anticoagulant protein S to the CCP1 of the ß-chain provides C4BP with the ability to strongly bind apoptotic and necrotic cells in order to prevent inflammation arising from activation of complement by these cells. Further, C4BP interacts strongly with various types of amyloid and enhances fibrillation of islet amyloid polypeptide secreted from pancreatic beta cells, which may attenuate pro-inflammatory and cytotoxic effects of this amyloid. Full deficiency of C4BP has not been identified but non-synonymous alterations in its sequence have been found in haemolytic uremic syndrome and recurrent pregnancy loss. Furthermore, C4BP is bound by several bacterial pathogens, notably Streptococcus pyogenes, which due to inhibition of complement and enhancement of bacterial adhesion to endothelial cells provides these bacteria with a survival advantage in the host. Thus, depending on the context, C4BP has a protective or detrimental role in the organism.
Asunto(s)
Aborto Espontáneo/inmunología , Síndrome Hemolítico Urémico Atípico/inmunología , Proteína de Unión al Complemento C4b/metabolismo , Complicaciones Hematológicas del Embarazo/inmunología , Aborto Espontáneo/genética , Animales , Síndrome Hemolítico Urémico Atípico/genética , Activación de Complemento/genética , Proteína de Unión al Complemento C4b/genética , Proteína de Unión al Complemento C4b/inmunología , Femenino , Predisposición Genética a la Enfermedad , Homeostasis , Humanos , Mutación/genética , Polimorfismo Genético , Embarazo , Complicaciones Hematológicas del Embarazo/genéticaRESUMEN
The complement, coagulation, and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross-talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to Streptococcus pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the ß-chain and protein S binds plasminogen much stronger than the main isoform containing the ß-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the ß-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, whereas the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation.
Asunto(s)
Proteína de Unión al Complemento C4b/metabolismo , Inflamación/metabolismo , Plasminógeno/metabolismo , Streptococcus pneumoniae/metabolismo , Proteína de Unión al Complemento C4b/genética , Fibrinógeno/metabolismo , Fibrinolisina/metabolismo , Fibrinólisis/genética , Humanos , Inflamación/patología , Lisina/metabolismo , Plasminógeno/genética , Activadores Plasminogénicos/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas , Proteína S/metabolismo , Streptococcus pneumoniae/patogenicidad , Resonancia por Plasmón de Superficie , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
INTRODUCTION: The C4b binding protein (C4bp) α/ß-chain C-terminal effectively induces polymerization during protein synthesis. Using this fragment and the single-domain antibody EG2, which targets the epidermal growth factor receptor (EGFR), we generated the novel multimeric antibody EG2-C4bpα. We radiolabeled EG2-C4bpα with (99m)Tc and evaluated its targeting efficiency and pharmacokinetics in tumor xenografts. METHODS: EGFR expression and EGFR-EG2-C4bpα binding was evaluated in A431 and OCM-1 cells by Western blotting and flow cytometry, respectively. EG2-C4bpα was radiolabeled with [(99m)Tc(CO)3(OH2)3](+) using a tricarbonyl vial followed by purification on a PD-10 column. In vitro studies with (99m)Tc-EG2-C4bpα were performed in A431 and/or OCM-1 cells. Single photon emission computed tomography (SPECT) imaging and biodistribution studies were carried out in (99m)Tc-EG2-C4bpα-injected mice bearing A431- and OCM-1-derived tumors. EGFR immunofluorescent staining in A431 and OCM-1 tumors was performed. RESULTS: A431 cells showed higher EGFR expression levels than OCM-1 cells, and flow cytometry confirmed EG2-C4bpα bound more A431 cells than OCM-1 cells. (99m)Tc-EG2-C4bpα was successfully prepared with radiochemical yields of 30.3-50.4%. The binding affinity of (99m)Tc-EG2-C4bpα to A431 cells was approximately 20 nM. (99m)Tc-EG2-C4bpα specifically bound A431 cells and this binding was blocked by 41% in the presence of 50 nM excess unlabeled EG2-C4bpα. In vivo radioactivity uptake in A431 tumors was detected 2h after (99m)Tc-EG2-C4bpα administration and sustained up to 18h. The highest ratio of A431 tumor-to-muscle and tumor-to-blood was 3.69 ± 0.48 at 10h and 0.77 ± 0.14 at 20 h, respectively. Excess unlabeled EG2-C4bpα blocked radioactivity uptake in A431 tumors by 55% at 10h. (99m)Tc-EG2-C4bpα was barely detectable in OCM-1 tumors, and biodistribution analysis confirmed that radioactivity uptake was significantly lower than in A431 tumors. CONCLUSIONS: (99m)Tc-EG2-C4bpα specifically and efficiently targets EGFR over-expressing tumors suggesting that EG2-C4bpα may be a promising antibody alternative for future diagnostic application and potential radioimmunotherapy. However, the high activity in the blood and liver, and the relative low ratio of tumor-to-blood should be noticed and improved.
Asunto(s)
Anticuerpos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Compuestos de Organotecnecio/farmacología , Radiofármacos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Animales , Anticuerpos/química , Western Blotting , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Compuestos de Organotecnecio/química , Compuestos de Organotecnecio/farmacocinética , Radioquímica , Radiofármacos/química , Radiofármacos/farmacocinética , Proteínas Recombinantes de Fusión/química , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.