RESUMEN
Recombinant human factor H (hFH) has potential for treating diseases linked to aberrant complement regulation including C3 glomerulopathy (C3G) and dry age-related macular degeneration. Murine FH (mFH), produced in the same host, is useful for pre-clinical investigations in mouse models of disease. An abundance of FH in plasma suggests high doses, and hence microbial production, will be needed. Previously, Pichia pastoris produced useful but modest quantities of hFH. Herein, a similar strategy yielded miniscule quantities of mFH. Since FH has 40 disulfide bonds, we created a P. pastoris strain containing a methanol-inducible codon-modified gene for protein-disulfide isomerase (PDI) and transformed this with codon-modified DNA encoding mFH under the same promoter. What had been barely detectable yields of mFH became multiple 10s of mg/L. Our PDI-overexpressing strain also boosted hFH overproduction, by about tenfold. These enhancements exceeded PDI-related production gains reported for other proteins, all of which contain fewer disulfide-stabilized domains. We optimized fermentation conditions, purified recombinant mFH, enzymatically trimmed down its (non-human) N-glycans, characterised its functions in vitro and administered it to mice. In FH-knockout mice, our de-glycosylated recombinant mFH had a shorter half-life and induced more anti-mFH antibodies than mouse serum-derived, natively glycosylated, mFH. Even sequential daily injections of recombinant mFH failed to restore wild-type levels of FH and C3 in mouse plasma beyond 24 hours after the first injection. Nevertheless, mFH functionality appeared to persist in the glomerular basement membrane because C3-fragment deposition here, a hallmark of C3G, remained significantly reduced throughout and beyond the ten-day dosing regimen.
Asunto(s)
Complemento C3/inmunología , Complemento C3/metabolismo , Factor H de Complemento/biosíntesis , Factor H de Complemento/deficiencia , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Expresión Génica , Inmunomodulación , Ratones , Ratones Noqueados , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Levaduras/genética , Levaduras/metabolismoRESUMEN
Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D2O/H2O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein-protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which 'matchout labelled' proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D2O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system.
Asunto(s)
Deuterio/química , Escherichia coli/genética , Ingeniería Genética/métodos , Difracción de Neutrones , Pichia/genética , Proteínas Recombinantes/genética , Dispersión del Ángulo Pequeño , Recuento de Células , Células Cultivadas , Factor H de Complemento/biosíntesis , Factor H de Complemento/química , Factor H de Complemento/genética , Factor H de Complemento/aislamiento & purificación , Expresión Génica , Marcaje Isotópico , Proteínas de Unión a Maltosa/biosíntesis , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (Physcomitrella patens). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial P. patens culture after a multistep purification process. This glycosylation-optimized factor H showed full in vitro complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.
Asunto(s)
Síndrome Hemolítico Urémico Atípico/tratamiento farmacológico , Bryopsida , Proteínas del Sistema Complemento , Enfermedades del Sistema Inmune/tratamiento farmacológico , Animales , Bryopsida/metabolismo , Factor H de Complemento/biosíntesis , Factor H de Complemento/metabolismo , Factor H de Complemento/uso terapéutico , Glicosilación , Humanos , RatonesRESUMEN
PURPOSE: To investigate how potentially functional genetic variants are coinherited on each of four common complement factor H (CFH) and CFH-related gene haplotypes and to measure expression of these genes in eye and liver tissues. METHODS: We sequenced the CFH region in four individuals (one homozygote for each of four common CFH region haplotypes) to identify all genetic variants. We studied associations between the haplotypes and AMD phenotypes in 2157 cases and 1150 controls. We examined RNA-seq profiles in macular and peripheral retina and retinal pigment epithelium/choroid/sclera (RCS) from eight eye donors and three liver samples. RESULTS: The haplotypic coinheritance of potentially functional variants (including missense variants, novel splice sites, and the CFHR3-CFHR1 deletion) was described for the four common haplotypes. Expression of the short and long CFH transcripts differed markedly between the retina and liver. We found no expression of any of the five CFH-related genes in the retina or RCS, in contrast to the liver, which is the main source of the circulating proteins. CONCLUSIONS: We identified all genetic variants on common CFH region haplotypes and described their coinheritance. Understanding their functional effects will be key to developing and stratifying AMD therapies. The small scale of our expression study prevented us from investigating the relationships between CFH region haplotypes and their expression, and it will take time and collaboration to develop epidemiologic-scale studies. However, the striking difference between systemic and ocular expression of complement regulators shown in this study suggests important implications for the development of intraocular and systemic treatments.
Asunto(s)
Coroides/metabolismo , Regulación de la Expresión Génica , Degeneración Macular/genética , ARN/genética , Epitelio Pigmentado de la Retina/metabolismo , Esclerótica/metabolismo , Coroides/patología , Factor H de Complemento/biosíntesis , Factor H de Complemento/genética , Femenino , Variación Genética , Genotipo , Haplotipos , Humanos , Degeneración Macular/metabolismo , Degeneración Macular/patología , Masculino , Persona de Mediana Edad , Epitelio Pigmentado de la Retina/patología , Esclerótica/patologíaRESUMEN
PURPOSE: To correlate a genetic risk score based on age-related macular degeneration (AMD) susceptibility genes with the risk of AMD in the second eye. METHODS: This is a retrospective, open cohort study consisting of 891 unilateral AMD patients, who were followed for at least 12 months and recruited from three institutes. DNAs were genotyped using Illumina OmniExpress, HumanOmni2.5-8, and/or HumanExome. Survival analyses and Cox proportional hazard models were used to examine the association between 11 AMD susceptibility genes and the duration until second-eye involvement in 499 samples from Kyoto University, which were replicated in two other cohorts. Genetic risk score (GRS) was also evaluated. RESULTS: The ARMS2 rs10490924 recessive model (hazard ratio [HR]meta = 2.04; Pmeta = 3.4 × 10⻳) and CFH rs800292 additive model (HRmeta = 1.77; Pmeta = 0.013) revealed significant associations with second-eye involvement. The dominant model of TNFRSF10A rs13278062, VEGFA rs943080, and CFI rs4698775 showed consistent effects across three datasets (I² = 0%; HRmeta = 1.46, 1.30, 1.51, respectively). The GRS using these five single nucleotide polymorphisms (SNPs) was also significantly associated (HRmeta [per score] = 2.42; P = 2.2 × 10â»5; I² = 0%). After 10 years from the first visit, the patients within the top 10% by GRS showed a 51% hazard rate, in contrast to 2.3% among patients within the lowest 10% by GRS. CONCLUSIONS: We demonstrated that the GRS using ARMS2, CFH, TNFRSF10A, VEGFA, and CFI was significantly associated with second-eye involvement. Genetic risk has high predictive ability for second-eye involvement of AMD.
Asunto(s)
Factor H de Complemento/genética , Predicción , Regulación de la Expresión Génica , Degeneración Macular/genética , Proteínas/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Factor A de Crecimiento Endotelial Vascular/genética , Alelos , Factor H de Complemento/biosíntesis , Femenino , Estudios de Seguimiento , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Técnicas de Genotipaje , Humanos , Incidencia , Japón/epidemiología , Degeneración Macular/epidemiología , Degeneración Macular/metabolismo , Masculino , Fenotipo , Polimorfismo de Nucleótido Simple , Proteínas/metabolismo , ARN/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Estudios Retrospectivos , Factores de Riesgo , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
We have previously reported that in vitro HCV infection of cells of hepatocyte origin attenuates complement system at multiple steps, and attenuation also occurs in chronically HCV infected liver, irrespective of the disease stage. However, none of these regulations alone completely impaired complement pathways. Modulation of the upstream proteins involved in proteolytic processing of the complement cascade prior to convertase formation is critical in promoting the function of the complement system in response to infection. Here, we examined the regulation of C2 complement expression in hepatoma cells infected in vitro with cell culture grown virus, and validated our observations using randomly selected chronically HCV infected patient liver biopsy specimens. C2 mRNA expression was significantly inhibited, and classical C3 convertase (C4b2a) decreased. In separate experiments for C3 convertase function, C3b deposition onto bacterial membrane was reduced using HCV infected patient sera as compared to uninfected control, suggesting impaired C3 convertase. Further, iC3b level, a proteolytically inactive form of C3b, was lower in HCV infected patient sera, reflecting impairment of both C3 convertase and Factor I activity. The expression level of Factor I was significantly reduced in HCV infected liver biopsy specimens, while Factor H level remained unchanged or enhanced. Together, these results suggested that inhibition of C3 convertase activity is an additional cumulative effect for attenuation of complement system adopted by HCV for weakening innate immune response.
Asunto(s)
Complemento C2/biosíntesis , Convertasas de Complemento C3-C5/metabolismo , Complemento C3b/biosíntesis , Factor H de Complemento/biosíntesis , Regulación de la Expresión Génica , Hepacivirus , Hepatitis C Crónica/metabolismo , Inmunidad Innata , Hígado/metabolismo , Femenino , Hepatitis C Crónica/patología , Humanos , Hígado/patología , Hígado/virología , Masculino , ARN Mensajero/biosíntesisRESUMEN
Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4(-/-) OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4(-/-) or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4(-/-) OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.
Asunto(s)
Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Haplotipos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/metabolismo , Transportadoras de Casetes de Unión a ATP/deficiencia , Animales , Membrana Celular/metabolismo , Complemento C3b/metabolismo , Factor H de Complemento/biosíntesis , Predisposición Genética a la Enfermedad/genética , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/patología , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Methamphetamine (METH) is a new type of drug with strong tolerance and addiction. However, the molecular mechanisms underlying the processes of METH addiction are still not fully understood. To determine possible biomarkers and mechanisms that are responsible for METH addiction, a 2-DE based proteomics approach was used to evaluate the changes in protein expression of the serum in Chinese patients addicted to METH, which to the best of our knowledge is the first study of its kind. We identified five proteins that were markedly altered and complement factor H (CFH), the most stably up-expressed protein in each 2-DE experiment, was further studied using the rat conditioned place preference (CPP) model to detect any changes to its expression in the sera and six brain regions of interest. We report, for the first time, that CFH was positive related to METH addiction.
Asunto(s)
Trastornos Relacionados con Anfetaminas/sangre , Factor H de Complemento/biosíntesis , Metanfetamina/administración & dosificación , Proteoma/análisis , Adulto , Animales , Biomarcadores/sangre , Encéfalo/metabolismo , Condicionamiento Clásico/efectos de los fármacos , Electroforesis en Gel Bidimensional , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Suero , Adulto JovenRESUMEN
PURPOSE: Genetic associations and the presence of complement components within pathological structures of age-related macular degeneration (AMD) have generated the hypothesis that AMD is caused by chronic local complement activation. Since the majority of activity in the common terminal pathway results from engagement of the amplification loop, the alternative pathway has been proposed as a logical therapeutic target. We recently generated a factor H (fH)-based complement inhibitor (CR2-fH) with the capacity to be "targeted" to sites of complement C3 activation. We asked whether the human therapeutic (TT30) is effective in a mouse model of AMD. METHODS: Choroidal neovascularization (CNV) was induced by argon laser photocoagulation of Bruch's membrane. Every other day, mice received intravenous injections of TT30 or vehicles, and after 6 days, the presence or absence of CNV and CNV-related changes were evaluated. Area of CNV, photoreceptor cell function, gene expression for complement components and cytokines, vascular endothelial growth factor (VEGF) protein levels, and TT30 bioavailability were determined. RESULTS: CNV development, which has previously been shown to require local complement activation, could be reduced by intravenous TT30 delivery. Specific inhibition of the alternative pathway not only reduced angiogenesis in CNV, but also ameliorated changes in several associated disease-related biomarkers, including diminished retinal function and molecular events known to be involved in AMD such as VEGF production. After intravenous injection, TT30 localized to CNV lesion sites in the retinal pigmented epithelium-choroid. CONCLUSION: Systemic administration of TT30 was found to reduce CNV pathology. These data may open new avenues for novel systemic AMD treatment strategies.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Neovascularización Coroidal/prevención & control , Factor H de Complemento/uso terapéutico , Vía Alternativa del Complemento/efectos de los fármacos , Rayos Láser/efectos adversos , Receptores de Complemento 3d/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Biomarcadores , Neovascularización Coroidal/patología , Factor H de Complemento/biosíntesis , Electrorretinografía , Humanos , Inmunohistoquímica , Ligandos , Ratones , Ratones Endogámicos C57BL , Plásmidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Complemento 3d/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: Genetic variation in complement factor H (CFH) has been implicated as a major risk factor for age-related macular degeneration (AMD). The reduction in CFH amount or its complement-modulating activity may lead to inadequate control of complement-driven inflammation at the outer retina. We explored the effect of photo-oxidative stress and inflammatory cytokine on the expression of CFH in retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were exposed to blue light in the presence of interferon-γ (IFN-γ). CFH expression in cell lysate was examined by Western blot and the secretory CFH in culture medium was analyzed by ELISA. RPE cells were treated with vitamin C and exogenous superoxide dismutase mimetic (Tempol) before photo-oxidative treatments. The intracellular reactive oxygen species were examined by flow cytometry. RESULTS: IFN-γ increased CFH expression in RPE and the expression was suppressed significantly under concomitant blue light illumination. The secretory CFH level also decreased significantly under blue light illumination, which was related to the decreased intracellular mRNA and protein expressions of CFH. The suppression was mediated through an oxidative mechanism, and was particularly related to superoxide anion generation. The suppression of CFH expression in RPE under blue light illumination was abrogated by vitamin C and Tempol. CONCLUSIONS: Photo-oxidative stress reduces the ability of IFN-γ to increase CFH expression in RPE. Apart from reducing the oxidative damage, vitamin C reduces the suppression of CFH under photo-oxidative stress. These results suggest a new perspective of the interaction between oxidative stress and inflammation, and provide a potential novel treatment strategy for age-related macular degeneration.
Asunto(s)
Factor H de Complemento/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Degeneración Macular/genética , Estrés Oxidativo/fisiología , ARN Mensajero/genética , Epitelio Pigmentado de la Retina/metabolismo , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Western Blotting , Supervivencia Celular , Células Cultivadas , Factor H de Complemento/biosíntesis , Óxidos N-Cíclicos/farmacología , Estudios de Seguimiento , Variación Genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Líquido Intracelular/metabolismo , Degeneración Macular/metabolismo , Degeneración Macular/patología , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Marcadores de SpinRESUMEN
Human complement factor H (FH), an abundant 155-kDa plasma glycoprotein with 40 disulphide bonds, regulates the alternative-pathway complement cascade. Mutations and single nucleotide polymorphisms in the FH gene predispose to development of age-related macular degeneration, atypical haemolytic uraemic syndrome and dense deposit disease. Supplementation with FH variants protective against disease is an enticing therapeutic prospect. Current sources of therapeutic FH are restricted to human blood plasma highlighting a need for recombinant material. Previously FH expression in cultured plant, mammalian or insect cells yielded protein amounts inadequate for full characterisation, and orders of magnitude below therapeutic usefulness. Here, the V62,Y402 variant of FH has been produced recombinantly (rFH) in Pichia pastoris cells. Codon-optimisation proved essential whilst exploitation of the yeast mating α-factor peptide ensured secretion. We thereby produced multiple 10s-of-milligram of rFH. Following endoglycosidase H digestion of N-linked glycans, rFH (with eight residual N-acetylglucosamine moieties) was purified on heparin-affinity resin and anion-exchange chromatography. Full-length rFH was verified by mass spectrometry and Western blot using monoclonal antibodies to the C-terminus. Recombinant FH is a single non-aggregated species (by dynamic light scattering) and fully functional in biochemical and biological assays. An additional version of rFH was produced in which eight N-glycosylation sequons were ablated by Asn-Gln substitutions resulting in a glycan-devoid product. Successful production of rFH in this potentially very highly expressing system makes production of therapeutically useful quantities economically viable. Furthermore, ease of genetic manipulation in P. pastoris would allow production of engineered FH versions with enhanced pharmacokinetic and pharmacodynamic properties.
Asunto(s)
Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Animales , Western Blotting , Complemento C3b/metabolismo , Factor H de Complemento/biosíntesis , Factor H de Complemento/química , Factor H de Complemento/genética , Electroforesis en Gel de Poliacrilamida , Eritrocitos/efectos de los fármacos , Genes Sintéticos , Hemólisis/efectos de los fármacos , Humanos , Luz , Degeneración Macular , Espectrometría de Masas , Pichia/genética , Polimorfismo de Nucleótido Simple , Proteínas Recombinantes/genética , Dispersión de Radiación , Ovinos , Resonancia por Plasmón de SuperficieRESUMEN
The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma.
Asunto(s)
Bryopsida/metabolismo , Proteínas Recombinantes/biosíntesis , Bryopsida/genética , Cromatografía Liquida , Factor H de Complemento/biosíntesis , Factor H de Complemento/química , Humanos , Espectrometría de Masas , Fenotipo , Plantas Modificadas GenéticamenteRESUMEN
Congenital and acquired deficiencies of complement regulatory proteins are associated with pathologic complement activation in several renal diseases. To elucidate the mechanisms by which renal tubular epithelial cells (TECs) control the complement system, we examined the expression of complement regulatory proteins by the cells. We found that Crry is the only membrane-bound complement regulator expressed by murine TECs, and its expression is concentrated on the basolateral surface. Consistent with the polarized localization of Crry, less complement activation was observed when the basolateral surface of TECs was exposed to serum than when the apical surface was exposed. Furthermore, greater complement activation occurred when the basolateral surface of TECs from Crry(-/-)fB(-/-) mice was exposed to normal serum compared with TECs from wild-type mice. Complement activation on the apical and basolateral surfaces was also greater when factor H, an alternative pathway regulatory protein found in serum, was blocked from interacting with the cells. Finally, we injected Crry(-/-)fB(-/-) and Crry(+/+)fB(-/-) mice with purified factor B (an essential protein of the alternative pathway). Spontaneous complement activation was seen on the tubules of Crry(-/-)fB(-/-) mice after injection with factor B, and the mice developed acute tubular injury. These studies indicate that factor H and Crry regulate complement activation on the basolateral surface of TECs and that factor H regulates complement activation on the apical surface. However, congenital deficiency of Crry or reduced expression of the protein on the basolateral surface of injured cells permits spontaneous complement activation and tubular injury.
Asunto(s)
Factor H de Complemento/fisiología , Proteínas Inactivadoras de Complemento/fisiología , Células Epiteliales/inmunología , Túbulos Renales/inmunología , Receptores de Complemento/fisiología , Animales , Células Cultivadas , Factor H de Complemento/biosíntesis , Factor H de Complemento/deficiencia , Proteínas Inactivadoras de Complemento/deficiencia , Vía Alternativa del Complemento/inmunología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Túbulos Renales/citología , Túbulos Renales/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/inmunología , Receptores de Complemento/biosíntesis , Receptores de Complemento/deficiencia , Receptores de Complemento 3bRESUMEN
Inflammatory liver disorders are often associated with a potentially tissue damaging complement activation directly at the main site of complement protein synthesis. As hepatocytes may be the primary target of complement attack, we investigated the expression and protective capacity of soluble and membrane-bound complement regulatory proteins in primary human hepatocytes (PHH). Isolated PHHs were analyzed for their basal and cytokine-induced complement regulator expression by cytofluorometry, rtPCR, confocal laser microscopy and ELISA. Susceptibility to complement-mediated cell lysis was investigated with cytotoxicity tests. In contrast to previous reports, PHHs expressed CD46, CD55, CD59, soluble CD59 (sCD59) and factor H (fH), but not CD35. A low basal expression of CD55 was strongly enhanced by IFN-gamma, IL-1 beta and TNF-alpha. The expression of CD59 could be augmented by IL-1 beta, IL-6 and TNF-alpha but was suppressed by IFN-gamma. CD46 expression was not significantly altered. PHHs synthesized fH and sCD59 and fH was detected on PHH surface after exposure to IL-1 beta. Inhibition experiments revealed that CD59 was most effective in protecting PHHs from complement attack. These data clearly indicate that PHHs are protected by multiple complement regulatory proteins, which are controlled by proinflammatory cytokines. CD59 appears to be pivotal in protecting PHHs against complement-mediated lysis.
Asunto(s)
Antígenos CD59/inmunología , Proteínas del Sistema Complemento/inmunología , Hepatocitos/inmunología , Antígenos CD/inmunología , Antígenos CD59/biosíntesis , Células Cultivadas , Factor H de Complemento/biosíntesis , Proteínas del Sistema Complemento/biosíntesis , Proteínas del Sistema Complemento/farmacología , Citocinas/inmunología , Citotoxicidad Inmunológica , Hepatitis/inmunología , Hepatitis/patología , Hepatocitos/metabolismo , Hepatocitos/patología , HumanosRESUMEN
Factor H is the primary soluble regulator of activation of the alternative pathway of complement. It prevents activation of complement on host cells and tissues upon association with C3b and surface polyanions such as sialic acids, heparin, and other glycosaminoglycans. Here we show that interaction with polyanions causes self-association forming tetramers of the 155,000 Da glycosylated protein. Monomeric human factor H is an extended flexible protein that exhibits an apparent size of 330,000 Da, relative to globular standards, during gel filtration chromatography in the absence of polyanions. In the presence of dextran sulfate (5000 Da) or heparin an intermediate species of apparent m.w. 700,000 and a limit species of m.w. 1,400,000 were observed by gel filtration. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a Mr of 607,000, corresponding to a tetramer, in the presence of less than a 2-fold molar excess of dextran sulfate. Increasing concentrations of dextran sulfate increased binding of factor H to zymosan-C3b 4.5-fold. This result was accompanied by an increase in both the decay accelerating and cofactor activity of factor H on these cells. An expressed fragment encompassing the C-terminal polyanion binding site (complement control protein domains 18-20) also exhibited polyanion-induced self-association, suggesting that the C-terminal ends of factor H mediate self-association. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H, and the resulting regulation of complement activation, may involve formation of dimers and tetramers of factor H.
Asunto(s)
Polímeros/química , Aniones/química , Aniones/metabolismo , Sitios de Unión/genética , Sitios de Unión/inmunología , Cromatografía en Gel , Factor H de Complemento/biosíntesis , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Sulfato de Dextran/química , Dimerización , Regulación de la Expresión Génica/inmunología , Humanos , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Polielectrolitos , Polímeros/metabolismo , Estructura Terciaria de Proteína/genética , TermodinámicaRESUMEN
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.
Asunto(s)
Factor H de Complemento/biosíntesis , Factor H de Complemento/química , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Estrés Oxidativo , Epitelio Pigmentado Ocular/metabolismo , Acetilación , Citocinas/metabolismo , Ambiente , Proteína Forkhead Box O3 , Predisposición Genética a la Enfermedad , Humanos , Inflamación , Interferón gamma/metabolismo , Polimorfismo Genético , Unión Proteica , Transducción de SeñalRESUMEN
Human plasma protein complement factor H (FH) is an inhibitor of the spontaneously activated alternative complement pathway. An allotypic variant of FH, 402His, has been associated with age-related macular degeneration, the leading cause of blindness in the elderly. Crystals of FH domains 6-8 (FH678) containing 402His have been grown in the presence of a polyanionic sucrose octasulfate ligand (an analogue of the natural glycosaminoglycan ligands of FH) using both native and selenomethionine-derivatized protein. Native data sets diffracting to 2.3 A and SeMet data sets of up to 2.8 A resolution have been collected. An anomalous difference Patterson map reveals self- and cross-peaks from two incorporated Se atoms. The corresponding selenium substructure has been solved.
Asunto(s)
Proteínas Recombinantes/química , Sacarosa/análogos & derivados , Factor H de Complemento/biosíntesis , Factor H de Complemento/química , Factor H de Complemento/genética , Factor H de Complemento/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Regulación de la Expresión Génica , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Sacarosa/química , Sacarosa/aislamiento & purificación , Sacarosa/metabolismoRESUMEN
Complement activation is thought to be involved in the pathogenesis of age-related macular degeneration (AMD), in part because certain gene polymorphisms in complement factor H (CFH), an important regulator of the alternative complement activation pathway, are high risk factors for AMD. How CFH is regulated locally at the retina/choroid interface and how this contributes to AMD development remain unknown. In the present study, we have confirmed that CFH was detectable by immunohistochemistry in the choroid, and at low levels in the RPE cell and interphotoreceptor matrix, but appeared to be concentrated in dense patches in Bruch's membrane. In vitro, cultured human and mouse RPE cells expressed high levels of CFH as evidenced by immunohistochemistry and western blot. Using a stabilized mouse RPE cell line, we confirmed that RPE cells constitutively synthesise CFH. Synthesis of CFH was not affected by a short-term (2 h) photoreceptor outer segment (POS) treatment. However, long-term (24-48 h) treatment of RPE cells with oxidised POS (ox-POS) but not normal POS (n-POS) markedly down-regulated CFH mRNA expression. Phagocytosis of both ox-POS and n-POS appeared to reduce intracellular CFH protein expression in RPE cultures. Synthesis of CFH by cultured RPE cells was also reduced at the mRNA level by the proinflammatory cytokines TNF-alpha and IL-6. Other cytokines tested including IFN-gamma, IL-1alpha and IL-4 showed no effect on either CFH protein or mRNA levels. Our results support the view that RPE cells synthesise and express CFH and are probably a major local source of this protein at the retina/choroid interface, secreting CFH into the interphotoreceptor matrix as well as Bruch's membrane. Prolonged phagocytosis of POS, particularly if modified by oxidative processes as occurs in inflammation, appears to markedly impair synthesis and secretion of CFH, with potential loss of important regulatory functions in counteracting the pro-inflammatory effects of activated complement.
Asunto(s)
Factor H de Complemento/biosíntesis , Epitelio Pigmentado Ocular/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Anciano , Animales , Lámina Basal de la Coroides/química , Línea Celular , Células Cultivadas , Coroides/química , Proteínas Inactivadoras del Complemento C3b/análisis , Factor H de Complemento/análisis , Citocinas/metabolismo , Regulación hacia Abajo/fisiología , Humanos , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal/métodos , Oxidación-Reducción , Fagocitosis/fisiología , Epitelio Pigmentado Ocular/citología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The objective of this study was to explore the role of classical, lectin, and alternative pathways of complement activation in laser-induced choroidal neovascularization (CNV). The classical and alternative pathways were blocked in C57BL/6 mice by small interfering RNAs (siRNA) directed against C1q and factor B, respectively. C4(-/-) mice developed CNV similar to their wild-type controls and inhibition of C1q by siRNA had no effect on the development of CNV. In contrast, CNV was significantly inhibited (p < 0.001) in C5(-/-) mice and C57BL/6 mice treated with factor B siRNA. Inhibition of the alternative pathway by factor B siRNA resulted in decreased levels of membrane attack complex and angiogenic factors-vascular endothelial growth factor and TGF-beta2. Furthermore, factor B was up-regulated in complement sufficient C57BL/6 mice at day 1 postlaser and remained elevated at day 7. Significantly reduced levels of factor H were observed at day 3 in these animals. In conclusion, our results demonstrate that activation of the factor B-dependent alternative pathway, but not the classical or lectin pathways, was essential for the development of CNV in mouse model of laser-induced CNV. Thus, specific blockade of the alternative pathway may represent a therapeutically relevant strategy for the inhibition of CNV.
Asunto(s)
Neovascularización Coroidal/inmunología , Factor B del Complemento/fisiología , Factor H de Complemento/fisiología , Vía Alternativa del Complemento/inmunología , Animales , Neovascularización Coroidal/genética , Neovascularización Coroidal/prevención & control , Complemento C1q/antagonistas & inhibidores , Complemento C1q/biosíntesis , Complemento C1q/genética , Complemento C4/deficiencia , Complemento C4/genética , Complemento C5/deficiencia , Complemento C5/genética , Factor B del Complemento/antagonistas & inhibidores , Factor B del Complemento/biosíntesis , Factor B del Complemento/genética , Factor H de Complemento/antagonistas & inhibidores , Factor H de Complemento/biosíntesis , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Vía Alternativa del Complemento/genética , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Inyecciones Intravenosas , Rayos Láser , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/administración & dosificación , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta2 , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy with manifestations of hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies have shown that mutations in complement regulatory proteins predispose to non-Shiga toxin-associated HUS (non-Stx-HUS). We undertook genetic analysis on membrane cofactor protein (MCP), complement factor H (CFH), and factor I (IF) in 156 patients with non-Stx-HUS. Fourteen, 11, and 5 new mutational events were found in MCP, CFH, and IF, respectively. Mutation frequencies were 12.8%, 30.1%, and 4.5% for MCP, CFH, and IF, respectively. MCP mutations resulted in either reduced protein expression or impaired C3b binding capability. MCP-mutated patients had a better prognosis than CFH-mutated and nonmutated patients. In MCP-mutated patients, plasma treatment did not impact the outcome significantly: remission was achieved in around 90% of both plasma-treated and plasma-untreated acute episodes. Kidney transplantation outcome was favorable in patients with MCP mutations, whereas the outcome was poor in patients with CFH and IF mutations due to disease recurrence. This study documents that the presentation, the response to therapy, and the outcome of the disease are influenced by the genotype. Hopefully this will translate into improved management and therapy of patients and will provide the way to design tailored treatments.