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Peter F Zipfel - One of the best experts on this subject based on the ideXlab platform.

  • Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors
    Frontiers Media S.A., 2019
    Co-Authors: Peter F Zipfel, Stefan Michelfelder, Juliana Parsons, Oguz Top, Lennard L. Bohlender, Phillipp Kopp, Christian Busch-steenberg, Sebastian N. W. Hoernstein, Karsten Häffner
    Abstract:

    The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH

  • Data_Sheet_1_Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors.docx
    2019
    Co-Authors: Oguz Top, Peter F Zipfel, Stefan Michelfelder, Juliana Parsons, Lennard L. Bohlender, Phillipp Kopp, Christian Busch-steenberg, Sebastian N. W. Hoernstein, Karsten Häffner, Ralf Reski
    Abstract:

    The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH.

  • successful treatment of deap hus with eculizumab
    Pediatric Nephrology, 2014
    Co-Authors: Damien Noone, Peter F Zipfel, Fred G Pluthero, Michael Kirschfink, Aoife M Waters, Denis F Geary, Christoph Licht
    Abstract:

    Background Deficiency of complement factor H-related (CFHR) proteins and CFH autoantibody-positive hemolytic uremic syndrome (DEAP-HUS) represents a unique subgroup of complement-mediated atypical HUS (aHUS). Autoantibodies to the C-terminus of CFH block CFH surface recognition and mimic mutations found in the genetic form of (CFH-mediated) aHUS. CFH autoantibodies are found in 10–15 % of aHUS patients and occur—so far unexplained—almost exclusively in the background of CFHR1 or CFHR3/CFHR1 deletions.

  • Complement Factor H-Related Proteins CFHR2 and CFHR5 Represent Novel Ligands for the Infection-Associated CRASP Proteins of Borrelia burgdorferi
    2013
    Co-Authors: Corinna Siegel, Reinhard Wallich, Barbara Uzonyi, Peter F Zipfel, Christine Skerka, Teresia Hallstrom, Brian Stevenson, Michael Karas, Hannes Eberhardt, Peter Kraiczy
    Abstract:

    Background: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complementmediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi. Methodology/Principal Findings: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amount

  • Serum proteins display differential binding capabilities to CRASP-3 and CRASP-5.
    2013
    Co-Authors: Corinna Siegel, Reinhard Wallich, Barbara Uzonyi, Christine Skerka, Teresia Hallstrom, Brian Stevenson, Tobias Beckhaus, Michael Karas, Hannes Eberhardt, Peter F Zipfel
    Abstract:

    (A) The binding capabilites of serum proteins to B. garinii strains G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were assessed in the presence of increasing salt concentrations. Spirochetes were incubated in NHS plus EDTA, washed fourfold with PBSA containing 0.05% Tween20. Cells were then resuspended in PBSA containing 450 mM NaCl, incubated for 15 min at room temperature, and sedimented by centrifugation. The steps were repeated with increasing concentrations of NaCl. Strong binding proteins were finally eluted using 0.1 M glycine buffer (e). The supernatants obtained from the last wash fraction (150 mM NaCl), fractions from the incubation reactions (450, 750, 1050, 1350 mM NaCl), and the eluate fraction were then separated by Glycine-SDS-PAGE and transferred to nitrocellulose. Membranes were probed with polyclonal anti-CFHR1 antiserum to detect CFH and CFHR proteins. Mobilities of molecular mass standards are shown to the left of the panels. (B) The binding capability of CFH to G1/pCRASP-3 and G1/pCRASP-5 was further analyzed by flow cytometry. The binding of CFH to G1/pCRASP-3 and G1/pCRASP-5 is shown by the solid line while the grey shaded histogramm represents the binding of control strain G1/pKFSS1 (control). Borrelial cells were incubated with 4 µg CFH. The x-acis shows the fluorescence on a log10 scale and the the y-acis represents the numbers of events. The isotype control (no CFH added) has been omitted for easier visualization.

Mihály Józsi - One of the best experts on this subject based on the ideXlab platform.

  • Atypical Hemolytic Uremic Syndrome-Associated Variants and Autoantibodies Impair Binding of Factor H and Factor H-Related Protein 1 to Pentraxin 3
    The Journal of Immunology, 2012
    Co-Authors: Anne Kopp, Stefanie Strobel, Pilar Sánchez-corral, Agustín Tortajada, Santiago Rodríguez De Córdoba, Zoltán Prohászka, Margarita López-trascasa, Mihály Józsi
    Abstract:

    Atypical hemolytic uremic syndrome (aHUS) is a renal disease associated with complement alternative pathway dysregulation and is characterized by endothelial injury. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule expressed by endothelial cells and upregulated under inflammatory conditions. PTX3 activates complement, but it also binds the complement inhibitor factor H. In this study, we show that native factor H, factor H-like protein 1, and factor H-related protein 1 (CFHR1) bind to PTX3 and that PTX3-bound factor H and factor H-like protein 1 maintain their complement regulatory activities. PTX3, when bound to extracellular matrix, recruited functionally active factor H. Residues within short consensus repeat 20 of factor H that are relevant for PTX3 binding were identified using a peptide array. aHUS-associated factor H mutations within this binding site caused a reduced factor H binding to PTX3. Similarly, seven of nine analyzed anti-factor H autoantibodies isolated from aHUS patients inhibited the interaction between factor H and PTX3, and five autoantibodies also inhibited PTX3 binding to CFHR1. Moreover, the aHUS-associated CFHR1*B variant showed reduced binding to PTX3 in comparison with CFHR1*A. Thus, the interactions of PTX3 with complement regulators are impaired by certain mutations and autoantibodies affecting factor H and CFHR1, which could result in an enhanced local complement-mediated inflammation, endothelial cell activation, and damage in aHUS.

  • factor h related protein 4 activates complement by serving as a platform for the assembly of alternative pathway c3 convertase via its interaction with c3b protein
    Journal of Biological Chemistry, 2012
    Co-Authors: Mario Hebecker, Mihály Józsi
    Abstract:

    Human complement factor H-related protein (CFHR) 4 belongs to the factor H family of plasma glycoproteins that are composed of short consensus repeat (SCR) domains. Although factor H is a well known inhibitor of the alternative complement pathway, the functions of the CFHR proteins are poorly understood. CFHR4 lacks SCRs homologous to the complement inhibitory domains of factor H and, accordingly, has no significant complement regulatory activities. We have previously shown that CFHR4 binds C-reactive protein via its most N-terminal SCR, which leads to classical complement pathway activation. CFHR4 binds C3b via its C terminus, but the significance of this interaction is unclear. Therefore, we set out to clarify the functional relevance of C3b binding by CFHR4. Here, we report a novel role for CFHR4 in the complement system. CFHR4 serves as a platform for the assembly of an alternative pathway C3 convertase by binding C3b. This is based on the sustained ability of CFHR4-bound C3b to bind factor B and properdin, leading to an active convertase that generates C3a and C3b from C3. The CFHR4-C3bBb convertase is less sensitive to the factor H-mediated decay compared with the C3bBb convertase. CFHR4 mutants containing exchanges of conserved residues within the C-terminal C3b-binding site showed significantly reduced C3b binding and alternative pathway complement activation. In conclusion, our results suggest that, in contrast to the complement inhibitor factor H, CFHR4 acts as an enhancer of opsonization by promoting complement activation.

  • factor h and factor h related protein 1 bind to human neutrophils via complement receptor 3 mediate attachment to candida albicans and enhance neutrophil antimicrobial activity
    Journal of Immunology, 2010
    Co-Authors: Josephine Losse, Peter F Zipfel, Mihály Józsi
    Abstract:

    The host complement system plays an important role in protection against infections. Several human-pathogenic microbes were shown to acquire host complement regulators, such as factor H (CFH), that downregulate complement activation at the microbial surface and protect the pathogens from the opsonic and lytic effects of complement. Because CFH can also bind to host cells, we addressed the role of CFH and CFH-related proteins as adhesion ligands in host-pathogen interactions. We show that the CFH family proteins CFH, CFH-like protein 1 (CFHL1), CFH-related protein (CFHR) 1, and CFHR4 long isoform bind to human neutrophil granulocytes and to the opportunistic human-pathogenic yeast Candida albicans. Two major binding sites, one within the N-terminus and one in the C-terminus of CFH, were found to mediate binding to neutrophils. Complement receptor 3 (CD11b/CD18; alpha(M)beta2 integrin) was identified as the major cellular receptor on neutrophils for CFH, CFHL1, and CFHR1, but not for CFHR4 long isoform. CFH and CFHR1 supported cell migration. Furthermore, CFH, CFHL1, and CFHR1 increased attachment of neutrophils to C. albicans. Adhesion of neutrophils to plasma-opsonized yeasts was reduced when CFH binding was inhibited by specific Abs or when using CFH-depleted plasma. Yeast-bound CFH and CFHR1 enhanced the generation of reactive oxygen species and the release of the antimicrobial protein lactoferrin by human neutrophils, and resulted in a more efficient killing of the pathogen. Thus, CFH and CFHR1, when bound on the surface of C. albicans, enhance antimicrobial activity of human neutrophils.

  • autoimmune forms of thrombotic micorangiopathy and membranoproliferative glomerulonephritis indications for a disease spectrum and common pathogenic principles
    Molecular Immunology, 2009
    Co-Authors: Christine Skerka, Stefanie Strobel, Barbara Uzonyi, Peter F Zipfel, Christoph Licht, Michael Mengel, Mihály Józsi
    Abstract:

    There is increasing evidence that Thrombotic Thrombocytopenic Purpura (TTP), atypical Hemolytic Uremic Syndrome (aHUS) and Membranoproliferative Glomerulonephritis (MPGN), especially subtype II (also termed Dense Deposit Disease) represent a spectrum of related disorders. Thrombi are common for all three disorders, develop in different microvascular beds and appear relevant for organ dysfunction. TTP not only develops primarily at neurological sites, but also in the kidney and aHUS develops primarily in the kidneys. In TTP thrombi formation occurs subsequently to the release of multimers of von Willebrand Factor (vWF) and in HUS (both typical and atypical) to endothelial cell damage (via toxins or complement dysregulation). In MPGN thrombus formation occurs in the kidney, however, the cause for thrombi development is less clear. In addition autoimmune forms, in which acquired inhibitors in form of autoantibodies are de novo generated, exist for all three disorders. However, the autoantibodies are directed against different antigens. In TTP against ADAMTS 13, the vWF cleaving protease and in the DEAP-HUS (Deficient for CFHR1 and CFHR3 proteins and autoantibody positive) group against the major complement regulator Factor H. Autoantibodies in MPGN are termed C3 Nephritic Factor (C3NeF) and are directed against a neoepitope of the complement C3 convertase C3bBb. Apparently C3NeF stabilizes this convertase and this results in C3 amplification and complement activation. Based on the existence of acquired immune inhibitors and the shared thrombus formation in TTP, aHUS (DEAP-HUS) and MPGN we here address the hypothesis if the three autoimmune forms represent a spectrum of related diseases and share a common pathogenic principle.

  • human complement factor h related protein 4 binds and recruits native pentameric c reactive protein to necrotic cells
    Molecular Immunology, 2009
    Co-Authors: Michael Mihlan, Mario Hebecker, Peter F Zipfel, Hansmartin Dahse, Steffi Halbich, Markus Huberlang, Regine Dahse, Mihály Józsi
    Abstract:

    Human complement factor H-related protein 4 (CFHR4) is a plasma glycoprotein which appears in two isoforms. CFHR4 is a member of the factor H protein family, and shares structural similarity and sequence homology with the other CFHR proteins and with the complement regulator factor H. Given the structural and sequence similarity, we hypothesized that similar to factor H, CFHR4 binds to C-reactive protein (CRP). We have recombinantly expressed the two CFHR4 isoforms and analyzed their binding to both native and denatured, monomeric CRP. Here, we show that both CFHR4 isoforms bind in the presence of calcium to native pentameric CRP, but not to modified CRP. This is in contrast to factor H, which binds to modified CRP independent of calcium. Comparison of the two CFHR4 isoforms and a recombinant CFHR4 fragment for CRP binding indicates that the first domain of CFHR4 is relevant for this interaction. Interaction of the native proteins was demonstrated by co-precipitation of CFHR4 and CRP from serum of sepsis patients with elevated CRP levels. CFHR4 bound to necrotic cells and was localized in necrotic tumor tissue as demonstrated by immunohistological analyses. In addition, CFHR4 facilitated binding of native CRP to the surface of necrotic cells. Altogether these data identify CFHR4 as a novel ligand for native CRP, and suggest a role for CFHR4 in opsonization of necrotic cells.

Gregory S. Hageman - One of the best experts on this subject based on the ideXlab platform.

  • Protective chromosome 1q32 haplotypes mitigate risk for age-related macular degeneration associated with the CFH-CFHR5 and ARMS2/HTRA1 loci
    'Springer Science and Business Media LLC', 2021
    Co-Authors: Chris M. Pappas, Moussa A. Zouache, Stacie Matthews, Caitlin D. Faust, Jill L. Hageman, Brandi L. Williams, Burt T. Richards, Gregory S. Hageman
    Abstract:

    Abstract Background Single-variant associations with age-related macular degeneration (AMD), one of the most prevalent causes of irreversible vision loss worldwide, have been studied extensively. However, because of a lack of refinement of these associations, there remains considerable ambiguity regarding what constitutes genetic risk and/or protection for this disease, and how genetic combinations affect this risk. In this study, we consider the two most common and strongly AMD-associated loci, the CFH-CFHR5 region on chromosome 1q32 (Chr1 locus) and ARMS2/HTRA1 gene on chromosome 10q26  (Chr10 locus). Results By refining associations within the CFH-CFHR5 locus, we show that all genetic protection against the development of AMD in this region is described by the combination of the amino acid-altering variant CFH I62V (rs800292) and genetic deletion of CFHR3/1. Haplotypes based on CFH I62V, a CFHR3/1 deletion tagging SNP and the risk variant CFH Y402H are associated with either risk, protection or neutrality for AMD and capture more than 99% of control- and case-associated chromosomes. We find that genetic combinations of CFH-CFHR5 haplotypes (diplotypes) strongly influence AMD susceptibility and that individuals with risk/protective diplotypes are substantially protected against the development of disease. Finally, we demonstrate that AMD risk in the ARMS2/HTRA1 locus is also mitigated by combinations of CFH-CFHR5 haplotypes, with Chr10 risk variants essentially neutralized by protective CFH-CFHR5 haplotypes. Conclusions Our study highlights the importance of considering protective CFH-CFHR5 haplotypes when assessing genetic susceptibility for AMD. It establishes a framework that describes the full spectrum of AMD susceptibility using an optimal set of single-nucleotide polymorphisms with known functional consequences. It also indicates that protective or preventive complement-directed therapies targeting AMD driven by CFH-CFHR5 risk haplotypes may also be effective when AMD is driven by ARMS2/HTRA1 risk variants

  • Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions.
    PloS one, 2016
    Co-Authors: Jonathan P. Hannan, Jennifer Laskowski, Joshua M. Thurman, Gregory S. Hageman, V. Michael Holers
    Abstract:

    Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation.

  • mapping the complement factor h related protein 1 CFHR1 c3b c3d interactions
    PLOS ONE, 2016
    Co-Authors: Jonathan P. Hannan, Jennifer Laskowski, Joshua M. Thurman, Gregory S. Hageman, Michael V Holers
    Abstract:

    Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation.

  • CFHR1 blocks other CFH-interaction sites on C3b.
    2016
    Co-Authors: Jonathan P. Hannan, Jennifer Laskowski, Joshua M. Thurman, Gregory S. Hageman, Michael V Holers
    Abstract:

    (A) The capacity of CFHR1 to compete with CFHL-1-binding to plate-bound C3b is shown. OD values at 405 nm were expressed as a percentage of CFHL-1 binding where CFHL-1 incubated in the absence of CFHR1 was calculated as 100%. (B) Heparin sepharose is able to pull-down CFHR1 (Left), but is unable to pull-down C3d on its own (Centre). However, when both CFHR1 and C3d are present, heparin-conjugated beads are able to pull-down both CFHR1 and C3d (Right). (C) Proposed mechanism of CFH regulation of C3b on a non-activating surface and CFHR1-driven deregulation of CFH on an activating surface. (Left) CFH engages C3b at two sites located within SCR1-4 and SCR19-20, while simultaneously binding to surface anionic markers at sites located within SCR6-8 and SCR20. SCR20 of CFH has also been reported to additionally bind to C3d. (Right) CFHR1 can also form a ternary complex by dually engaging C3d/C3b and GAGs. CFHR1 competes with CFH SCR19-20 for binding to the TED, and also sterically inhibits engagement of SCR1-7. CFHR1 is represented in its various characterized physiologic oligomeric states, but additionally forms hetero-dimers with CFHR2 and CFHR5 [22, 23].

  • Domain composition of the CFH gene family.
    2016
    Co-Authors: Jonathan P. Hannan, Jennifer Laskowski, Joshua M. Thurman, Gregory S. Hageman, Michael V Holers
    Abstract:

    CFH comprises 20 SCR modules, and the CFH gene splice variant, CFHL-1, encompasses 7 SCR modules. CFHL-1 is invariant from the N-terminal seven SCRs of CFH apart from the C-terminal four residues (indicated). CFHR1-5 range from 4 to 9 SCRs. The SCR domains of each CFHR protein are aligned with the most homologous domains of CFH. C3b/C3d and GAG/sialic-binding regions are indicated. Two forms of CFHR4 are found in circulation, a long 9 SCR form of the protein, CFHR4a, and a shorter 5 SCR form, CFHR4b.

Santiago Rodríguez De Córdoba - One of the best experts on this subject based on the ideXlab platform.

  • high complement factor h related fhr 3 levels are associated with the atypical hemolytic uremic syndrome risk allele cfhr3 b
    Frontiers in Immunology, 2018
    Co-Authors: Santiago Rodríguez De Córdoba, Richard B Pouw, Irene Gomez Delgado, Alberto Lopez Lera, Diana Wouters, Taco W Kuijpers, Pilar Sanchezcorral
    Abstract:

    Dysregulation of the complement alternative pathway (AP) is a major pathogenic mechanism in atypical hemolytic-uremic syndrome (aHUS). Genetic or acquired defects in factor H (FH), the main AP regulator, are major aHUS drivers that associate with a poor prognosis. FH activity has been suggested to be downregulated by homologous FH-related (FHR) proteins, including FHR-3 and FHR-1. Hence, their relative levels in plasma could be disease-relevant. The genes coding for FH, FHR-3, and FHR-1 (; CFH, CFHR3; , and; CFHR1; , respectively) are polymorphic and located adjacent to each other on human chromosome 1q31.3. We have previously shown that haplotype; CFH(H3)-CFHR3*B-CFHR1*B; associates with aHUS and reduced FH levels. In this study, we used a specific enzyme-linked immunosorbent assay to quantify FHR-3 in plasma samples from controls and patients with aHUS genotyped for the three known; CFHR3; alleles (; CFHR3*A, CFHR3*B; , and; CFHR3*Del; ). In the 218 patients carrying at least one copy of; CFHR3; , significant differences between; CFHR3; genotype groups were found, with; CFHR3*A/Del; patients having the lowest FHR-3 concentration (0.684-1.032 µg/mL),; CFHR3*B/Del; and; CFHR3*A/A; patients presenting intermediate levels (1.437-2.201 µg/mL), and; CFHR3*A/B; and; CFHR3*B/B; patients showing the highest concentration (2.330-4.056 µg/mL) (; p; < 0.001). These data indicate that; CFHR3*A; is a low-expression allele, whereas; CFHR3*B; , associated with increased risk of aHUS, is a high-expression allele. Our study reveals that the aHUS-risk haplotype; CFH(H3)-CFHR3*B-CFHR1*B; generates twofold more FHR-3 than the non-risk; CFH(H1)-CFHR3*A-CFHR1*A; haplotype. In addition, FHR-3 levels were higher in patients with aHUS than in control individuals with the same; CFHR3; genotype. These data suggest that increased plasma levels of FHR-3, altering the balance between FH and FHR-3, likely impact the FH regulatory functions and contribute to the development of aHUS.

  • table_1_High Complement Factor H-Related (FHR)-3 Levels Are Associated With the Atypical Hemolytic-Uremic Syndrome-Risk Allele CFHR3*B.doc
    2018
    Co-Authors: Richard B Pouw, Santiago Rodríguez De Córdoba, Irene Gomez Delgado, Alberto Lopez Lera, Diana Wouters, Taco W Kuijpers, Pilar Sánchez-corral
    Abstract:

    Dysregulation of the complement alternative pathway (AP) is a major pathogenic mechanism in atypical hemolytic-uremic syndrome (aHUS). Genetic or acquired defects in factor H (FH), the main AP regulator, are major aHUS drivers that associate with a poor prognosis. FH activity has been suggested to be downregulated by homologous FH-related (FHR) proteins, including FHR-3 and FHR-1. Hence, their relative levels in plasma could be disease-relevant. The genes coding for FH, FHR-3, and FHR-1 (CFH, CFHR3, and CFHR1, respectively) are polymorphic and located adjacent to each other on human chromosome 1q31.3. We have previously shown that haplotype CFH(H3)–CFHR3*B–CFHR1*B associates with aHUS and reduced FH levels. In this study, we used a specific enzyme-linked immunosorbent assay to quantify FHR-3 in plasma samples from controls and patients with aHUS genotyped for the three known CFHR3 alleles (CFHR3*A, CFHR3*B, and CFHR3*Del). In the 218 patients carrying at least one copy of CFHR3, significant differences between CFHR3 genotype groups were found, with CFHR3*A/Del patients having the lowest FHR-3 concentration (0.684–1.032 µg/mL), CFHR3*B/Del and CFHR3*A/A patients presenting intermediate levels (1.437–2.201 µg/mL), and CFHR3*A/B and CFHR3*B/B patients showing the highest concentration (2.330–4.056 µg/mL) (p 

  • a novel atypical hemolytic uremic syndrome associated hybrid CFHR1 cfh gene encoding a fusion protein that antagonizes factor h dependent complement regulation
    Journal of The American Society of Nephrology, 2015
    Co-Authors: Elisabetta Valoti, Agustín Tortajada, Santiago Rodríguez De Córdoba, Marta Alberti, Jesus Garciafernandez, Sara Gastoldi, Luca Besso, Elena Bresin, Giuseppe Remuzzi, Marina Noris
    Abstract:

    Genomic aberrations affecting the genes encoding factor H (FH) and the five FH-related proteins (FHRs) have been described in patients with atypical hemolytic uremic syndrome (aHUS), a rare condition characterized by microangiopathic hemolytic anemia, thrombocytopenia, and ARF. These genomic rearrangements occur through nonallelic homologous recombinations caused by the presence of repeated homologous sequences in CFH and CFHR1-R5 genes. In this study, we found heterozygous genomic rearrangements among CFH and CFHR genes in 4.5% of patients with aHUS. CFH/CFHR rearrangements were associated with poor clinical prognosis and high risk of post-transplant recurrence. Five patients carried known CFH/CFHR1 genes, but we found a duplication leading to a novel CFHR1/CFH hybrid gene in a family with two affected subjects. The resulting fusion protein contains the first four short consensus repeats of FHR1 and the terminal short consensus repeat 20 of FH. In an FH-dependent hemolysis assay, we showed that the hybrid protein causes sheep erythrocyte lysis. Functional analysis of the FHR1 fraction purified from serum of heterozygous carriers of the CFHR1/CFH hybrid gene indicated that the FHR1/FH hybrid protein acts as a competitive antagonist of FH. Furthermore, sera from carriers of the hybrid CFHR1/CFH gene induced more C5b-9 deposition on endothelial cells than control serum. These results suggest that this novel genomic hybrid mediates disease pathogenesis through dysregulation of complement at the endothelial cell surface. We recommend that genetic screening of aHUS includes analysis of CFH and CFHR rearrangements, particularly before a kidney transplant.

  • Atypical Hemolytic Uremic Syndrome-Associated Variants and Autoantibodies Impair Binding of Factor H and Factor H-Related Protein 1 to Pentraxin 3
    The Journal of Immunology, 2012
    Co-Authors: Anne Kopp, Stefanie Strobel, Pilar Sánchez-corral, Agustín Tortajada, Santiago Rodríguez De Córdoba, Zoltán Prohászka, Margarita López-trascasa, Mihály Józsi
    Abstract:

    Atypical hemolytic uremic syndrome (aHUS) is a renal disease associated with complement alternative pathway dysregulation and is characterized by endothelial injury. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule expressed by endothelial cells and upregulated under inflammatory conditions. PTX3 activates complement, but it also binds the complement inhibitor factor H. In this study, we show that native factor H, factor H-like protein 1, and factor H-related protein 1 (CFHR1) bind to PTX3 and that PTX3-bound factor H and factor H-like protein 1 maintain their complement regulatory activities. PTX3, when bound to extracellular matrix, recruited functionally active factor H. Residues within short consensus repeat 20 of factor H that are relevant for PTX3 binding were identified using a peptide array. aHUS-associated factor H mutations within this binding site caused a reduced factor H binding to PTX3. Similarly, seven of nine analyzed anti-factor H autoantibodies isolated from aHUS patients inhibited the interaction between factor H and PTX3, and five autoantibodies also inhibited PTX3 binding to CFHR1. Moreover, the aHUS-associated CFHR1*B variant showed reduced binding to PTX3 in comparison with CFHR1*A. Thus, the interactions of PTX3 with complement regulators are impaired by certain mutations and autoantibodies affecting factor H and CFHR1, which could result in an enhanced local complement-mediated inflammation, endothelial cell activation, and damage in aHUS.

  • characterization of complement factor h related cfhr proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome
    Blood, 2009
    Co-Authors: Cynthia Abarrateguigarrido, Santiago Rodríguez De Córdoba, Ruben Martinezbarricarte, Margarita Lopeztrascasa, Pilar Sanchezcorral
    Abstract:

    The factor H–related protein family (CFHR) is a group of minor plasma proteins genetically and structurally related to complement factor H (fH). Notably, deficiency of CFHR1/CFHR3 associates with protection against age-related macular degeneration and with the presence of anti-fH autoantibodies in atypical hemolytic uremic syndrome (aHUS). We have developed a proteomics strategy to analyze the CFHR proteins in plasma samples from controls, patients with aHUS, and patients with type II membranoproliferative glomerulonephritis. Here, we report on the identification of persons carrying novel deficiencies of CFHR1, CFHR3, and CFHR1/CFHR4A, resulting from point mutations in CFHR1 and CFHR3 or from a rearrangement involving CFHR1 and CFHR4 . Remarkably, patients with aHUS lacking CFHR1, but not those lacking CFHR3, present anti-fH autoantibodies, suggesting that generation of these antibodies is specifically related to CFHR1 deficiency. We also report the characterization of a novel CFHR1 polymorphism, resulting from a gene conversion event between CFH and CFHR1 , which strongly associates with aHUS. The risk allotype CFHR1*B, with greater sequence similarity to fH, may compete with fH, decreasing protection of cellular surfaces against complement damage. In summary, our comprehensive analyses of the CFHR proteins have improved our understanding of these proteins and provided further insights into aHUS pathogenesis.

Christine Skerka - One of the best experts on this subject based on the ideXlab platform.

  • IMMUNOBIOLOGY Factor H–related protein 1 (CFHR-1) inhibits complement C5 convertase activity
    2016
    Co-Authors: Christine Skerka
    Abstract:

    fragment in human chromosome 1 that encompasses the CFHR1 and CFHR3 genes represents a risk factor for hemo-lytic uremic syndrome (HUS) but has a protective effect in age-related macular degeneration (AMD). Here we identify CFHR1 as a novel inhibitor of the comple-ment pathway that blocks C5 convertase activity and interferes with C5b surface deposition and MAC formation. This activ-ity is distinct from complement factor H, and apparently factor H and CFHR1 con-trol complement activation in a sequen-tial manner. As both proteins bind to the same or similar sites at the cellular sur-faces, the gain of CFHR1 activity presum-ably is at the expense of CFH-mediated function (inhibition of the C3 convertase). In HUS, the absence of CFHR1 may result in reduced inhibition of terminal complex formation and in reduced protection of endothelial cells upon complement at-tack. These findings provide new insights into complement regulation on the cell surface and biosurfaces and likely define the role of CFHR1 in human diseases. (Blood. 2009;114:2439-2447

  • Represent Novel Ligands for the Infection-Associated
    2016
    Co-Authors: Barbara Uzonyi, Christine Skerka, Teresia Hallstrom, Corinna Siegel, Hannes Eberhardt
    Abstract:

    Complement factor H-related proteins CFHR2 and CFHR5 represent novel ligands for the infection-associated CRASP proteins of Borrelia burgdorfer

  • Complement Factor H-Related Proteins CFHR2 and CFHR5 Represent Novel Ligands for the Infection-Associated CRASP Proteins of Borrelia burgdorferi
    2013
    Co-Authors: Corinna Siegel, Reinhard Wallich, Barbara Uzonyi, Peter F Zipfel, Christine Skerka, Teresia Hallstrom, Brian Stevenson, Michael Karas, Hannes Eberhardt, Peter Kraiczy
    Abstract:

    Background: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complementmediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi. Methodology/Principal Findings: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amount

  • Binding of complement regulators to intact spirochetes.
    2013
    Co-Authors: Jasmin Schwab, Reinhard Wallich, Peter F Zipfel, Christine Skerka, Claudia Hammerschmidt, Dania Richter, Franz-rainer Matuschka, Peter Kraiczy
    Abstract:

    (A) Ligand affinity blotting was performed to detect binding of complement regulators. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 as well as B. duttonii LA1 were incubated in NHS-EDTA to prevent complement activation, washed extensively, and bound proteins were eluted using 100 mM glycine-HCl (pH 2.0). Both the last wash (W) and the eluate (E) fractions obtained from each strain were separated by 12.5% Laemmli-SDS-PAGE (detection of CFH, FHL1, FHR1, FHR2, and C1-Inh) or by 10% tris/tricine SDS-PAGE (C4Bp detection) and transferred to nitrocellulose. Membranes were probed either with a polyclonal anti-SCR1-4 antiserum for the detection of both CFH and FHL1, a polyclonal anti-CFHR1 antiserum for the detection of CFHR1 and CFHR2, a polyclonal anti-C4Bp antiserum, or with a polyclonal anti-C1-Inh antiserum. Binding of CFH (B) or C4Bp (C) to spirochetes (5×107 cells) was assessed by ELISA. Bound CFH was detected with a polyclonal goat anti-CFH antiserum and C4Bp was detected with a polyclonal sheep anti-C4Bp antiserum. Data represent means and SD from three separate experiments, each performed at least in triplicate. ***p

  • Human factor H-related protein 2 (CFHR2) regulates complement activation
    2013
    Co-Authors: Hannes U Eberhardt, Reinhard Wallich, Peter F Zipfel, Qian Chen, Andrea Hartmann, Denise Buhlmann, Peter Hortschansky, Markus J. Kemper, Christine Skerka
    Abstract:

    Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs). Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I