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Peter F Zipfel - One of the best experts on this subject based on the ideXlab platform.
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Data_Sheet_1_Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors.docx
2019Co-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 ReskiAbstract: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.
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Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors
Frontiers Media S.A., 2019Co-Authors: Peter F Zipfel, Stefan Michelfelder, Juliana Parsons, Oguz Top, Lennard L. Bohlender, Phillipp Kopp, Christian Busch-steenberg, Sebastian N. W. Hoernstein, Karsten HäffnerAbstract: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
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the mfhr1 fusion protein is a novel synthetic multitarget Complement Inhibitor with therapeutic potential
Journal of The American Society of Nephrology, 2018Co-Authors: Stefan Michelfelder, Peter F Zipfel, Friedericke Fischer, Astrid Waldin, Kim V Horle, Martin Pohl, Juliana Parsons, Ralf Reski, Eva L Decker, Christine SkerkaAbstract:The Complement system is essential for host defense, but uncontrolled Complement system activation leads to severe, mostly renal pathologies, such as atypical hemolytic uremic syndrome or C3 glomerulopathy. Here, we investigated a novel combinational approach to modulate Complement activation by targeting C3 and the terminal pathway simultaneously. The synthetic fusion protein MFHR1 links the regulatory domains of Complement factor H (FH) with the C5 convertase/C5b-9 Inhibitory fragment of the FH-related protein 1. In vitro, MFHR1 showed cofactor and decay acceleration activity and inhibited C5 convertase activation and C5b-9 assembly, which prevented C3b deposition and reduced C3a/C5a and C5b-9 generation. Furthermore, this fusion protein showed the ability to escape deregulation by FH-related proteins and form multimeric complexes with increased Inhibitory activity. In addition to substantially inhibiting alternative and classic pathway activation, MFHR1 blocked hemolysis mediated by serum from a patient with aHUS expressing truncated FH. In FH-/- mice, MFHR1 administration augmented serum C3 levels, reduced abnormal glomerular C3 deposition, and ameliorated C3 glomerulopathy. Taking the unique design of MFHR1 into account, we suggest that the combination of proximal and terminal cascade inhibition together with the ability to form multimeric complexes explain the strong Inhibitory capacity of MFHR1, which offers a novel basis for Complement therapeutics.
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the Complement Inhibitor factor h generates an anti inflammatory and tolerogenic state in monocyte derived dendritic cells
Journal of Immunology, 2016Co-Authors: Rut Olivar, Anna M Blom, Peter F Zipfel, Ana Luque, Sonia Cardenasbrito, Mar Naranjogomez, Francesc E Borras, Santiago Rodriguez De Cordoba, Josep M AranAbstract:The activation of the Complement system is a key initiating step in the protective innate immune-inflammatory response against injury, although it may also cause harm if left unchecked. The structurally related soluble Complement Inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of Complement activation, respectively, attenuating the activity of the C3/C5 convertases and, consequently, avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study, we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly, FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture, resembling immature MoDCs, lower expression of the maturation marker CD83 and the costimulatory molecules CD40, CD80, and CD86, decreased production of key proinflammatory Th1-cytokines (IL-12, TNF-α, IFN-γ, IL-6, and IL-8), and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover, FH-treated MoDCs show low Ag uptake and, when challenged with LPS, display reduced CCR7 expression and chemotactic migration, impaired CD4(+) T cell alloproliferation, inhibition of IFN-γ secretion by the allostimulated T cells, and, conversely, induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus, this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity, transplantation, and autoimmunity.
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plasminogen is a Complement Inhibitor
Journal of Biological Chemistry, 2012Co-Authors: Diana Barthel, Peter F Zipfel, Susann SchindlerAbstract:Plasminogen is a 92-kDa single chain glycoprotein that circulates in plasma as a zymogen and when converted to proteolytically active plasmin dissolves preformed fibrin clots and extracellular matrix components. Here, we characterize the role of plasmin(ogen) in the Complement cascade. Plasminogen binds the central Complement protein C3, the C3 cleavage products C3b and C3d, and C5. Plasminogen binds to C3, C3b, C3d, and C5 via lysine residues, and the interaction is ionic strength-dependent. Plasminogen and Factor H bind C3b; however, the two proteins bind to different sites and do not compete for binding. Plasminogen affects Complement action in multiple ways. Plasminogen enhanced Factor I-mediated C3b degradation in the presence of the cofactor Factor H. Plasminogen when activated to plasmin inhibited Complement as demonstrated by hemolytic assays using either rabbit or sheep erythrocytes. Similarly, plasmin either in the fluid phase or attached to surfaces inhibited Complement that was activated via the alternative and classical pathways and cleaved C3b to fragments of 68, 40, 30, and 17 kDa. The C3b fragments generated by plasmin differ in size from those generated by the Complement protease Factor I, suggesting that plasmin-mediated C3b cleavage fragments lack effector function. Plasmin also cleaved C5 to products of 65, 50, 30, and 25 kDa. Thus, plasmin(ogen) regulates both Complement and coagulation, the two central cascade systems of a vertebrate organism. This Complement-Inhibitory activity of plasmin provides a new explanation why pathogenic microbes utilize plasmin(ogen) for immune evasion and tissue penetration.
Anna M Blom - One of the best experts on this subject based on the ideXlab platform.
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the Complement Inhibitor factor h generates an anti inflammatory and tolerogenic state in monocyte derived dendritic cells
Journal of Immunology, 2016Co-Authors: Rut Olivar, Anna M Blom, Peter F Zipfel, Ana Luque, Sonia Cardenasbrito, Mar Naranjogomez, Francesc E Borras, Santiago Rodriguez De Cordoba, Josep M AranAbstract:The activation of the Complement system is a key initiating step in the protective innate immune-inflammatory response against injury, although it may also cause harm if left unchecked. The structurally related soluble Complement Inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of Complement activation, respectively, attenuating the activity of the C3/C5 convertases and, consequently, avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study, we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly, FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture, resembling immature MoDCs, lower expression of the maturation marker CD83 and the costimulatory molecules CD40, CD80, and CD86, decreased production of key proinflammatory Th1-cytokines (IL-12, TNF-α, IFN-γ, IL-6, and IL-8), and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover, FH-treated MoDCs show low Ag uptake and, when challenged with LPS, display reduced CCR7 expression and chemotactic migration, impaired CD4(+) T cell alloproliferation, inhibition of IFN-γ secretion by the allostimulated T cells, and, conversely, induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus, this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity, transplantation, and autoimmunity.
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streptococcus pneumoniae phosphoglycerate kinase is a novel Complement Inhibitor affecting the membrane attack complex formation
Journal of Biological Chemistry, 2014Co-Authors: Anna M Blom, Kristian Riesbeck, Simone Bergmann, Marcus Fulde, Vaibhav AgarwalAbstract:The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that causes infections ranging from acute otitis media to life-threatening invasive disease. Pneumococci have evolved several strategies to circumvent the host immune response, in particular the Complement attack. The pneumococcal glycolytic enzyme phosphoglycerate kinase (PGK) is both secreted and bound to the bacterial surface and simultaneously binds plasminogen and its tissue plasminogen activator tPA. In the present study we demonstrate that PGK has an additional role in modulating the Complement attack. PGK interacted with the membrane attack complex (MAC) components C5, C7, and C9, thereby blocking the assembly and membrane insertion of MAC resulting in significant inhibition of the hemolytic activity of human serum. Recombinant PGK interacted in a dose-dependent manner with these terminal pathway proteins, and the interactions were ionic in nature. In addition, PGK inhibited C9 polymerization both in the fluid phase and on the surface of sheep erythrocytes. Interestingly, PGK bound several MAC proteins simultaneously. Although C5 and C7 had partially overlapping binding sites on PGK, C9 did not compete with either one for PGK binding. Moreover, PGK significantly inhibited MAC deposition via both the classical and alternative pathway at the pneumococcal surface. Additionally, upon activation plasmin(ogen) bound to PGK cleaved the central Complement protein C3b thereby further modifying the Complement attack. In conclusion, our data demonstrate for the first time to our knowledge a novel pneumococcal Inhibitor of the terminal Complement cascade aiding Complement evasion by this important pathogen.
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binding of Complement Inhibitor c4b binding protein to a highly virulent s pyogenes m1 strain is mediated by protein h and enhances adhesion to and invasion of endothelial cells
Journal of Biological Chemistry, 2013Co-Authors: David Ermert, Vaibhav Agarwal, Antonin Weckel, Ingamaria Frick, Lars Bjorck, Anna M BlomAbstract:Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the Complement Inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonisation with Complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of 125I-C4b. Further, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains, or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18 amino acid long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6x10-7 M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonisation but also for invasion of endothelial cells by Streptococcus pyogenes. (Less)
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sushi domain containing protein 4 susd4 inhibits Complement by disrupting the formation of the classical c3 convertase
The FASEB Journal, 2013Co-Authors: Emelie Holmquist, Björn Nodin, Marcin Okroj, Karin Jirström, Anna M BlomAbstract:Recently discovered Sushi domain-containing protein 4 (SUSD4) contains several Sushi or Complement control protein domains; therefore, we hypothesized that it may act as Complement Inhibitor. Two isoforms of human SUSD4, fused to the Fc part of human IgG, were recombinantly expressed in Chinese hamster ovary (CHO) cells. The secreted soluble isoform of SUSD4 (SUSD4b) inhibited the classical and lectin Complement pathways by 50% at a concentration of 0.5 μM. This effect was due to the fact that 1 μM SUSD4b inhibited the formation of the classical C3 convertase by 90%. The membrane-bound isoform (SUSD4a) inhibited the classical and alternative Complement pathways when expressed on the surface of CHO cells but not when expressed as a soluble, truncated protein. In all functional studies, we used known Complement Inhibitors as positive controls, while Coxsackie adenovirus receptor, which has no effect on Complement, expressed with Fc tag, was a negative control. We also studied the mRNA expression of both isoforms of SUSD4 in a panel of human tissues using quantitative PCR and primarily found SUSD4a in esophagus and brain, while SUSD4b was highly expressed in esophagus, ovary, and heart. Overall, our results show that SUSD4 is a novel Complement Inhibitor with restricted expression.-Holmquist, E., Okroj, M., Nodin, B., Jirstrom, K. Blom, A. M. Sushi domain-containing protein 4 (SUSD4) inhibits Complement by disrupting the formation of the classical C3 convertase. (Less)
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enolase of streptococcus pneumoniae binds human Complement Inhibitor c4b binding protein and contributes to Complement evasion
Journal of Immunology, 2012Co-Authors: Vaibhav Agarwal, Sven Malm, Kristian Riesbeck, Simone Bergmann, Sven Hammerschmidt, Anna M BlomAbstract:Streptococcus pneumoniae (pneumococcus) is a pathogen that causes severe local and life-threatening invasive diseases, which are associated with high mortality rates. Pneumococci have evolved several strategies to evade the host immune system, including Complement to disseminate and to survive in various host niches. Thus, pneumococci bind Complement Inhibitors such as C4b-binding protein (C4BP) and factor H via pneumococcal surface protein C, thereby inhibiting the classical and alternative Complement pathways. In this study, we identified the pneumococcal glycolytic enzyme enolase, a nonclassical cell surface and plasminogen-binding protein, as an additional pneumococcal C4BP-binding protein. Furthermore, we demonstrated that human, but not mouse, C4BP bound pneumococci. Recombinant enolase bound in a dose-dependent manner C4BP purified from plasma, and the interaction was reduced by increasing ionic strength. Enolase recruited C4BP and plasminogen, but not factor H, from human serum. Moreover, C4BP and plasminogen bound to different domains of enolase as they did not compete for the interaction with enolase. In direct binding assays with recombinant C4BP mutants lacking individual domains, two binding sites for enolase were identified on the Complement control protein (CCP) domain 1/CCP2 and CCP8 of the C4BP α-chains. C4BP bound to the enolase retained its cofactor activity as determined by C4b degradation. Furthermore, in the presence of exogenously added enolase, an increased C4BP binding to and subsequently decreased C3b deposition on pneumococci was observed. Taken together, pneumococci specifically interact with human C4BP via enolase, which represents an additional mechanism of human Complement control by this versatile pathogen.
John D Lambris - One of the best experts on this subject based on the ideXlab platform.
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The alternative Complement pathway regulates pathological angiogenesis in the retina
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2014Co-Authors: J. Harry Sweigard, John D Lambris, Ryoji Yanai, Philipp Gaissert, Magali Saint-geniez, Keiko Kataoka, Aristomenis Thanos, Gregory L. Stahl, Kip M. ConnorAbstract:A defining feature in proliferative retinopathies is the formation of pathological neovessels. In these diseases, the balance between neovessel formation and regression determines blindness, making the modulation of neovessel growth highly desirable. The role of the immune system in these retinopathies is of increasing interest, but it is not completely understood. We investigated the role of the alternative Complement pathway during the formation and resolution of aberrant neovascularization. We used alternative Complement pathway-deficient (Fb(-/-)) mice and age- and strain-matched control mice to assess neovessel development and regression in an oxygen-induced retinopathy (OIR) mouse model. In the control mice, we found increased transcription of Fb after OIR treatment. In the Fb(-/-) mice, we prepared retinal flatmounts and identified an increased number of neovessels, peaking at postnatal day 17 (P17; P=0.001). Subjecting human umbilical vein endothelial cells (HUVECs) to low oxygen, mimicking a characteristic of neovessels, decreased the expression of the Complement Inhibitor Cd55. Finally, using laser capture microdissection (LCM) to isolate the neovessels after OIR, we found decreased expression of Cd55 (P=0.005). Together, our data implicate the alternative Complement pathway in facilitating neovessel clearance by down-regulating the Complement Inhibitor Cd55 specifically on neovessels, allowing for their targeted removal while leaving the established vasculature intact.-Sweigard, J. H., Yanai, R., Gaissert, P., Saint-Geniez, M., Kataoka, K., Thanos, A., Stahl, G. L., Lambris, J. D., Connor, K. M. The alternative Complement pathway regulates pathological angiogenesis in the retina.
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ornithodoros moubata Complement Inhibitor is an equally effective c5 Inhibitor in pigs and humans
Journal of Immunology, 2011Co-Authors: Andreas Barrattdue, Miles A Nunn, John D Lambris, Olga Lissina, Ebbe Billmann Thorgersen, Julie Katrine Lindstad, Anne Pharo, Tom Eirik MollnesAbstract:Experimental evidence suggests that C inhibition and more particularly combined inhibition of C and the TLR coreceptor CD14 may be of therapeutic benefit in sepsis and other inflammatory conditions. A barrier to the testing and further development of many Inhibitors is that their activity is species specific. Pig is a relevant species for experimental models of human disease, and this study undertakes a comprehensive comparison of the Inhibitory efficacy of the C5 Inhibitor Ornithodoros moubata C Inhibitor (OmCI) in human and porcine whole blood ex vivo models of Escherichia coli-induced sepsis. The effect of OmCI on Complement activity in pigs undergoing E. coli sepsis was also examined. Porcine and human serum, and whole blood anticoagulated with lepirudin, was incubated with E. coli and the effect of OmCI investigated. The ex vivo results were virtually identical in pig and human. OmCI completely ablated the activity of all three C pathways at 0.64 μM. E. coli-induced C activation and expression of CD11b (wCD11R3 in the pig), was abolished ex vivo at 0.32 μM OmCI. Combining anti-CD14 and OmCI reduced the formation of IL-8 and TNF-α more potently than the single Inhibitors. OmCI also efficiently bound E. coli-induced leukotriene B(4) in pig and human plasma. In support of our ex vivo findings, in vivo the activity of all C pathways was inhibited at 0.6 mg OmCI/kg pig. In conclusion, OmCI efficiently inhibited pig and human C activation, has accompanying anti-inflammatory effects and is a promising candidate Inhibitor for further in vivo studies of sepsis.
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synthesis and activity of thioether containing analogues of the Complement Inhibitor compstatin
ACS Chemical Biology, 2011Co-Authors: Patrick J Knerr, Apostolia Tzekou, Daniel Ricklin, Hui Chen, Wilfred A Van Der Donk, John D LambrisAbstract:Disulfide bonds are essential for the structural stability and biological activity of many bioactive peptides. However, these bonds are labile to reducing agents, which can limit the therapeutic utility of such peptides. Substitution of a disulfide bond with a reduction-resistant cystathionine bridge is an attractive means of improving stability while imposing minimal structural perturbation to the peptide. We have applied this approach to the therapeutic Complement Inhibitor compstatin, a disulfide-containing peptide currently in clinical trials for age-related macular degeneration, in an effort to maintain its potent activity while improving its biological stability. Thioether-containing compstatin analogues were produced via solid-phase peptide synthesis utilizing orthogonally protected cystathionine amino acid building blocks and solid-supported peptide cyclization. Overall, the affinity of these analogues for their biological target and potent inhibition of Complement activation were largely maintain...
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novel analogues of the therapeutic Complement Inhibitor compstatin with significantly improved affinity and potency
Molecular Immunology, 2011Co-Authors: Paola Magotti, Daniel Ricklin, Yiannis N Kaznessis, Ioannis Kourtzelis, John D LambrisAbstract:Compstatin is a 13-residue disulfide-bridged peptide that inhibits a key step in the activation of the human Complement system. Compstatin and its derivatives have shown great promise for the treatment of many clinical disorders associated with unbalanced Complement activity. To obtain more potent compstatin analogues, we have now performed an N-methylation scan of the peptide backbone and amino acid substitutions at position 13. One analogue (Ac-I[CVW(Me)QDW-Sar-AHRC](NMe)I-NH(2)) displayed a 1000-fold increase in both potency (IC(50) = 62 nM) and binding affinity for C3b (K(D) = 2.3 nM) over that of the original compstatin. Biophysical analysis using surface plasmon resonance and isothermal titration calorimetry suggests that the improved binding originates from more favorable free conformation and stronger hydrophobic interactions. This study provides a series of significantly improved drug leads for therapeutic applications in Complement-related diseases, and offers new insights into the structure-activity relationships of compstatin analogues.
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structure kinetic relationship analysis of the therapeutic Complement Inhibitor compstatin
Journal of Molecular Recognition, 2009Co-Authors: Paola Magotti, Daniel Ricklin, Yiannis N Kaznessis, John D LambrisAbstract:Compstatin is a 13-residue peptide that inhibits activation of the Complement system by binding to the central component C3 and its fragments C3b and C3c. A combination of theoretical and experimental approaches has previously allowed us to develop analogs of the original compstatin peptide with up to 264-fold higher activity; one of these analogs is now in clinical trials for the treatment of age-related macular degeneration (AMD). Here we used functional assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to assess the effect of modifications at three key residues (Trp-4, Asp-6, Ala-9) on the affinity and activity of compstatin and its analogs, and we correlated our findings to the recently reported co-crystal structure of compstatin and C3c. The KD values for the panel of tested analogs ranged from 10−6 to 10−8 M. These differences in binding affinity could be attributed mainly to differences in dissociation rather than association rates, with a >4-fold range in kon values (2–10 × 105 M−1 s−1) and a koff variation of >35-fold (1–37 × 10−2 s−1) being observed. The stability of the C3b-compstatin complex seemed to be highly dependent on hydrophobic effects at position 4, and even small changes at position 6 resulted in a loss of complex formation. Induction of a β-turn shift by an A9P modification resulted in a more favorable entropy but a loss of binding specificity and stability. The results obtained by the three methods utilized here were highly correlated with regard to the activity/affinity of the analogs. Thus, our analyses have identified essential structural features of compstatin and provided important information to support the development of analogs with improved efficacy. Copyright © 2009 John Wiley & Sons, Ltd.
Pampee P Young - One of the best experts on this subject based on the ideXlab platform.
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brown recluse spider bite mediated hemolysis clinical features a possible role for Complement Inhibitor therapy and reduced rbc surface glycophorin a as a potential biomarker of venom exposure
PLOS ONE, 2013Co-Authors: Eric A Gehrie, Hui Nian, Pampee P YoungAbstract:Background The venom of Loxosceles reclusa (Brown Recluse spider) can cause a severe, life-threatening hemolysis in humans for which no therapy is currently available in the USA beyond supportive measures. Because this hemolysis is uncommon, relatively little is known about its clinical manifestation, diagnosis, or management. Here, we aimed to clarify the clinical details of envenomation, to determine the efficacy of the Complement Inhibitor eculizumab to prevent the hemolysis in vitro, and to investigate markers of exposure to Brown Recluse venom. Study Design and Methods We performed a 10-year chart review of cases of Brown Recluse spider bite-mediated hemolysis at our institution. We also designed an in vitro assay to test the efficacy of eculizumab to inhibit hemolysis of venom exposed red blood cells. Finally, we compared levels of CD55, CD59 and glycophorin A on venom exposed versus venom-naive cells. Results Most victims of severe Brown Recluse spider mediated hemolysis at our institution are children and follow an unpredictable clinical course. Brown Recluse spider bite mediated hemolysis is reduced by 79.2% (SD=18.8%) by eculizumab in vitro. Erythrocyte glycophorin A, but not CD55 or CD59, is reduced after red blood cells are incubated with venom in vitro. Conclusion Taken together, our laboratory data and clinical observations indicate that L. reclusa venom exposure results in non-specific antibody and Complement fixation on red blood cells, resulting in Complement mediated hemolysis that is curtailed by the Complement Inhibitor eculizumab in vitro. Glycophorin A measurement by flow cytometry may help to identify victims of L. reclusa envenomation.
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brown recluse spider bite mediated hemolysis clinical features a possible role for Complement Inhibitor therapy and reduced rbc surface glycophorin a as a potential biomarker of venom exposure
PLOS ONE, 2013Co-Authors: Eric A Gehrie, Hui Nian, Pampee P YoungAbstract:Background The venom of Loxosceles reclusa (Brown Recluse spider) can cause a severe, life-threatening hemolysis in humans for which no therapy is currently available in the USA beyond supportive measures. Because this hemolysis is uncommon, relatively little is known about its clinical manifestation, diagnosis, or management. Here, we aimed to clarify the clinical details of envenomation, to determine the efficacy of the Complement Inhibitor eculizumab to prevent the hemolysis in vitro, and to investigate markers of exposure to Brown Recluse venom. Study Design and Methods We performed a 10-year chart review of cases of Brown Recluse spider bite-mediated hemolysis at our institution. We also designed an in vitro assay to test the efficacy of eculizumab to inhibit hemolysis of venom exposed red blood cells. Finally, we compared levels of CD55, CD59 and glycophorin A on venom exposed versus venom-naive cells. Results Most victims of severe Brown Recluse spider mediated hemolysis at our institution are children and follow an unpredictable clinical course. Brown Recluse spider bite mediated hemolysis is reduced by 79.2% (SD=18.8%) by eculizumab in vitro. Erythrocyte glycophorin A, but not CD55 or CD59, is reduced after red blood cells are incubated with venom in vitro. Conclusion Taken together, our laboratory data and clinical observations indicate that L. reclusa venom exposure results in non-specific antibody and Complement fixation on red blood cells, resulting in Complement mediated hemolysis that is curtailed by the Complement Inhibitor eculizumab in vitro. Glycophorin A measurement by flow cytometry may help to identify victims of L. reclusa envenomation.
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Brown Recluse spider bite mediated hemolysis: clinical features, a possible role for Complement Inhibitor therapy, and reduced RBC surface glycophorin A as a potential biomarker of venom exposure.
Public Library of Science (PLoS), 2026Co-Authors: Eric A Gehrie, Hui Nian, Pampee P YoungAbstract:The venom of Loxosceles reclusa (Brown Recluse spider) can cause a severe, life-threatening hemolysis in humans for which no therapy is currently available in the USA beyond supportive measures. Because this hemolysis is uncommon, relatively little is known about its clinical manifestation, diagnosis, or management. Here, we aimed to clarify the clinical details of envenomation, to determine the efficacy of the Complement Inhibitor eculizumab to prevent the hemolysis in vitro, and to investigate markers of exposure to Brown Recluse venom.We performed a 10-year chart review of cases of Brown Recluse spider bite-mediated hemolysis at our institution. We also designed an in vitro assay to test the efficacy of eculizumab to inhibit hemolysis of venom exposed red blood cells. Finally, we compared levels of CD55, CD59 and glycophorin A on venom exposed versus venom-naïve cells.Most victims of severe Brown Recluse spider mediated hemolysis at our institution are children and follow an unpredictable clinical course. Brown Recluse spider bite mediated hemolysis is reduced by 79.2% (SD=18.8%) by eculizumab in vitro. Erythrocyte glycophorin A, but not CD55 or CD59, is reduced after red blood cells are incubated with venom in vitro.Taken together, our laboratory data and clinical observations indicate that L. reclusa venom exposure results in non-specific antibody and Complement fixation on red blood cells, resulting in Complement mediated hemolysis that is curtailed by the Complement Inhibitor eculizumab in vitro. Glycophorin A measurement by flow cytometry may help to identify victims of L. reclusa envenomation
Jos A. G. Van Strijp - One of the best experts on this subject based on the ideXlab platform.
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Identification of a staphylococcal Complement Inhibitor with broad host specificity in equid Staphylococcus aureus strains.
Journal of Biological Chemistry, 2018Co-Authors: Nienke W. M. De Jong, Maartje Ruyken, Brandon L. Garcia, Jos A. G. Van Strijp, Manouk Vrieling, G. Koop, Matt Brettmann, Piet C. Aerts, Mark A. Holmes, Ewan M. HarrisonAbstract:Staphylococcus aureus is a versatile pathogen capable of causing a broad range of diseases in many different hosts. S. aureus can adapt to its host through modification of its genome (e.g. by acquisition and exchange of mobile genetic elements that encode host-specific virulence factors). Recently, the prophage φSaeq1 was discovered in S. aureus strains from six different clonal lineages almost exclusively isolated from equids. Within this phage, we discovered a novel variant of staphylococcal Complement Inhibitor (SCIN), a secreted protein that interferes with activation of the human Complement system, an important line of host defense. We here show that this equine variant of SCIN, eqSCIN, is a potent blocker of equine Complement system activation and subsequent phagocytosis of bacteria by phagocytes. Mechanistic studies indicate that eqSCIN blocks equine Complement activation by specific inhibition of the C3 convertase enzyme (C3bBb). Whereas SCIN-A from human S. aureus isolates exclusively inhibits human Complement, eqSCIN represents the first animal-adapted SCIN variant that functions in a broader range of hosts (horses, humans, and pigs). Binding analyses suggest that the human-specific activity of SCIN-A is related to amino acid differences on both sides of the SCIN-C3b interface. These data suggest that modification of this phage-encoded Complement Inhibitor plays a role in the host adaptation of S. aureus and are important to understand how this pathogen transfers between different hosts.
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the innate immune modulators staphylococcal Complement Inhibitor and chemotaxis Inhibitory protein of staphylococcus aureus are located on β hemolysin converting bacteriophages
Journal of Bacteriology, 2006Co-Authors: Willem J B Van Wamel, Maartje Ruyken, Suzan H M Rooijakkers, Kok P M Van Kessel, Jos A. G. Van StrijpAbstract:Two newly discovered immune modulators, chemotaxis Inhibitory protein of Staphylococcus aureus (CHIPS) and staphylococcal Complement Inhibitor (SCIN), cluster on the conserved 3′ end of β-hemolysin (hlb)-converting bacteriophages (βC-φs). Since these βC-φs also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3′ end of βC-φ represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinical Staphylococcus aureus strains and 5 classical laboratory strains, we show that 90% of S. aureus strains carry a βC-φ with an IEC. Seven IEC variants were discovered, carrying different combinations of chp, sak, or sea (or sep), always in the same 5′-to-3′ orientation and on the 3′ end of a βC-φ. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated in S. aureus R5 (broad phage host). All IEC-carrying bacteriophages integrated into hlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different βI-φs. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred among S. aureus strains by a diverse group of β-hemolysin-converting bacteriophages.
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the innate immune modulators staphylococcal Complement Inhibitor and chemotaxis Inhibitory protein of staphylococcus aureus are located on β hemolysin converting bacteriophages
Journal of Bacteriology, 2006Co-Authors: Willem J B Van Wamel, Maartje Ruyken, Suzan H M Rooijakkers, Kok P M Van Kessel, Jos A. G. Van StrijpAbstract:Two newly discovered immune modulators, chemotaxis Inhibitory protein of Staphylococcus aureus (CHIPS) and staphylococcal Complement Inhibitor (SCIN), cluster on the conserved 3' end of beta-hemolysin (hlb)-converting bacteriophages (betaC-phis). Since these betaC-phis also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3' end of betaC-phi represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinical Staphylococcus aureus strains and 5 classical laboratory strains, we show that 90% of S. aureus strains carry a betaC-phi with an IEC. Seven IEC variants were discovered, carrying different combinations of chp, sak, or sea (or sep), always in the same 5'-to-3' orientation and on the 3' end of a betaC-phi. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated in S. aureus R5 (broad phage host). All IEC-carrying bacteriophages integrated into hlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different betaI-phis. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred among S. aureus strains by a diverse group of beta-hemolysin-converting bacteriophages.