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John P. Atkinson - One of the best experts on this subject based on the ideXlab platform.
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Lack of Evidence from Studies of Soluble Protein Fragments that Knops Blood Group Polymorphisms in Complement Receptor-Type 1 Are Driven by Malaria
2012Co-Authors: Patience B. Tetteh-quarcoo, Richard E. Hauhart, John P. Atkinson, Haydyn D T Mertens, Christoph Q Schmidt, Waihong Tham, Alan F Cowman, Arthur Rowe, Alexandra J. Rowe, Paul N BarlowAbstract:Complement receptor-type 1 (CR1, CD35) is the immune-adherence receptor, a Complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McCa/b may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on Complement Control Protein modules (CCPs) 15–25 of its ectodomain. These eleven modules encompass a region (CCPs 15–17) key to rosetting, opsonin recognition and Complement regulation, as well as the Knops blood group polymorphisms in CCPs 24–25. All four CR1 15–25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15–25 variants had virtually indistinguishable affinities for immobilized Complement fragments C3b (KD = 0.8–1.1 µM) and C4b (KD = 5.0–5.3 µM). They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in Complement-regulatory functionality, nor interactions with P. falciparum Proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.
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plasmodium falciparum uses a key functional site in Complement receptor type 1 for invasion of human erythrocytes
Blood, 2011Co-Authors: Richard E. Hauhart, John P. Atkinson, Christoph Q Schmidt, Mara Guariento, Waihong Tham, Patience B Tettehquarcoo, Sash Lopaticki, Paul N BarlowAbstract:The Plasmodium falciparum adhesin PfRh4 binds to Complement receptor type-1 (CR1) on human erythrocytes and mediates a glycophorin-independent invasion pathway. CR1 is a Complement regulator and immune-adherence receptor on erythrocytes required for shuttling of C3b/C4b-opsonized particles to liver and spleen for phagocytosis. Using recombinant CR1 constructs, we mapped the recognition site for PfRh4 to Complement Control Protein modules 1 to 3 (CCP1-3) at the membrane-distal amino terminus of CR1. This region of CR1 binds to C4b and C3b and accelerates decay of both classic pathway and alternative pathway C3 and C5 convertases. CCP1-3 competed for PfRh4 binding to erythroid CR1 and inhibited the PfRh4-CR1 invasion pathways across a wide range of P falciparum strains. PfRh4 did not bind significantly to other CR1 constructs, including CCP15-17, which is 85% identical to CCP1-3. PfRh4 binding to CR1 did not affect its C3b/C4b binding capability, and we show evidence for a ternary complex between CCP1-3, C4b, and PfRh4. PfRh4 binding specifically inhibited CR1's convertase decay-accelerating activity, whereas there was no effect on factor H-mediated decay-accelerating activity. These results increase our understanding of the functional implications of CR1 engagement with PfRh4 and highlight the interplay between Complement regulation and infection.
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membrane cofactor Protein mutations in atypical hemolytic uremic syndrome ahus fatal stx hus c3 glomerulonephritis and the hellp syndrome
Blood, 2007Co-Authors: Celia J Fang, Kathryn M Liszewski, Timothy H.j. Goodship, Veronique Fremeauxbacchi, Gaia Pianetti, Marina Noris, John P. AtkinsonAbstract:The hemolytic uremic syndrome (HUS) is a triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies demonstrate that heterozygous mutations of membrane cofactor Protein (MCP;CD46) predispose to atypical HUS (aHUS), which is not associated with exposure to Shiga toxin (Stx). Among the initial 25 MCP mutations in patients with aHUS were 2, R69W and A304V, that were expressed normally and for which no dysfunction was found. The R69W mutation is in Complement Control Protein module 2, while A304V is in the hydrophobic transmembrane domain. In addition to 3 patients with aHUS, the A304V mutation was identified in 1 patient each with fatal Stx-HUS, the HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and glomerulonephritis with C3 deposits. A major goal was to assess if these putative mutations lead to defective Complement regulation. Permanent cell lines expressing the mutated Proteins were Complement "challenged," and membrane Control of C3 fragment deposition was monitored. Both the R69W and A304V MCP mutations were deficient in their ability to Control the alternative pathway of Complement activation on a cell surface, illustrating the importance of modeling transmembrane Proteins in situ.
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membrane cofactor Protein mutations in atypical hemolytic uremic syndrome ahus fatal stx hus c3 glomerulonephritis and the hellp syndrome
Blood, 2007Co-Authors: Celia J Fang, Kathryn M Liszewski, Timothy H.j. Goodship, Veronique Fremeauxbacchi, Gaia Pianetti, Marina Noris, John P. AtkinsonAbstract:The hemolytic uremic syndrome (HUS) is a triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies demonstrate that heterozygous mutations of membrane cofactor Protein (MCP;CD46) predispose to atypical HUS (aHUS), which is not associated with exposure to Shiga toxin (Stx). Among the initial 25 MCP mutations in patients with aHUS were 2, R69W and A304V, that were expressed normally and for which no dysfunction was found. The R69W mutation is in Complement Control Protein module 2, while A304V is in the hydrophobic transmembrane domain. In addition to 3 patients with aHUS, the A304V mutation was identified in 1 patient each with fatal Stx-HUS, the HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and glomerulonephritis with C3 deposits. A major goal was to assess if these putative mutations lead to defective Complement regulation. Permanent cell lines expressing the mutated Proteins were Complement "challenged," and membrane Control of C3 fragment deposition was monitored. Both the R69W and A304V MCP mutations were deficient in their ability to Control the alternative pathway of Complement activation on a cell surface, illustrating the importance of modeling transmembrane Proteins in situ.
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Synergy between Two Active Sites of Human Complement Receptor Type 1 (CD35) in Complement Regulation: Implications for the Structure of the Classical Pathway C3 Convertase and Generation of More Potent Inhibitors
Journal of Immunology, 2005Co-Authors: Malgorzata Krych-goldberg, Richard E. Hauhart, Tina Porzukowiak, John P. AtkinsonAbstract:The extracellular domain of the Complement receptor type 1 (CR1; CD35) consists entirely of 30 Complement Control Protein repeats (CCPs). CR1 has two distinct functional sites, site 1 (CCPs 1–3) and two copies of site 2 (CCPs 8–10 and CCPs 15–17). In this report we further define the structural requirements for decay-accelerating activity (DAA) for the classical pathway (CP) C3 and C5 convertases and, using these results, generate more potent decay accelerators. Previously, we demonstrated that both sites 1 and 2, tandemly arranged, are required for efficient DAA for C5 convertases. We show that site 1 dissociates the CP C5 convertase, whereas the role of site 2 is to bind the C3b subunit. The intervening CCPs between two functional sites are required for optimal DAA, suggesting that a spatial orientation of the two sites is important. DAA for the CP C3 convertase is increased synergistically if two copies of site 1, particularly those carrying DAA-increasing mutations, are contained within one Protein. DAA in such constructs may exceed that of long homologous repeat A (CCPs 1–7) by up to 58-fold. To explain this synergy, we propose a dimeric structure for the CP C3 convertase on cell surfaces. We also extended our previous studies of the amino acid requirements for DAA of site 1 and found that the CCP 1/CCP 2 junction is critical and that Phe 82 may contact the C3 convertases. These observations increase our understanding of the mechanism of DAA. In addition, a more potent decay-accelerating form of CR1 was generated.
Anna M Blom - One of the best experts on this subject based on the ideXlab platform.
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novel structure of the n terminal helical domain of biba a group b streptococcus immunogenic bacterial adhesin
Acta Crystallographica Section D-biological Crystallography, 2020Co-Authors: Kartik Manne, Anna M Blom, Debasish Chattopadhyay, Vaibhav Agarwal, Baldeep Khare, Srinivas Chakravarthy, Chungyu Chang, Hung Tonthat, Sthanam V L NarayanaAbstract:BibA, a group B streptococcus (GBS) surface Protein, has been shown to protect the pathogen from phagocytic killing by sequestering a Complement inhibitor: C4b-binding Protein (C4BP). Here, the X-ray crystallographic structure of a GBS BibA fragment (BibA126-398) and a low-resolution small-angle X-ray scattering (SAXS) structure of the full-length N-terminal domain (BibA34-400) are described. The BibA126-398 fragment crystal structure displayed a novel and predominantly helical structure. The tertiary arrangement of helices forms four antiparallel three-helix-bundle-motif repeats, with one long helix from a bundle extending into the next. Multiple mutations on recombinant BibA34-400 delayed the degradation of the Protein, and circular dichroism spectroscopy of BibA34-400 suggested a similar secondary-structure composition to that observed in the crystallized BibA126-398 fragment. A model was generated for the 92 N-terminal residues (BibA34-125) using structural similarity prediction programs, and a BibA34-400 model was generated by combining the coordinates of BibA34-126 and BibA126-398. The X-ray structure of BibA126-398 and the model of BibA34-400 fitted well into the calculated SAXS envelope. One possible binding site for the BibA N-terminal domain was localized to the N-terminal CCP (Complement-Control Protein) domains of the C4BP α-chain, as indicated by the decreased binding of BibA to a ΔCCP1 C4BP α-chain mutant. In summary, it is suggested that the GBS surface Protein BibA, which consists of three antiparallel α-helical-bundle motifs, is unique and belongs to a new class of Gram-positive surface adhesins.
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c4b binding Protein the good the bad and the deadly novel functions of an old friend
Immunology Letters, 2016Co-Authors: David Ermert, Anna M BlomAbstract:C4b-binding Protein (C4BP) is best known as a potent soluble inhibitor of the classical and lectin pathways of the Complement system. This large 500 kDa multimeric plasma glycoProtein is expressed mainly in the liver but also in lung and pancreas. It consists of several identical 75 kDa α-chains and often also one 40 kDa β-chain, both of which are mainly composed of Complement Control Protein (CCP) domains. Structure-function studies revealed that one crucial binding site responsible for inhibition of Complement is located to CCP1-3 of the α-chain. Binding of anticoagulant Protein S to the CCP1 of the β-chain provides C4BP with the ability to strongly bind apoptotic and necrotic cells in order to prevent inflammation arising from activation of Complement by these cells. Further, C4BP interacts strongly with various types of amyloid and enhances fibrillation of islet amyloid polypeptide secreted from pancreatic beta cells, which may attenuate pro-inflammatory and cytotoxic effects of this amyloid. Full deficiency of C4BP has not been identified but non-synonymous alterations in its sequence have been found in haemolytic uremic syndrome and recurrent pregnancy loss. Furthermore, C4BP is bound by several bacterial pathogens, notably Streptococcus pyogenes, which due to inhibition of Complement and enhancement of bacterial adhesion to endothelial cells provides these bacteria with a survival advantage in the host. Thus, depending on the context, C4BP has a protective or detrimental role in the organism.
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fine mapping of the interaction between c4b binding Protein and outer membrane Proteins liga and ligb of pathogenic leptospira interrogans
PLOS Neglected Tropical Diseases, 2015Co-Authors: Leandro Carvalho Dantas Breda, Anna M Blom, Angela S Barbosa, Chinglin Hsieh, Monica Marcela Castiblanco Valencia, Ludmila Bezerra Da Silva, Chang Yungfu, Lourdes IsaacAbstract:The Complement system consists of more than 40 Proteins that participate in the inflammatory response and in pathogen killing. Complement inhibitors are necessary to avoid the excessive consumption and activation of this system on host cells. Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Pathogenic leptospires are able to escape from Complement activation by binding to host Complement inhibitors Factor H [FH] and C4b-binding Protein (C4BP) while non-pathogenic leptospires are rapidly killed in the presence of fresh serum. In this study, we demonstrate that Complement Control Protein domains (CCP) 7 and 8 of C4BP α-chain interact with the outer membrane Proteins LcpA, LigA and LigB from the pathogenic leptospire L. interrogans. The interaction between C4BP and LcpA, LigA and LigB is sensitive to ionic strength and inhibited by heparin. We fine mapped the LigA and LigB domains involved in its binding to C4BP and heparin and found that both interactions are mediated through the bacterial immunoglobulin-like (Big) domains 7 and 8 (LigA7-8 and LigB7-8) of both LigA and LigB and also through LigB9-10. Therefore, C4BP and heparin may share the same binding sites on Lig Proteins.
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the novel Complement inhibitor human cub and sushi multiple domains 1 csmd1 Protein promotes factor i mediated degradation of c4b and c3b and inhibits the membrane attack complex assembly
The FASEB Journal, 2013Co-Authors: Astrid Escuderoesparza, Nikolina Kalchishkova, E Kurbasic, Wen Guo Jiang, Anna M BlomAbstract:CUB and Sushi multiple domains 1 (CSMD1) is a transmembrane Protein containing 15 consecutive Complement Control Protein (CCP) domains, which are characteristic for Complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized Complement inhibitors were used as positive Controls, whereas Coxsackie adenovirus receptor, a Protein with no effect on Complement, was a negative Control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the Controls. Fur...
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the c type lectin of the aggrecan g3 domain activates Complement
PLOS ONE, 2013Co-Authors: Camilla Melin Furst, Dick Heinegard, Matthias Morgelin, Kasper Vadstrup, Anna M BlomAbstract:Excessive Complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage Proteins are fragmented and released into the synovial fluid. Some of these Proteins and fragments activate Complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix Proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of Complement, via binding of C1q and C3, respectively. The Complement Control Protein (CCP) domain adjacent to the CLD showed no effect on Complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed Complement activation was attenuated due to binding of Complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal Complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint.
Paul N Barlow - One of the best experts on this subject based on the ideXlab platform.
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disease linked mutations in factor h reveal pivotal role of cofactor activity in self surface selective regulation of Complement activation
Journal of Biological Chemistry, 2017Co-Authors: Heather Kerr, Elisavet Makou, Andrew P Herbert, David J Kavanagh, Edwin K S Wong, Yi Yang, Kevin J Marchbank, Anna Richards, Paul N BarlowAbstract:Spontaneous activation enables the Complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by Complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble Protein containing 20 Complement-Control Protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing Complement-mediated hemolysis of ES PspCN, a CFH-binding Streptococcus pneumoniae Protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on ES Conversely, PspCN boosted the CA, on ES, of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants.
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solution structure of ccp modules 10 12 illuminates functional architecture of the Complement regulator factor h
Journal of Molecular Biology, 2012Co-Authors: Elisavet Makou, Haydyn D T Mertens, Mateusz Maciejewski, Dinesh C Soares, Ilias Matis, Christoph Q Schmidt, Andrew P Herbert, Dmitri I Svergun, Paul N BarlowAbstract:The 155-kDa plasma glycoProtein factor H (FH), which consists of 20 Complement Control Protein (CCP) modules, protects self-tissue but not foreign organisms from damage by the Complement cascade. Protection is achieved by selective engagement of FH, via CCPs 1–4, CCPs 6–8 and CCPs 19–20, with polyanion-rich host surfaces that bear covalently attached, activation-specific, fragments of Complement component C3. The role of intervening CCPs 9–18 in this process is obscured by lack of structural knowledge. We have concatenated new high-resolution solution structures of overlapping recombinant CCP pairs, 10–11 and 11–12, to form a three-dimensional structure of CCPs 10–12 and validated it by small-angle X-ray scattering of the recombinant triple‐module fragment. Superimposing CCP 12 of this 10–12 structure with CCP 12 from the previously solved CCP 12–13 structure yielded an S-shaped structure for CCPs 10–13 in which modules are tilted by 80–110° with respect to immediate neighbors, but the bend between CCPs 10 and 11 is counter to the arc traced by CCPs 11–13. Including this four-CCP structure in interpretation of scattering data for the longer recombinant segments, CCPs 10–15 and 8–15, implied flexible attachment of CCPs 8 and 9 to CCP 10 but compact and intimate arrangements of CCP 14 with CCPs 12, 13 and 15. Taken together with difficulties in recombinant production of module pairs 13–14 and 14–15, the aberrant structure of CCP 13 and the variability of 13–14 linker sequences among orthologues, a structural dependency of CCP 14 on its neighbors is suggested; this has implications for the FH mechanism.
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structural analysis of the c terminal region modules 18 20 of Complement regulator factor h fh
PLOS ONE, 2012Co-Authors: Hugh P Morgan, Haydyn D T Mertens, Dinesh C Soares, Christoph Q Schmidt, Andrew P Herbert, Dmitri I Svergun, Paul N Barlow, Mara Guariento, Jonathan P HannanAbstract:Factor H (FH) is a soluble regulator of the human Complement system affording protection to host tissues. It selectively inhibits amplification of C3b, the activation-specific fragment of the abundant Complement component C3, in fluid phase and on self-surfaces and accelerates the decay of the alternative pathway C3 convertase, C3bBb. We have determined the crystal structure of the three carboxyl-terminal Complement Control Protein (CCP) modules of FH (FH18–20) that bind to C3b, and which additionally recognize polyanionic markers specific to self-surfaces. These CCPs harbour nearly 30 disease-linked missense mutations. We have also deployed small-angle X-ray scattering (SAXS) to investigate FH18–20 flexibility in solution using FH18–20 and FH19–20 constructs. In the crystal lattice FH18–20 adopts a “J”-shape: A ∼122-degree tilt between the structurally highly similar modules 18 and 19 precedes an extended, linear arrangement of modules 19 and 20 as observed in previously determined structures of these two modules alone. However, under solution conditions FH18–20 adopts multiple conformations mediated by flexibility between CCPs 18 and 19. We also pinpoint the locations of disease-associated missense mutations on the module 18 surface and discuss our data in the context of the C3b:FH interaction.
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Lack of Evidence from Studies of Soluble Protein Fragments that Knops Blood Group Polymorphisms in Complement Receptor-Type 1 Are Driven by Malaria
2012Co-Authors: Patience B. Tetteh-quarcoo, Richard E. Hauhart, John P. Atkinson, Haydyn D T Mertens, Christoph Q Schmidt, Waihong Tham, Alan F Cowman, Arthur Rowe, Alexandra J. Rowe, Paul N BarlowAbstract:Complement receptor-type 1 (CR1, CD35) is the immune-adherence receptor, a Complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McCa/b may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on Complement Control Protein modules (CCPs) 15–25 of its ectodomain. These eleven modules encompass a region (CCPs 15–17) key to rosetting, opsonin recognition and Complement regulation, as well as the Knops blood group polymorphisms in CCPs 24–25. All four CR1 15–25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15–25 variants had virtually indistinguishable affinities for immobilized Complement fragments C3b (KD = 0.8–1.1 µM) and C4b (KD = 5.0–5.3 µM). They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in Complement-regulatory functionality, nor interactions with P. falciparum Proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.
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plasmodium falciparum uses a key functional site in Complement receptor type 1 for invasion of human erythrocytes
Blood, 2011Co-Authors: Richard E. Hauhart, John P. Atkinson, Christoph Q Schmidt, Mara Guariento, Waihong Tham, Patience B Tettehquarcoo, Sash Lopaticki, Paul N BarlowAbstract:The Plasmodium falciparum adhesin PfRh4 binds to Complement receptor type-1 (CR1) on human erythrocytes and mediates a glycophorin-independent invasion pathway. CR1 is a Complement regulator and immune-adherence receptor on erythrocytes required for shuttling of C3b/C4b-opsonized particles to liver and spleen for phagocytosis. Using recombinant CR1 constructs, we mapped the recognition site for PfRh4 to Complement Control Protein modules 1 to 3 (CCP1-3) at the membrane-distal amino terminus of CR1. This region of CR1 binds to C4b and C3b and accelerates decay of both classic pathway and alternative pathway C3 and C5 convertases. CCP1-3 competed for PfRh4 binding to erythroid CR1 and inhibited the PfRh4-CR1 invasion pathways across a wide range of P falciparum strains. PfRh4 did not bind significantly to other CR1 constructs, including CCP15-17, which is 85% identical to CCP1-3. PfRh4 binding to CR1 did not affect its C3b/C4b binding capability, and we show evidence for a ternary complex between CCP1-3, C4b, and PfRh4. PfRh4 binding specifically inhibited CR1's convertase decay-accelerating activity, whereas there was no effect on factor H-mediated decay-accelerating activity. These results increase our understanding of the functional implications of CR1 engagement with PfRh4 and highlight the interplay between Complement regulation and infection.
Kenji M Cunnion - One of the best experts on this subject based on the ideXlab platform.
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Clumping factor A interaction with Complement factor I increases C3b cleavage on the bacterial surface of Staphylococcus aureus, and decreases Complement-mediated phagocytosis
Infection and immunity, 2010Co-Authors: Pamela S Hair, Joan A Geoghegan, Timothy J Foster, Charlene G. Echague, Amber M. Sholl, Justin A. Watkins, Kenji M CunnionAbstract:The human Complement system is important in the immunological Control of Staphylococcus aureus infection. We showed previously that S. aureus surface Protein clumping factor A (ClfA), when expressed in recombinant form, bound Complement Control Protein factor I and increased factor I cleavage of C3b to iC3b. In the present study, we show that, compared to the results for the wild type, when isogenic ClfA-deficient S. aureus mutants were incubated in serum, they bound less factor I, generated less iC3b on the bacterial surface, and bound fewer C3 fragments. It has been shown previously that two amino acids in ClfA (P336 and Y338) are essential for fibrinogen binding. However, S. aureus expressing ClfA(P336A Y338S) was less virulent than ClfA-deficient strains in animal models. This suggested that ClfA contributed to S. aureus virulence by a mechanism different than fibrinogen binding. In the present study, we showed that S. aureus expressing ClfA(P336A Y338S) was more susceptible to Complement-mediated phagocytosis than a ClfA-null mutant or the wild type. Unlike ClfA, ClfA(P336A Y338S) did not enhance factor I cleavage of C3b to iC3b and inhibited the cofactor function of factor H. Fibrinogen enhanced factor I binding to ClfA and the S. aureus surface. Twenty clinical S. aureus strains all expressed ClfA and bound factor I. High levels of factor I binding by clinical strains correlated with poor phagocytosis. In summary, our results suggest that the interaction of ClfA with factor I contributes to S. aureus virulence by a Complement-mediated mechanism.
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staphylococcus aureus clumping factor a binds to Complement regulator factor i and increases factor i cleavage of c3b
The Journal of Infectious Diseases, 2008Co-Authors: Pamela S Hair, Timothy J Foster, Michael D Ward, John O Semmes, Kenji M CunnionAbstract:The human Complement system plays an important role in the Control of Staphylococcus aureus infection. For instance, we previously demonstrated that the central Complement component deposited on the organism’s surface, C3b, can be cleaved by the host Complement Control Protein, factor I, resulting in diminished phagocytosis of S. aureus. In the present study, we have identified clumping factor A (ClfA) from cell wall Proteins of S. aureus as a specific Protein bound by factor I. Recombinant ClfA (rClfA) containing the full-length A region (peptides 40 –559) also bound factor I. We identified an 50-kDa fragment of ClfA that is shed by S. aureus into growth medium. The shed ClfA fragment was derived from the A region of ClfA and bound factor I. rClfA and the shed ClfA fragment increased factor I cleavage of C3b into inactive C3b. Our findings describe a new S. aureus mechanism for modification of host Complement activities. Staphylococcus aureus is a highly successful pathogen, a common cause of community-associated infection, and the most common cause of severe nosocomial infection [1–3]. S. aureus infections cause consider
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staphylococcus aureus clumping factor a binds to Complement regulator factor i and increases factor i cleavage of c3b
The Journal of Infectious Diseases, 2008Co-Authors: Pamela S Hair, Timothy J Foster, Michael D Ward, John O Semmes, Kenji M CunnionAbstract:The human Complement system plays an important role in the Control of Staphylococcus aureus infection. For instance, we previously demonstrated that the central Complement component deposited on the organism's surface, C3b, can be cleaved by the host Complement Control Protein, factor I, resulting in diminished phagocytosis of S. aureus. In the present study, we have identified clumping factor A (ClfA) from cell wall Proteins of S. aureus as a specific Protein bound by factor I. Recombinant ClfA (rClfA) containing the full-length A region (peptides 40-559) also bound factor I. We identified an 50-kDa fragment of ClfA that is shed by S. aureus into growth medium. The shed ClfA fragment was derived from the A region of ClfA and bound factor I. rClfA and the shed ClfA fragment increased factor I cleavage of C3b into inactive C3b. Our findings describe a new S. aureus mechanism for modification of host Complement activities.
Girish J Kotwal - One of the best experts on this subject based on the ideXlab platform.
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inhibition of Complement and cd14 attenuates the escherichia coli induced inflammatory response in porcine whole blood
Infection and Immunity, 2009Co-Authors: Ebbe Billmann Thorgersen, Girish J Kotwal, Anne Pharo, K Haverson, Anne K Axelsen, Peter Gaustad, Georgia Sfyroera, Tom Eirik MollnesAbstract:The innate immune response is a double-edged sword in systemic inflammation and sepsis. UnControlled or inappropriate activation can damage and be lethal to the host. Several studies have investigated inhibition of downstream mediators, including tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Emerging evidence indicates that upstream inhibition is a better therapeutic approach for attenuating damaging immune activation. Therefore, we investigated inhibition of two central innate immune pathways, those of Complement and CD14/Toll-like receptor 4 (TLR4)/myeloid differentiation Protein 2 (MD-2), in a porcine in vitro model of Escherichia coli-induced inflammation. Porcine whole blood anticoagulated with lepuridin, which did not interfere with the Complement system, was incubated with E. coli lipopolysaccharide (LPS) or whole bacteria. Inhibitors of Complement and CD14 and thus the LPS CD14/TLR4/MD-2 receptor complex were tested to investigate the effect on the inflammatory response. A broad range of inflammatory readouts were used to monitor the effect. Anti-CD14 was found to saturate the CD14 molecule on granulocytes and completely inhibited LPS-induced proinflammatory cytokines in a dose-dependent manner. Anti-CD14 significantly reduced the levels of the E. coli-induced proinflammatory cytokines TNF-alpha and IL-1beta, but not IL-8, in a dose-dependent manner. No effect on bacterial clearance was seen. Vaccinia Complement Control Protein and smallpox inhibitor of Complement enzymes, two Orthopoxvirus-encoded Complement inhibitors, completely inhibited Complement activation. Furthermore, these agents almost completely inhibited the expression of wCD11R3, which is associated with CD18 as a beta2 integrin, on porcine granulocytes and decreased IL-8 levels significantly in a dose-dependent manner. As expected, Complement inhibition reduced bacterial clearance. We conclude that inhibition of Complement and CD14 attenuates E. coli-induced inflammation and might be used as a therapeutic regimen in gram-negative sepsis along with appropriate treatment with antibiotics.
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the structure of Complement c3b provides insights into Complement activation and regulation
Nature, 2006Co-Authors: Abdul A Ajees, Sthanam V L Narayana, K Gunasekaran, John E Volanakis, Girish J KotwalAbstract:The human Complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or Protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor Protein (MCP, CD46), Complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus Complement-Control Protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the Complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in Complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'Complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.
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vaccinia virus Complement Control Protein is capable of protecting xenoendothelial cells from antibody binding and killing by human Complement and cytotoxic cells
Transplantation, 2001Co-Authors: Futwan Almohanna, Ranjit S Parhar, Girish J KotwalAbstract:BACKGROUND Vaccinia virus Complement Control Protein (VCP) was the first secretory microbial Protein shown to have structural similarity to the family of Complement Control Proteins. VCP can block both the classical and alternate Complement pathways. Recently, VCP has been shown to bind to heparin, and this property contributes to separate functions, making the molecule a multifunctional Protein. METHODS VCP prepared from a natural infection of RK-13 cells with vaccinia virus was purified to homogeneity. Cultured pig aortic endothelial cells (PAECs) were mixed with human serum, anti-Gal alpha1,3 Gal antibody, neutrophils, or natural killer (NK) cells in the presence or absence of VCP and either direct binding of FITC-labeled antibody or killing by cytotoxic cells was estimated. RESULTS Our cell culture studies demonstrate that VCP blocks Complement-mediated killing of PAECs by human serum in a dose-dependent manner. We also demonstrate that VCP is capable of blocking Gal alpha1,3 Gal binding sites on PAECS. Surprisingly, VCP effectively blocked interactions between PAECs and cytotoxic cells such as human naive neutrophils and NK cells. CONCLUSION VCP is a novel Protein amongst the Complement Control Protein family and can, not only block xenorejection by inhibiting Complement but also by blocking killing by cytotoxic cells.
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crystal structure of a Complement Control Protein that regulates both pathways of Complement activation and binds heparan sulfate proteoglycans
Cell, 2001Co-Authors: Krishna H M Murthy, Paul N Barlow, Scott A Smith, Vannakambadi K Ganesh, K Judge, Nicholas P Mullin, Craig M Ogata, Girish J KotwalAbstract:Abstract Vaccinia virus Complement Control Protein (VCP) inhibits both pathways of Complement activation through binding the third and fourth components. A homolog of mammalian regulators of Complement activation, its ability to bind heparin endows VCP with additional activities of significance to viral infectivity. The structure of VCP reveals a highly extended molecule with a putative heparin recognition site at its C-terminal end. A second cluster of positive charges provides a possibly overlapping binding site for both heparin and Complement components. Experiments suggested by the structure indicate that VCP can bind heparin and Control Complement simultaneously. This, the structure of any intact regulator of Complement activation, along with attendant functional insights, will stimulate the design of new therapeutic inhibitors of Complement.
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the inflammation modulatory Protein imp of cowpox virus drastically diminishes the tissue damage by down regulating cellular infiltration resulting from Complement activation
Molecular and Cellular Biochemistry, 1998Co-Authors: Girish J Kotwal, Cathie G Miller, David E JustusAbstract:Vaccinia virus (VV) and other pathogenic poxviruses encode for a Complement Control Protein. The VV Complement Control Protein or VCP, was one of the first soluble microbial Proteins postulated to have an active role in the immunomodulation of the host defense. Since then, 2 other poxviruses, including variola virus and cowpox virus (CPV), were found to have corresponding Proteins. Based upon earlier studies which demonstrated the role of the CPV Complement Control Protein in modulating the specific tissue responses in BALB/c and congenic-matched C5-sufficient and C5-deficient mice [1], the CPV equivalent has been renamed the inflammation modulatory Protein (IMP), so as to specifically reflect its function. In this study, the in vivo cellular response of mice injected with CPV or a recombinant virus lacking the IMP sequence (CPV-IMP) was examined using a connective tissue air pouch model. Microscopic examination revealed that CPV-IMP caused a significant mononuclear cell infiltration into the connective tissue and adjacent dermal tissue of the skin. To characterize IMP`'s ability to regulate the observed cellular infiltration through both Complement derived and non-Complement derived chemotactic factors, footpad and skin connective tissue of C3 knockout mice and footpad of MIP-1 α knockout mice received injections of CPV and CPV-IMP. In comparison to the matched Control, significantly greater footpad specific swelling response was seen in C3 -/- mice injected with CPV. This indicates an important role for C3 in poxvirus pathogenesis. However, MIP-1 alpha -/- mice injected with CPV-IMP recovered earlier than mice injected with CPV alone. This indicates that the function of IMP in vivo in mice with a complete repertoire of immune components is to limit cellular infiltration by down regulating the Complement derived chemotactic analphylotoxins, thereby modulating the inflammatory response contributing to a diminished tissue pathology and preservation of viral habitat.