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Michael K Pangburn - One of the best experts on this subject based on the ideXlab platform.
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salo a novel Classical Pathway complement inhibitor from saliva of the sand fly lutzomyia longipalpis
Scientific Reports, 2016Co-Authors: Viviana P Ferreira, Michael K Pangburn, Vladimir Fazito Vale, Maha Abdeladhim, Antonio Ferreira Mendessousa, Iliano V Coutinhoabreu, Manoochehr Rasouli, Elizabeth A Brandt, Claudio Meneses, Kolyvan Ferreira LimaAbstract:Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the Classical Pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the Classical Pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the Classical Pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin Pathway of complement. In conclusion our data shows that SALO is a specific Classical Pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement Classical Pathway-mediated pathogenic effects in human diseases.
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Role of the C3b-binding site on C4b-binding protein in regulating Classical Pathway C5 convertase.
Molecular Immunology, 2006Co-Authors: Nenoo Rawal, Michael K PangburnAbstract:Abstract A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of Classical Pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC 50 for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC 50 for inhibiting formation of the soluble C3 convertase (5 nM) is 50–80-fold below the normal serum concentration of C4BP (250–400 nM), C4BP in blood effectively prevents formation of Classical Pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.
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Formation of high affinity C5 convertase of the Classical Pathway of complement.
The Journal of biological chemistry, 2003Co-Authors: Nenoo Rawal, Michael K PangburnAbstract:Abstract C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of Classical Pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6–9 μm). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 μm) well below the normal concentration of C5 in blood (0.37 μm). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative Pathway convertase indicates that the 6–9-fold greater catalytic rate of the Classical Pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this Pathway.
Ronald D. Gorham - One of the best experts on this subject based on the ideXlab platform.
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Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models.
Frontiers in immunology, 2018Co-Authors: Seline A. Zwarthoff, Suzan H. M. Rooijakkers, Maartje Ruyken, Evelien T.m. Berends, Sanne Mol, Piet C. Aerts, Mihály Józsi, Carla J. C. De Haas, Ronald D. GorhamAbstract:Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative Pathway C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by alternative Pathway (AP) convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases (C3bBb(C3b)n) is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a newly-developed Classical Pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases, but also inhibit formation of a functional Classical Pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.
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functional characterization of alternative and Classical Pathway c3 c5 convertase activity and inhibition using purified models
Frontiers in Immunology, 2018Co-Authors: Seline A. Zwarthoff, Suzan H. M. Rooijakkers, Maartje Ruyken, Evelien T.m. Berends, Sanne Mol, Piet C. Aerts, Mihály Józsi, Carla J. C. De Haas, Ronald D. GorhamAbstract:Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative Pathway C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by alternative Pathway (AP) convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases (C3bBb(C3b)n) is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a newly-developed Classical Pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases, but also inhibit formation of a functional Classical Pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.
Nenoo Rawal - One of the best experts on this subject based on the ideXlab platform.
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Activation of complement component C5: comparison of C5 convertases of the lectin Pathway and the Classical Pathway of complement.
The Journal of biological chemistry, 2008Co-Authors: Nenoo Rawal, Rema Rajagopalan, Veena Prakash SalviAbstract:Although the initiating complex of lectin Pathway (called M1 in this study) generates C3/C5 convertases similar to those assembled by the initiating complex (C1) of the Classical Pathway, activation of complement component C5 via the lectin Pathway has not been examined. In the present study kinetic analysis of lectin Pathway C3/C5 convertases assembled on two surfaces (zymosan and sheep erythrocytes coated with mannan (E(Man))) revealed that the convertases (ZymM1,C4b,C2a and E(Man)M1,C4b,C2a) exhibited a similar but weak affinity for the substrate, C5 indicated by a high K(m) (2.73-6.88 microm). Very high affinity C5 convertases were generated when the low affinity C3/C5 convertases were allowed to deposit C3b by cleaving native C3. These C3b-containing convertases exhibited K(m) (0.0086-0.0075 microm) well below the normal concentration of C5 in blood (0.37 microm). Although kinetic parameters, K(m) and k(cat), of the lectin Pathway C3/C5 convertases were similar to those reported for Classical Pathway C3/C5 convertases, studies on the ability of C4b to bind C2 indicated that every C4b deposited on zymosan or E(Man) was capable of forming a convertase. These findings differ from those reported for the Classical Pathway C3/C5 convertase, where only one of four C4b molecules deposited formed a convertase. The potential for four times more amplification via the lectin Pathway than the Classical Pathway in the generation of C3/C5 convertases and production of pro-inflammatory products, such as C3a, C4a, and C5a, implies that activation of complement via the lectin Pathway might be a more prominent contributor to the pathology of inflammatory reactions.
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Role of the C3b-binding site on C4b-binding protein in regulating Classical Pathway C5 convertase.
Molecular Immunology, 2006Co-Authors: Nenoo Rawal, Michael K PangburnAbstract:Abstract A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of Classical Pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC 50 for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC 50 for inhibiting formation of the soluble C3 convertase (5 nM) is 50–80-fold below the normal serum concentration of C4BP (250–400 nM), C4BP in blood effectively prevents formation of Classical Pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.
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Formation of high affinity C5 convertase of the Classical Pathway of complement.
The Journal of biological chemistry, 2003Co-Authors: Nenoo Rawal, Michael K PangburnAbstract:Abstract C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of Classical Pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6–9 μm). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 μm) well below the normal concentration of C5 in blood (0.37 μm). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative Pathway convertase indicates that the 6–9-fold greater catalytic rate of the Classical Pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this Pathway.
Seline A. Zwarthoff - One of the best experts on this subject based on the ideXlab platform.
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Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models.
Frontiers in immunology, 2018Co-Authors: Seline A. Zwarthoff, Suzan H. M. Rooijakkers, Maartje Ruyken, Evelien T.m. Berends, Sanne Mol, Piet C. Aerts, Mihály Józsi, Carla J. C. De Haas, Ronald D. GorhamAbstract:Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative Pathway C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by alternative Pathway (AP) convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases (C3bBb(C3b)n) is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a newly-developed Classical Pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases, but also inhibit formation of a functional Classical Pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.
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functional characterization of alternative and Classical Pathway c3 c5 convertase activity and inhibition using purified models
Frontiers in Immunology, 2018Co-Authors: Seline A. Zwarthoff, Suzan H. M. Rooijakkers, Maartje Ruyken, Evelien T.m. Berends, Sanne Mol, Piet C. Aerts, Mihály Józsi, Carla J. C. De Haas, Ronald D. GorhamAbstract:Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative Pathway C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by alternative Pathway (AP) convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases (C3bBb(C3b)n) is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a newly-developed Classical Pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases, but also inhibit formation of a functional Classical Pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.
V J Benedi - One of the best experts on this subject based on the ideXlab platform.
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Activation of the Complement Classical Pathway (C1q Binding) by Mesophilic Aeromonas hydrophila Outer Membrane Protein
Infection and immunity, 1998Co-Authors: Susana Merino, Sebastian Alberti, V J Benedi, María Mercedes Nogueras, Alicia Aguilar, Xavier Rubires, Juan M TomasAbstract:The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement Classical Pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355-3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates the Classical Pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the Classical Pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the Classical Pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups.
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c1q binding and activation of the complement Classical Pathway by klebsiella pneumoniae outer membrane proteins
Infection and Immunity, 1993Co-Authors: Sebastian Alberti, G Marques, Silvia Camprubi, Susana Merino, Juan M Tomas, F Vivanco, V J BenediAbstract:The mechanisms of killing of Klebsiella pneumoniae serum-sensitive strains in nonimmune serum by the complement Classical Pathway have been studied. The bacterial cell surface components that bind C1q more efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterial species. These two outer membrane proteins were isolated from a representative serum-sensitive strain. We have demonstrated that in their purified form, they bind C1q and activate the Classical Pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the Classical Pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted in factor D). Binding of C1q to other components of the bacterial outer membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of the Classical Pathway by this lipopolysaccharide was also much less efficient than activation by the two outer membrane proteins. The antibody-independent binding of C1q to serum-sensitive strains was independent of the presence of capsular polysaccharide, while strains possessing lipopolysaccharide O antigen bind less C1q and are resistant to complement-mediated killing.