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Michael K Pangburn - One of the best experts on this subject based on the ideXlab platform.

  • Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase.
    Molecular Immunology, 2006
    Co-Authors: Nenoo Rawal, Michael K Pangburn
    Abstract:

    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.

  • Formation of high affinity C5 convertase of the classical pathway of complement.
    The Journal of biological chemistry, 2003
    Co-Authors: Nenoo Rawal, Michael K Pangburn
    Abstract:

    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.

  • Formation of High-Affinity C5 Convertases of the Alternative Pathway of Complement
    Journal of immunology (Baltimore Md. : 1950), 2001
    Co-Authors: Nenoo Rawal, Michael K Pangburn
    Abstract:

    Cleavage of C5 by C5 convertase is the last enzymatic step in the complement activation cascade leading to the formation of the cytolytic proteolytically activated form of C5 (C5b)-9 complex. In the present study, we examined the effect of the density of C3b (the proteolytically activated form of C3) on the function of the noncatalytic subunit of natural surface-bound forms of the enzyme. A comparison of the kinetic parameters of C5 convertases assembled on three surfaces (zymosan, rabbit erythrocytes, and sheep erythrocytes) were similar and revealed that the average Km decreased ∼28-fold (5.2–0.18 μM) when the density of C3b was increased from ∼18,000 to 400,000 C3b/cell. Very-high-affinity C5 convertases were generated when preformed C3 convertases were allowed to self amplify by giving them excess C3. These convertases exhibited Km from 0.016 to 0.074 μM, well below the normal plasma concentration of C5 in blood (0.37 μM). The results suggest that in serum convertases formed with monomeric C3b will be relatively inefficient in capturing C5 but will continue to cleave C3 opsonizing the cell surface for phagocytosis, whereas convertases formed with C3b-C3b complexes in areas of high C3b density will primarily cleave C5. The catalytic rate of these convertases approaches maximum velocity, thereby switching the enzyme from cleavage of C3 to cleavage of C5, and production of the cytolytic C5b-9 complex.

  • Structure/function of C5 convertases of complement.
    International immunopharmacology, 2001
    Co-Authors: Nenoo Rawal, Michael K Pangburn
    Abstract:

    C5 convertases are serine proteases that cleave both C3 and C5. Alternative pathway C3/C5 convertases formed with monomeric C3b (C3b,Bb) because of their weak interaction with C5 primarily cleave C3 thereby opsonizing the cell surface with C3b. In contrast, C3/C5 convertases formed with a high density of C3b/cell exhibit higher affinities for C5 as indicated by Km values well below the physiological concentration of C5 in blood. These C3/C5 convertases bind C5 efficiently and cleave it at a velocity approaching Vmax thereby switching the enzyme from C3 cleavage to production of the cytolytic C5b-9 complex. Studies of the structure of C3/C5 convertases have postulated that C4b-C3b and C3b-C3b dimers from high affinity C5 binding sites while indel studies have shown two binding sites in C5 for the convertase in addition to the C5 cleavage site. Together, these studies indicate that with increasing deposition of C3b on the surface, C3b complexes are formed which through multivalent attachment bind the substrate C5 with higher affinities, thereby converting the low affinity C3/C5 convertases to high affinity C5 convertases. The process underlying the formation of high affinity C5 convertases during complement activation is discussed.

  • Functional Role of the Noncatalytic Subunit of Complement C5 Convertase
    Journal of immunology (Baltimore Md. : 1950), 2000
    Co-Authors: Nenoo Rawal, Michael K Pangburn
    Abstract:

    The C5 convertase is a serine protease that consists of two subunits: a catalytic subunit which is bound in a Mg 2+ -dependent complex to a noncatalytic subunit. To understand the functional role of the noncatalytic subunit, we have determined the C5-cleaving properties of the cobra venom factor-dependent C5 convertase (CVF,Bb) made with CVF purified from the venom of Naja naja (CVF n ) and Naja haje (CVF h ) and compared them to those for two C3b-dependent C5 convertases (ZymC3b,Bb and C3b,Bb). A comparison of the kinetic parameters indicated that although the four C5 convertases (CVF n ,Bb, ZymC3b,Bb, CVF h ,Bb, and C3b,Bb) had similar catalytic rate constants ( k cat = 0.004–0.012 s −1 ) they differed 700-fold in their affinity for the substrate as indicated by the K m values (CVF n ,Bb = 0.036 μM, ZymC3b,Bb = 1.24 μM, CVF h ,Bb = 14.0 μM, and C3b,Bb = 24 μM). Analysis of binding interactions between C5 and the noncatalytic subunits (CVF h or C3b, or CVF n ) using the BIAcore, revealed dissociation binding constants ( K d ) that were similar to the K m values of the respective enzymes. The kinetic and binding data demonstrate that the binding site for C5 resides in the noncatalytic subunit of the enzyme, the affinity for the substrate is solely determined by the noncatalytic subunit and the catalytic efficiency of the enzyme appears not to be influenced by the nature of this subunit.

Nenoo Rawal - One of the best experts on this subject based on the ideXlab platform.

  • Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP).
    Molecular immunology, 2009
    Co-Authors: Nenoo Rawal, Rema Rajagopalan, Veena Prakash Salvi
    Abstract:

    The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E(Man)) was examined. While the C4BP concentration for inhibiting 50% (IC(50)) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05nM), approximately 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81nM) and E(Man) (42.66nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000-431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven alpha-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79+/-0.60) when the C4b density on E(Man) was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a approximately 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.

  • Activation of complement component C5: comparison of C5 convertases of the lectin pathway and the classical pathway of complement.
    The Journal of biological chemistry, 2008
    Co-Authors: Nenoo Rawal, Rema Rajagopalan, Veena Prakash Salvi
    Abstract:

    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.

  • Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase.
    Molecular Immunology, 2006
    Co-Authors: Nenoo Rawal, Michael K Pangburn
    Abstract:

    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.

  • Formation of high affinity C5 convertase of the classical pathway of complement.
    The Journal of biological chemistry, 2003
    Co-Authors: Nenoo Rawal, Michael K Pangburn
    Abstract:

    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.

  • Formation of High-Affinity C5 Convertases of the Alternative Pathway of Complement
    Journal of immunology (Baltimore Md. : 1950), 2001
    Co-Authors: Nenoo Rawal, Michael K Pangburn
    Abstract:

    Cleavage of C5 by C5 convertase is the last enzymatic step in the complement activation cascade leading to the formation of the cytolytic proteolytically activated form of C5 (C5b)-9 complex. In the present study, we examined the effect of the density of C3b (the proteolytically activated form of C3) on the function of the noncatalytic subunit of natural surface-bound forms of the enzyme. A comparison of the kinetic parameters of C5 convertases assembled on three surfaces (zymosan, rabbit erythrocytes, and sheep erythrocytes) were similar and revealed that the average Km decreased ∼28-fold (5.2–0.18 μM) when the density of C3b was increased from ∼18,000 to 400,000 C3b/cell. Very-high-affinity C5 convertases were generated when preformed C3 convertases were allowed to self amplify by giving them excess C3. These convertases exhibited Km from 0.016 to 0.074 μM, well below the normal plasma concentration of C5 in blood (0.37 μM). The results suggest that in serum convertases formed with monomeric C3b will be relatively inefficient in capturing C5 but will continue to cleave C3 opsonizing the cell surface for phagocytosis, whereas convertases formed with C3b-C3b complexes in areas of high C3b density will primarily cleave C5. The catalytic rate of these convertases approaches maximum velocity, thereby switching the enzyme from cleavage of C3 to cleavage of C5, and production of the cytolytic C5b-9 complex.

Dennis E. Hourcade - One of the best experts on this subject based on the ideXlab platform.

  • Properdin and Complement Activation: A Fresh Perspective
    Current drug targets, 2008
    Co-Authors: Dennis E. Hourcade
    Abstract:

    The C3 convertases are the major proteases of the complement cascade and are assembled at the site of complement activation via several different pathways. Properdin's functional role in stabilizing the alternative pathway convertase has been long established; however, new evidence demonstrates that properdin can also bind to certain microbial surfaces, and provide a platform for de novo convertase assembly. Therefore, properdin participates in two distinct mechanisms for complement activation: the alternative pathway and a properdin-directed pathway. Previous work had implicated the alternative pathway in the initiation and/or progression of several autoimmune diseases and in the host defense against certain bacterial pathogens. Those conclusions were based on evidence that cannot distinguish effects of the alternative pathway from effects of the properdin-directed pathway. With the identification of the new role for properdin in C3 convertase assembly there became a pressing need to reassess the mechanisms of complement activation, determine the specific role of properdin in each of these pathways, and explore the new therapeutic avenues that could arise.

  • a corresponding tyrosine residue in the c2 factor b type a domain is a hot spot in the decay acceleration of the complement c3 convertases
    Journal of Biological Chemistry, 2003
    Co-Authors: Lisa Kuttnerkondo, Megan P Dybvig, Lynne M Mitchell, Nasima Muqim, Edward M Medof, John P. Atkinson, Dennis E. Hourcade
    Abstract:

    Abstract The cleavage of C3 by the C3 convertases (C3bBb and C4b2a) determines whether complement activation proceeds. Dissociation (decay acceleration) of these central enzymes by the regulators decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP) controls their function. In a previous investigation, we obtained evidence implicating the α4/5 region of the type A domain of Bb (especially Tyr338) in decay acceleration of C3bBb and proposed this site as a potential interaction point with DAF and long homologous repeat A of CR1. Because portions of only two DAF complement control protein domains (CCPs), CCP2 and CCP3, are necessary to mediate its decay of the CP C3 convertase (as opposed to portions of at least three CCPs in all other cases, e.g. CCPs 1–3 of CR1), DAF/C4b2a provides the simplest structural model for this reaction. Therefore, we examined the importance of the C2 α4/5 site on decay acceleration of C4b2a. Functional C4b2a complexes made with the C2 Y327A mutant, the C2 homolog to factor B Y338A, were highly resistant to DAF, C4BP, and long homologous repeat A of CR1, whereas C2 substitutions in two nearby residues (N324A and L328A) resulted in partial resistance. Our new findings indicate that the α4/5 region of C2a is critical to decay acceleration mediated by DAF, C4BP, and CR1 and suggest that decay acceleration of C4b2a and C3bBb requires interaction of the convertase α4/5 region with a CCP2/CCP3 site of DAF or structurally homologous sites of CR1 and C4BP.

  • A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the complement C3 convertases.
    Journal of Biological Chemistry, 2003
    Co-Authors: Lisa Kuttner-kondo, Megan P Dybvig, Lynne M Mitchell, Nasima Muqim, M. Edward Medof, John P. Atkinson, Dennis E. Hourcade
    Abstract:

    Abstract The cleavage of C3 by the C3 convertases (C3bBb and C4b2a) determines whether complement activation proceeds. Dissociation (decay acceleration) of these central enzymes by the regulators decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP) controls their function. In a previous investigation, we obtained evidence implicating the α4/5 region of the type A domain of Bb (especially Tyr338) in decay acceleration of C3bBb and proposed this site as a potential interaction point with DAF and long homologous repeat A of CR1. Because portions of only two DAF complement control protein domains (CCPs), CCP2 and CCP3, are necessary to mediate its decay of the CP C3 convertase (as opposed to portions of at least three CCPs in all other cases, e.g. CCPs 1–3 of CR1), DAF/C4b2a provides the simplest structural model for this reaction. Therefore, we examined the importance of the C2 α4/5 site on decay acceleration of C4b2a. Functional C4b2a complexes made with the C2 Y327A mutant, the C2 homolog to factor B Y338A, were highly resistant to DAF, C4BP, and long homologous repeat A of CR1, whereas C2 substitutions in two nearby residues (N324A and L328A) resulted in partial resistance. Our new findings indicate that the α4/5 region of C2a is critical to decay acceleration mediated by DAF, C4BP, and CR1 and suggest that decay acceleration of C4b2a and C3bBb requires interaction of the convertase α4/5 region with a CCP2/CCP3 site of DAF or structurally homologous sites of CR1 and C4BP.

  • characterization of the active sites in decay accelerating factor
    Journal of Immunology, 2001
    Co-Authors: Lisa Kuttnerkondo, Lynne M Mitchell, Dennis E. Hourcade, Edward M Medof
    Abstract:

    Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2–4). Previous modeling on the nuclear magnetic resonance structure of CCP15–16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2–3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2–3 of DAF were oriented according to the crystal structure of CCP1–2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.

  • Decay accelerating activity of complement receptor type 1 (CD35). Two active sites are required for dissociating C5 convertases.
    The Journal of biological chemistry, 1999
    Co-Authors: Malgorzata Krych-goldberg, Richard E. Hauhart, Dennis E. Hourcade, V. B. Subramanian, B. M. Yurcisin, Dan L. Crimmins, John P. Atkinson
    Abstract:

    Abstract The goal of this study was to identify the site(s) in CR1 that mediate the dissociation of the C3 and C5 convertases. To that end, truncated derivatives of CR1 whose extracellular part is composed of 30 tandem repeating modules, termed complement control protein repeats (CCPs), were generated. Site 1 (CCPs 1–3) alone mediated the decay acceleration of the classical and alternative pathway C3 convertases. Site 2 (CCPs 8–10 or the nearly identical CCPs 15–17) had one-fifth the activity of site 1. In contrast, for the C5 convertase, site 1 had only 0.5% of the decay accelerating activity, while site 2 had no detectable activity. Efficient C5 decay accelerating activity was detected in recombinants that carried both site 1 and site 2. The activity was reduced if the intervening repeats between site 1 and site 2 were deleted. The results indicate that, for the C5 convertases, decay accelerating activity is mediated primarily by site 1. A properly spaced site 2 has an important auxiliary role, which may involve its C3b binding capacity. Moreover, using homologous substitution mutagenesis, residues important in site 1 for dissociating activity were identified. Based on these results, we generated proteins one-fourth the size of CR1 but with enhanced decay accelerating activity for the C3 convertases.

Suzan H. M. Rooijakkers - One of the best experts on this subject based on the ideXlab platform.

  • Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models.
    Frontiers in immunology, 2018
    Co-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. Gorham
    Abstract:

    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.

  • Molecular insights into the surface-specific arrangement of complement C5 convertase enzymes.
    BMC biology, 2015
    Co-Authors: Evelien T.m. Berends, Maartje Ruyken, Ronald D. Gorham, Piet C. Aerts, Carla J. C. De Haas, Jasper A. Soppe, Hatice Orhan, Piet Gros, Suzan H. M. Rooijakkers
    Abstract:

    Background Complement is a large protein network in plasma that is crucial for human immune defenses and a major cause of aberrant inflammatory reactions. The C5 convertase is a multi-molecular protease complex that catalyses the cleavage of native C5 into its biologically important products. So far, it has been difficult to study the exact molecular arrangement of C5 convertases, because their non-catalytic subunits (C3b) are covalently linked to biological surfaces through a reactive thioester. Through development of a highly purified model system for C5 convertases, we here aim to provide insights into the surface-specific nature of these important protease complexes.

  • Convertase Inhibitory Properties of Staphylococcal Extracellular Complement-binding Protein
    The Journal of biological chemistry, 2010
    Co-Authors: Ilse Jongerius, Brandon L. Garcia, Jos A. G. Van Strijp, Brian V. Geisbrecht, Suzan H. M. Rooijakkers
    Abstract:

    Abstract The human pathogen Staphylococcus aureus secretes several complement evasion molecules to combat the human immune response. Extracellular complement-binding protein (Ecb) binds to the C3d domain of C3 and thereby blocks C3 convertases of the alternative pathway and C5 convertases via all complement pathways. Inhibition of C5 convertases results in complete inhibition of C5a generation and subsequent neutrophil migration. Here, we show that binding of Ecb to the C3d domain of C3b is crucial for inhibition of C5 convertases. Ecb does not interfere with substrate binding to convertases but prevents formation of an active convertase enzyme.

  • Staphylococcal Complement Inhibitor Modulates Phagocyte Responses by Dimerization of Convertases
    The Journal of Immunology, 2009
    Co-Authors: Ilse Jongerius, Jos A. G. Van Strijp, Maartje Ruyken, Manon Puister, Suzan H. M. Rooijakkers
    Abstract:

    The human pathogen Staphylococcus aureus produces several complement-evasion molecules that enable the bacterium to withstand the host immune response. The human-specific staphylococcal complement inhibitor (SCIN) blocks the central C3 convertase enzymes that trigger critical complement functions, such as C3b deposition, phagocytosis, and C5a generation. SCIN effectively blocks the conversion of C3 by alternative pathway C3 convertases (C3bBb), but also induces dimerization of these enzymes. In this study, we show that formation of dimeric convertases by SCIN is important for S. aureus immune evasion because it modulates complement recognition by phagocytic receptors. Dimeric, but not monomeric, SCIN convertases showed an impaired binding to complement receptor 1 and the complement receptor of the Ig superfamily. The dimerization site of SCIN is essential for its strong antiphagocytic properties. These studies provide critical insights into the unique immune-evasion strategies used by S. aureus.

  • Staphylococcal complement evasion by various convertase-blocking molecules
    Journal of Experimental Medicine, 2007
    Co-Authors: Ilse Jongerius, Maartje Ruyken, Jos A. G. Van Strijp, Jörg Köhl, Manoj K. Pandey, Kok P. M. Van Kessel, Suzan H. M. Rooijakkers
    Abstract:

    To combat the human immune response, bacteria should be able to divert the effectiveness of the complement system. We identify four potent complement inhibitors in Staphylococcus aureus that are part of a new immune evasion cluster. Two are homologues of the C3 convertase modulator staphylococcal complement inhibitor (SCIN) and function in a similar way as SCIN. Extracellular fibrinogen-binding protein (Efb) and its homologue extracellular complement-binding protein (Ecb) are identified as potent complement evasion molecules, and their inhibitory mechanism was pinpointed to blocking C3b-containing convertases: the alternative pathway C3 convertase C3bBb and the C5 convertases C4b2aC3b and C3b2Bb. The potency of Efb and Ecb to block C5 convertase activity was demonstrated by their ability to block C5a generation and C5a-mediated neutrophil activation in vitro. Further, Ecb blocks C5a-dependent neutrophil recruitment into the peritoneal cavity in a mouse model of immune complex peritonitis. The strong antiinflammatory properties of these novel S. aureus –derived convertase inhibitors make these compounds interesting drug candidates for complement-mediated diseases.

M. Edward Medof - One of the best experts on this subject based on the ideXlab platform.

  • A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the complement C3 convertases.
    Journal of Biological Chemistry, 2003
    Co-Authors: Lisa Kuttner-kondo, Megan P Dybvig, Lynne M Mitchell, Nasima Muqim, M. Edward Medof, John P. Atkinson, Dennis E. Hourcade
    Abstract:

    Abstract The cleavage of C3 by the C3 convertases (C3bBb and C4b2a) determines whether complement activation proceeds. Dissociation (decay acceleration) of these central enzymes by the regulators decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP) controls their function. In a previous investigation, we obtained evidence implicating the α4/5 region of the type A domain of Bb (especially Tyr338) in decay acceleration of C3bBb and proposed this site as a potential interaction point with DAF and long homologous repeat A of CR1. Because portions of only two DAF complement control protein domains (CCPs), CCP2 and CCP3, are necessary to mediate its decay of the CP C3 convertase (as opposed to portions of at least three CCPs in all other cases, e.g. CCPs 1–3 of CR1), DAF/C4b2a provides the simplest structural model for this reaction. Therefore, we examined the importance of the C2 α4/5 site on decay acceleration of C4b2a. Functional C4b2a complexes made with the C2 Y327A mutant, the C2 homolog to factor B Y338A, were highly resistant to DAF, C4BP, and long homologous repeat A of CR1, whereas C2 substitutions in two nearby residues (N324A and L328A) resulted in partial resistance. Our new findings indicate that the α4/5 region of C2a is critical to decay acceleration mediated by DAF, C4BP, and CR1 and suggest that decay acceleration of C4b2a and C3bBb requires interaction of the convertase α4/5 region with a CCP2/CCP3 site of DAF or structurally homologous sites of CR1 and C4BP.

  • Structure/function studies of human decay-accelerating factor
    Immunology, 2000
    Co-Authors: William G. Brodbeck, Carolyn Mold, Lisa Kuttner-kondo, M. Edward Medof
    Abstract:

    The decay-accelerating factor (DAF) contains four complement control protein repeats (CCPs) with a single N-linked glycan positioned between CCPs 1 and 2. In previous studies we found that the classical pathway regulatory activity of DAF resides in CCPs 2 and 3 while its alternative pathway regulatory activity resides in CCPs 2, 3 and 4. Molecular modelling of the protein predicted that a positively charged surface area on CCPs 2 and 3 (including KKK125–127) and nearby exposed hydrophobic residues (L147F148) on CCP3 may function as ligand-binding sites. To assess the roles of the N-linked glycan and the above two sets of amino acids in the function of DAF, we mutated N61 to Q, KKK125–127 to TTT and L147F148 to SS. Following expression of the mutated cDNAs in Chinese hamster ovary cells, the glycosylphosphatidylinositol (GPI)-anchored mutant proteins were affinity purified and their functions were assessed. In initial assays, the proteins were incorporated into sheep and rabbit erythrocytes and the effects of the mutations on regulation of classical and alternative C3 convertase activity were quantified by measuring C3b deposition. Since DAF also functions on C5 convertases, comparative haemolytic assays of cells bearing each mutant protein were performed. Finally, to establish if spatial orientation between DAF and the convertases on the cell surface played any role in the observed effects, fluid-phase C3a generation assays were performed. All three assays gave equivalent results and showed that the N-linked glycan of DAF is not involved in its regulatory function; that L147F148 in a hydrophobic area of CCP3 is essential in both classical and alternative pathway C3 convertase regulation; and that KKK125–127 in the positively charged pocket between CCPs 2 and 3 is necessary for the regulatory activity of DAF on the alternative pathway C3 convertase but plays a lesser role in its activity on the classical pathway enzyme.

  • Decay acceleration of the complement alternative pathway C3 convertase.
    Immunopharmacology, 1999
    Co-Authors: Dennis E. Hourcade, L M Mitchell, M. Edward Medof
    Abstract:

    An ELISA-based method is described for analyzing the mechanism by which the decay of the alternative pathway C3 convertase is accelerated by C3 regulatory proteins. Using this assay, we show that human decay-accelerating factor (DAF) and factor H are active on mature convertase complexes (C3bBb) but not on their nascent precursor (C3bB). This finding has implications on the mechanisms of action of these two regulators. The complement convertases cleave the serum protein C3, and the resulting C3b activation fragments covalently attach to nearby targets where they direct antigen selection, immune clearance, and cell lysis. Several proteins, including the membrane protein DAF, and the serum protein factor H, limit convertase activity by promoting their irreversible dissociation. An understanding of the biochemical mechanisms providing for their activities would be helpful for the therapeutic control of the complement response.

  • localization of classical and alternative pathway regulatory activity within the decay accelerating factor
    Journal of Immunology, 1996
    Co-Authors: William G. Brodbeck, Carolyn Mold, M. Edward Medof, Jason L Sperry
    Abstract:

    Decay-accelerating factor (DAF) is a cell-associated C regulatory protein that protects host cells from autologous C attack. It functions intrinsically in host cell surface membranes to rapidly dissociate autologous classical and alternative pathway C3 convertases whenever these amplifying enzymes assemble on host cell surfaces. It is composed of four contiguous approximately 70 amino acid long regions termed short consensus repeats (SCRs) that share homology with similar units in other C3 convertase regulatory proteins. It is attached to the cell surface membrane by a glycoinositol phospholipid (GPI) anchor that is added posttranslationally. In this study, we prepared rGPI-anchored DAF proteins devoid of individual SCRs. We then incorporated the GPI-anchored products into sheep erythrocyte (Esh) hemolytic intermediates and examined their abilities to intrinsically regulate classical or alternative pathway activation. We found that classical pathway C3 convertase regulatory function resides within SCR-2 and SCR-3, while alternative pathway C3 convertase regulatory function resides within SCR-2, -3, and -4. Functional comparisons of the variant DAF proteins in fluid phase C3 activation assays established that the differences reflect domain-specific interactions rather than changes in the spatial arrangement of SCRs above the cell surface. In accordance with these findings, we found that variant DAF molecules containing SCR-1, -2, and -3, but not SCR-4, function to selectively inhibit classical pathway activation.