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C3-Convertase

The Experts below are selected from a list of 207 Experts worldwide ranked by ideXlab platform

M E Medof – 1st expert on this subject based on the ideXlab platform

  • Structure/function studies of human decay-accelerating factor
    Immunology, 2000
    Co-Authors: William G. Brodbeck, L. Kuttner-kondo, Carolyn Mold, M E 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: D E Hourcade, L M Mitchell, M E 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, M E Medof, Carolyn Mold, 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.

Dennis E Hourcade – 2nd expert on this subject based on the ideXlab platform

  • Structure-based mapping of DAF active site residues that accelerate the decay of C3 convertases
    Journal of Biological Chemistry, 2007
    Co-Authors: Lisa Kuttner-kondo, Lynne M Mitchell, Dennis E Hourcade, Nasima Muqim, Vernon E. Anderson, Dinesh C. Soares, Paul N. Barlow, M. Edward Medof

    Abstract:

    Abstract Focused complement activation on foreign targets depends on regulatory proteins that decay the bimolecular C3 convertases. Although this process is central to complement control, how the convertases engage and disassemble is not established. The second and third complement control protein (CCP) modules of the cell surface regulator, decay-accelerating factor (DAF, CD55), comprise the simplest structure mediating this activity. Positioning the functional effects of 31 substitution mutants of DAF CCP2 to -4 on partial structures was previously reported. In light of the high resolution crystal structure of the DAF four-CCP functional region, we now reexamine the effects of these and 40 additional mutations. Moreover, we map six monoclonal antibody epitopes and overlap their effects with those of the amino acid substitutions. The data indicate that the interaction of DAF with the convertases is mediated predominantly by two patches ∼13A apart, one centered around Arg69 and Arg96 on CCP2 and the other around Phe148 and Leu171 on CCP3. These patches on the same face of the adjacent modules bracket an intermodular linker of critical length (16A). Although the key DAF residues in these patches are present or there are conservative substitutions in all other C3 convertase regulators that mediate decay acceleration and/or provide factor I-cofactor activity, the linker region is highly conserved only in the former. Intra-CCP regions also differ. Linker region comparisons suggest that the active CCPs of the decay accelerators are extended, whereas those of the cofactors are tilted. Intra-CCP comparisons suggest that the two classes of regulators bind different regions on their respective ligands.

  • The Role of Properdin in the Assembly of the Alternative Pathway C3 Convertases of Complement
    Journal of Biological Chemistry, 2005
    Co-Authors: Dennis E Hourcade

    Abstract:

    Complement is a powerful host defense system that contributes to both innate and acquired immunity. There are three pathways of complement activation, the classical pathway, lectin pathway, and alternative pathway. Each generates a C3 convertase, a serine protease that cleaves the central complement protein, C3. Nearly all the biological consequences of complement are dependent on the resulting cleavage products. Properdin is a positive regulator of complement activation that stabilizes the alternative pathway convertases (C3bBb). Properdin is composed of multiple identical protein subunits, with each subunit carrying a separate ligand-binding site. Previous reports suggest that properdin function depends on multiple interactions between its subunits with its ligands. In this study I used surface plasmon resonance assays to examine properdin interactions with C3b and factor B. I demonstrated that properdin promotes the association of C3b with factor B and provides a focal point for the assembly of C3bBb on a surface. I also found that properdin binds to preformed alternative pathway C3 convertases. These findings support a model in which properdin, bound to a target surface via C3b, iC3b, or other ligands, can use its unoccupied C3b-binding sites as receptors for nascent C3b, bystander C3b, or pre-formed C3bB and C3bBb complexes. New C3bP and C3bBP intermediates can lead to in situ assembly of C3bBbP. The full stabilizing effect of properdin on C3bBb would be attained as properdin binds more than one ligand at a time, forming a lattice of properdin: ligand interactions bound to a surface scaffold.

  • 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, John P. Atkinson, Edward M Medof, Lynne M Mitchell, Megan P Dybvig, Nasima Muqim, 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.

Edward M Medof – 3rd expert 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 Kuttnerkondo, John P. Atkinson, Edward M Medof, Lynne M Mitchell, Megan P Dybvig, Nasima Muqim, 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.