Characterization of the active sites in decay-accelerating factor.
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.
Kuttner-Kondo
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Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
Mitchell
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Hourcade
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Medof
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry; D001665:Binding Sites / Q000276:immunology; D018958:CD55 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050577:Complement C3-C5 Convertases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003169:Complement Inactivator Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003171:Complement Pathway, Classical / Q000235:genetics; D014644:Genetic Variation / Q000276:immunology; D006801:Humans; D008253:Macaca mulatta; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry
D015703:Antigens, CD; C497857:CD46 protein, human; D018958:CD55 Antigens; D003169:Complement Inactivator Proteins; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D014764:Viral Proteins; C074554:herpesvirus saimiri protein, Saimiriine herpesvirus 2; D050577:Complement C3-C5 Convertases