Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.
Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM-Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2-terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway.
Matsushita
M
M
Department of Biochemistry, Fukushima Medical College, Japan.
Fujita
T
T
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003173:Complement C1s / Q000302:isolation & purification / Q000378:metabolism*; D003175:Complement C2 / Q000378:metabolism; D003181:Complement C4 / Q000378:metabolism; D003171:Complement Pathway, Classical / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000097:blood* / Q000302:isolation & purification
D002352:Carrier Proteins; D003175:Complement C2; D003181:Complement C4; D037241:Mannose-Binding Lectins; D012697:Serine Endopeptidases; D003173:Complement C1s; D008358:Mannose