Purification and characterization of a human membrane protein that activates the alternative complement pathway and allows the deposition of homologous complement C3.
A human myeloid cell subline, P39+, is found to be a target for human complement (C) via the alternative pathway and to allow the deposition of multiple C3 fragments on its membranes, though expressing the complement regulatory proteins decay-accelerating factor and membrane cofactor protein. The parent cell line, P39-, which is phenotypically similar to the P39+ subline, does not allow the deposition of homologous C3 fragments. In this study, we established a monoclonal antibody, M161 Ab, which reacted with P39+ but not P39- cells. This Ab recognized a 43-kD protein in P39+ cell lysate transblotted onto nitrocellulose. Using this Ab as a probe, we purified the 43-kD protein, namely, M161 antigen (Ag). M161 Ag had a basic isoelectric point (pI), 9.3-9.4 by chromatofocusing, and was precipitated as an insoluble material at the pI point. The purified M161 Ag was a single-chain protein and did not possess N- or O-linked carbohydrates. When the purified M161 Ag was transblotted onto nitrocellulose and incubated with Mg(2+)-EGTA serum, human C3 fragments were efficiently deposited on M161 Ag. The major species of the deposited C3 fragments was C3b. Furthermore, the C3 fragments bound to the M161 Ag were detached by 1 M hydroxylamine, suggesting that a covalent ester linkage sustains M161 Ag-C3b interaction. NH2-terminal amino acid analysis revealed that M161Ag is a novel membrane protein. Hence, it appeared that M161 Ag is a potent activator of human alternative complement pathway on human cells that activates homologous C3 and allows the deposition of C3b on itself. Thus, under some conditions, homeostasis of complement is maintained even on human cells, not only by the complement regulatory proteins, but also by membrane C3-activating molecules on which C3b is deposited.
Matsumoto
M
M
Department of Immunology, Center for Adult Diseases Osaka, Japan.
Yamashita
F
F
Iida
K
K
Tomita
M
M
Seya
T
T
D000911:Antibodies, Monoclonal; D002460:Cell Line; D003176:Complement C3 / Q000378:metabolism*; D003170:Complement Pathway, Alternative; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D008970:Molecular Weight; D010449:Peptide Mapping
D000911:Antibodies, Monoclonal; D003176:Complement C3; C091221:M161 antigen; D008565:Membrane Proteins