Purification and characterization of a human membrane protein that activates the alternative complement pathway and allows the deposition of homologous complement C3.

Abstract

SummaryA 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 Mg2+-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.

Mycoplasma fermentansLipoprotein M161Ag-Induced Cell Activation Is Mediated by Toll-Like Receptor 2: Role of N-Terminal Hydrophobic Portion in its Multiple Functions

“…After blocking with 10% skimmed milk for 1 h at 37°C, the sheets were washed three times with Dulbecco's PBS containing 0.05% Tween 20, and then incubated with either 25% EDTA-NHS or 25% Mg 2ϩ -EGTA-NHS for 20 min at 37°C. Deposited C3 fragments were detected with anti-human C3b/C3bi mAbs, HRP-labeled secondary Ab, and an ECL kit (Amersham Pharmacia Biotech AB) (42).…”

Complement Activation inMycoplasma fermentans-Induced Mycoplasma Clearance from Infected Cells: Probing of the Organism with Monoclonal Antibodies against M161Ag

“…MAbs were produced by the method of Köhler and Milstein (15). M161Ag, partially purified from P39(ϩ) cell lysates using mouse IgG-Sepharose, Q-Sepharose, and chromatofocusing columns as described previously (19), was mixed with TiterMax (CytRx Co., Norcross, Ga.) and injected subcutaneously into female BALB/c mice once every week for a total of three times. After 1 week, P39(ϩ) cells (8 ϫ 10 6 ) were administered intraperitoneally as a final booster.…”

“…MAbs against M161Ag (M161) and CD46 (M177) were produced and purified in our laboratory as described previously (19,34). Anti-human C3b MAb (C5G) and anti-CD55 MAb (IA10) were gifts from K. Iida (Takeda Chemical Industries) and T. Kinoshita (Osaka University), respectively (10,13).…”

“…Recently, we discovered a membrane-associated novel C3-activating protein in human tumor cell lines (18)(19)(20). Based on the genomic analysis, it was found to originate from Mycoplasma fermentans (21).…”

“…This protein, designated M161Ag, is a palmitoylated protein with a molecular mass of 43 kDa (21). It activates human complement via the alternative pathway, allowing the deposition of C3b and C3bi on human cells infected by M. fermentans and thus overcoming the functions of the complement regulatory proteins, CD46 and CD55, expressed on these cells (1,18,19). Interestingly, M161Ag efficiently promotes the production of interleukin 1␤ (IL-1␤), tumor necrosis factor alpha (TNF-␣), IL-6, IL-10, and IL-12 in human peripheral blood monocytes (21).…”