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*
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland; and
Department of Clinical Chemistry, Lund University, University Hospital, The Wallenberg Laboratory, Malmö, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain complement control protein 4 domain.
Functional tests showed that tumor cell-bound C4bp retained its
cofactor activity for factor I-mediated inactivation of C4b, thus
increasing the control of classical complement pathway activation on
the surfaces of these cells. These results demonstrate a novel
mechanism of complement regulation on cell surfaces, particularly on
those of malignant ovarian tumor cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fluid-phase inhibitors of complement that control the C3/C5 convertases include factor H (fH), fH-like protein 1, and C4b-binding protein (C4bp). C4bp has a similar decay-accelerating activity as DAF on the C3/C5 convertase of the classical pathway, and it can act as a cofactor for fI-mediated cleavage of C4b (6, 7). fH and fH-like protein 1 have analogous functions in regulating the C3bBb enzyme complex (8, 9, 10). The primary role of these soluble complement regulators is to prevent excessive activation and depletion of complement in the fluid phase.
C4bp is a large plasma protein with an apparent molecular mass
of 540–590 kDa in SDS-PAGE. It seems to be synthesized only in liver
and is secreted to plasma to maintain an average concentration of 160
µg/ml. About half of C4bp in blood is bound to the anticoagulant
protein S. This binding blocks the anticoagulant activity of protein S,
but has no known effect on the complement-regulatory activity of C4bp
(11). As protein S associates with negatively charged
lipid membranes, it has been proposed that this interaction could
direct C4bp to sites of cell injury on cell surfaces and thus protect
tissues from excessive complement activation. C4bp is composed of six
or seven 70-kDa -chains and one or no 45-kDa 
-chain, depending on
the isoform (12). Electron microscopic analysis of C4bp
has revealed a spider-like structure, in which the -chains and the
-chain are linked together in the central core by their
carboxyl-terminal domains (13). The -chains consist of
eight domains called either short consensus repeats or complement
control protein (CCP) units. These domains are common to all regulators
of complement activation, whose genes are encoded in the long arm of
chromosome 1 (14, 15). In C4bp, the amino-terminal CCP1
and CCP2 in the -chain are involved in the binding of C4b and are
critical for the functional activity (16). As a cofactor
for fI, C4bp promotes cleavage of the C4b molecule to C4c and C4d, thus
preventing formation of the C4b2a enzyme complex. C4b is composed of
(93 kDa)-, (75 kDa)-, and 
-chains (32 kDa) linked together
with disulfide bonds. The fI-mediated cleavages occur in the -chain
between R937 and T938 and
between R1317 and N1318.
The fragment between these cleavage sites is C4d (45 kDa), and the
remaining part of the molecule is called C4c (6, 17, 18).
Previously, we have observed that malignant glioblastoma cells (the H2 cell line) can bind the soluble complement alternative pathway regulator fH directly to their cell surfaces and thus inhibit complement activation (19). This mechanism is actually so efficient that even if all the known membrane complement regulators were blocked with mAbs, little or no lysis of the H2 cells could be observed after treatment with complement-activating Abs and serum. This was due to the fact that fH bound to the surface of H2 cells restricted the activation cascade at the C3 level. The principal mechanism whereby the fluid-phase regulators can protect cells from complement attack involves first, the binding of the regulator to C3b or C4b that have become covalently bound to the cell surface, and second, the reactions leading to the inactivation of the C3 convertases (cofactor and decay-accelerating functions). However, the ability of a regulator to directly bind to a cell surface can significantly increase the efficacy of complement inhibition, as the cell may become coated with a fluid-phase regulator also in the absence of initial complement activation. It has been demonstrated previously that in addition to H2 cells, fH can bind directly to a variety of other cells (20, 21, 22). Surface polyanions such as sialic acid have been shown to be important in promoting fH binding to cells, particularly after an initial deposition of C3b. Surface-bound fH thus directs subsequent events toward inhibition of complement activation (23, 24, 25).
In the present study, we screened malignant cell lines to find out
whether also C4bp, a functional analogue of fH in the classical
pathway, could bind to cell surfaces. As a model, we present SK-OV-3
and Caov-3 ovarian adenocarcinoma cell lines. We observed that C4bp
binds to these cells via -chain domains that are not involved in the
complement-regulatory activity. Demonstration of C4bp binding to these
cells in a functionally active form establishes a novel mechanism
whereby human tumor cells can regulate complement activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human C4bp was purified from plasma, as described previously
(26). Protein S was removed from the molecule during
purification. Polymeric recombinant mutant C4bp molecules lacking
individual short consensus repeat domains were constructed and
expressed essentially as described previously (27). The
mutants, denoted as 
CCP1, CCP1–2, CCP2, CCP3, CCP4,
CCP5, and CCP6, lack either CCP1, both -1 and -2, -2, -3, -4, -5,
or -6, respectively. Human C4b and fI were obtained from Calbiochem (La
Jolla, CA). C4bp, the deletion mutants, and C4b were labeled with
125I by the Iodogen method (28). A
PD-10 gel filtration column (Amersham Pharmacia Biotech, Uppsala,
Sweden) was used for separation of free iodine from the labeled
protein, and Veronal-buffered saline (pH 7.4) was used as a buffer.
Purities of the labeled proteins were verified by SDS-PAGE and
autoradiography. Four mAbs against the -chain of C4bp were used.
mAbs 104 and 96 recognize CCP1–2, mAb 102 is against CCP1, and mAb 67
against CCP4. These Abs have similar affinities for the C4bp -chain
(29). The GB24 mouse mAb against MCP was a kind gift from
J. Atkinson (Washington University School of Medicine, St. Louis, MO).
A rabbit polyclonal Ab against the C4bp -chain was from Calbiochem,
and mouse polyclonal Ab against the C4bp -chain was a kind gift from
S. R. de Córdoba (Madrid, Spain).
Cell lines
The ovarian adenocarcinoma cell lines CaOV-3, SK-OV-3, and SW626, and a teratocarcinoma cell line PA-1 were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 in RPMI 1640 medium (Life Technologies Laboratories, Paisley, U.K.) supplemented with 10% heat-inactivated FCS, 10 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The cells were detached from the culture flasks with 0.02% (w/w) EDTA and washed before use.
Binding of radiolabeled C4bp to cells
Binding of 125I-labeled C4bp (125I-C4bp) to cells was analyzed by incubating 2 x 105 cells/sample with 125I-C4bp (3–100 ng) in a 50-µl solution of GVBS (0.1% gelatin, Veronal-buffered saline, pH 7.4) at 37°C for 60 min. Diluted, undiluted, or concentrated GVBS was used as the sample buffer, depending on the experiment. Control samples for the background signal did not contain any cells, but were treated otherwise as cell samples. After incubation, the cell suspensions were transferred on top of a 250-µl column of 20% sucrose in GVBS (with the same dilution as in the sample buffer) in narrow 0.4-ml test tubes. The tubes were centrifuged for 5 min at 5600 x g to separate the cells and free protein. The tubes were frozen and cut at the apex to separate the cell pellet and the sample supernatant. The radioactivities of both were measured. The amount of binding of 125I-C4bp to cells was calculated from the radioactivity in the cell pellet after subtracting the mean background signal. All binding experiments were performed in duplicate or triplicate.
Cofactor activity test for cell-bound C4bp
To analyze the functional activity of cell-bound C4bp, its cofactor activity for fI-mediated cleavage of 125I-labeled C4b (125I-C4b) was tested. For C4bp binding, 3 x 106 cells were incubated with C4bp at a 50 µg/ml concentration in 300 µl RPMI 1640 medium (no FCS added) for 60 min at 37°C. The cells were then washed twice with PBS (pH 7.4) and divided into separate microtubes to have 3 x 105 cells/tube. To block the cofactor activity of cell surface MCP and/or C4bp, GB24 and mAb104 Abs, respectively, were added to the appropriate samples at a concentration of 33 µg/ml and incubated for 15 min at 37°C. Factor I (17 µg/ml) and 125I-C4b (6 µg/ml) were added, and the samples were incubated in a final volume of 60 µl for 100 min at 37°C. A positive control sample for C4b degradation contained 10 µg/ml C4bp, 17 µg/ml fI, and 6 µg/ml 125I-C4b in RPMI 1640. The negative control had 17 µg/ml fI and 6 µg/ml 125I-C4b in RPMI 1640. After incubation, the cell samples were centrifuged and the supernatants were subjected to SDS-PAGE analysis under reducing conditions. In addition to the actual cofactor activity test, control samples for each cell line were prepared to monitor for possible cofactor activity of C4bp dissociated from the cells during the Ab and 125I-C4b/fI incubations. These samples were treated similarly as the other samples until the 125I-C4b/fI incubation step. However, at this point, no 125I-C4b or fI was added, but the samples were incubated in RPMI 1640 with 33 µg/ml GB24 for 100 min under similar conditions as the actual test samples. The tubes were then centrifuged, and the supernatants of the control samples were removed and mixed with 125I-C4b (6 µg/ml) and fI (17 µg/ml), and incubated for another 100 min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To find out whether tumor cells could bind C4bp from the fluid
phase, binding experiments with radiolabeled C4bp were performed. Of
the various cell lines tested, SK-OV-3 adenocarcinoma cells bound C4bp
the most, and PA-1 cells the least. Caov-3 and SW626 cells bound
intermediately (Fig. 1
). To further examine the nature of the binding, inhibition tests with
unlabeled C4bp and BSA were performed. 125I-C4bp
binding to cells was inhibited by 40% when unlabeled C4bp was added
within a concentration range of 80–120 µg/ml into a sample buffer
that contained 0.3 µg/ml 125I-C4bp. Under
similar conditions, 100–500 µg/ml BSA had no significant effect
(0–10% inhibition of binding). The binding of C4bp to cells was
sensitive to an increasing salt concentration in the sample buffer. The
binding of 125I-C4bp increased 6-fold in GVBS
containing 70 mM NaCl, and decreased by 50% at 400 mM, when compared
with the binding in GVBS containing a physiological concentration of
NaCl (150 mM).
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Inhibition tests with Abs were performed to tentatively map the
binding site on C4bp. Different concentrations of either polyclonal Abs
or mAbs against C4bp were added to the sample buffer with
125I-C4bp and incubated for 15 min at 20°C
before the cells were added. As shown in Fig. 2A, a rabbit polyclonal Ab against the C4bp -chain
inhibited 125I-C4bp binding to SK-OV-3 cells.
When a polyclonal Ab against C4bp -chain was used at similar
concentrations, no inhibition of binding could be observed. Instead,
the Ab increased C4bp binding to the cells, most likely because of
oligomerization of C4bp. To further examine the binding site, the
effect of various mAbs against C4bp -chain was analyzed. mAb 102
(anti-C4bp -chain CCP1), mAb 96 (anti--chain CCP1–2),
and mAb 67 (anti--chain CCP4) all inhibited C4bp binding, but
with mAb 67 the inhibition was clearly strongest, resulting in a 62%
decrease in binding at a 30 µg/ml concentration. These results
suggest that CCP4 of the C4bp -chain plays a role in the binding of
C4bp to SK-OV-3 cells (Fig. 2B).
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To further characterize the interaction between C4bp and SK-OV-3
cells, a set of 125I-C4bp -chain deletion
mutants was tested in the binding experiments (Fig. 3). These tests were performed with 90 mM NaCl in the sample buffer. The
binding of CCP1 and CCP6 to cells was as strong or even slightly
stronger than that of the wild-type C4bp (6% binding of offered
125I-C4bp). The mutants that had CCP2, -3, -5, or
both -1 and -2 removed bound intermediately (2–3% binding). The only
mutant that bound clearly more weakly than any other protein was
CCP4 (0.8% binding). This suggests that CCP4 is the primary cell
surface binding site on C4bp, which is in accordance with the mAb
inhibition tests.
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To analyze whether binding of C4bp had any functional
consequences, cofactor activity tests for fI-mediated C4b degradation
were performed with C4bp-preincubated and control cells. The cofactor
activity of cell surface MCP (CD46) was blocked with the GB24 mAb. In
these experiments, SK-OV-3, Caov-3, and SW626 cells induced fI-mediated
cleavage of 125I-C4b in the sample solution, also
in the presence of GB24. This was seen in SDS-PAGE analysis of the
sample supernatants as a characteristic fI-mediated cleavage pattern of
C4b (Fig. 4A). In the samples that contained both GB24 and mAb104 that
blocks the cofactor function of C4bp, no cleavage of
125I-C4b was observed. PA-1 cells, preincubated
with C4bp, had a weaker cofactor activity than the other cell lines,
and it was completely inhibited by GB24 alone. To find out whether the
cofactor activity seen in these cell samples was actually caused by
cell-bound C4bp and not by C4bp that had become dissociated from the
cells during the experiment, the supernatants of control cell samples
were tested for their cofactor activity. SK-OV-3 and Caov-3 cells did
not release any cofactor activity for C4b inactivation to the
supernatants, whereas the SW626 cell supernatants had some cofactor
activity (Fig. 4B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain without prior complement activation and
C4b deposition. The primary binding site on C4bp was found to be on
CCP4 of the -chain. The cofactor activity of cell-bound C4bp for C4b
inactivation was retained, suggesting that this phenomenon directs
complement-regulatory activity to the cell surfaces. Direct binding of
C4bp to cell surfaces has not been reported before, and this finding
suggests that in ovarian tumor tissue C4bp can bind to cell surface
structures and lead to an increased control of the classical pathway
activation. Of the cell lines tested, the binding of C4bp was strongest to the SK-OV-3 adenocarcinoma cells. These cells bound appreciable amounts of C4bp under physiological ionic strength conditions. The other ovarian adenocarcinoma cells, Caov-3 and SW626, also bound C4bp, whereas with the PA-1 teratocarcinoma cells, the binding was negligible. The binding of C4bp to cells was sensitive to salt concentration in the sample buffer, suggesting that the interaction is of an electrostatic rather than of hydrophobic nature. Relatively high concentrations of unlabeled C4bp were required in the inhibition tests to compete out 125I-C4bp binding to cells. This suggests a large number of binding sites on cell surface, and in contrast, a possibly relatively low affinity interaction. Initial ligand-blotting experiments with 125I-C4bp have not revealed any protein ligand on the cell membranes (data not shown). Together these results suggest that the cell surfaces have diffusely distributed acceptors, such as carbohydrates or proteoglycans, which carry a large reservoir of negative charge, a possible attractant of C4bp molecules.
The cell surface binding sites on C4bp were found to be on the
-chain, as the polyclonal -chain-specific Ab inhibited the
binding, but the anti--chain Ab did not. Both the inhibition
tests with mAbs and the binding tests with C4bp -chain deletion
mutants indicated that CCP4 of the -chain is the most important
domain for the binding. Removal of CCP1 or CCP6 did not interfere with
the C4bp binding, ruling out these domains as possible binding sites.
The small inhibiting effect of the mAb 102 (anti-CCP1) could have
been due to steric hindrance. The other deletion mutants bound
intermediately, making it difficult to judge the importance of CCP2,
-3, and -5 for the binding. It is possible that these neighboring
domains also contribute to the binding. In contrast, in deletion
mutants, one cannot exclude changes in interdomain structure that may
affect C4bp binding to cells. In conclusion, while multiple
interactions between the cell surface and C4bp -chain CCPs 2 to 5
may exist, the CCP4 domain has the strongest activity.
The functional activity of cell-bound C4bp as a complement regulator was analyzed by testing its cofactor activity for fI-mediated inactivation of C4b. Cofactor activity tests showed that after binding of purified C4bp, SK-OV-3, Caov-3, and SW626 cells could degrade C4b under conditions in which the cofactor activity of MCP was blocked with the GB24 Ab. As all the cofactor activity for C4b degradation could be blocked by adding both GB24 and the anti-C4bp mAb104 to the sample buffer, the other cofactor in these samples must have been C4bp, and no other cofactors were present. To confirm that cell-bound C4bp was actually the principal cofactor and not free C4bp released into the sample supernatant, additional experiments were performed. The results did not show any cofactor activity in the supernatants of other than the SW626 cells. This suggests that with SK-OV-3 and Caov-3 cells, the binding interaction was strong enough to keep most C4bp associated with cells during the experiment, and that cell-bound C4bp retained its cofactor activity. With SW626, it is difficult to determine whether the cell-bound C4bp or dissociated C4bp is actually responsible for the cofactor activity. The results of the cofactor tests are in agreement with the binding results, as the release of cofactor activity to the SW626 sample supernatant suggests a weaker affinity of C4bp binding. Also, the PA-1 cells did not recruit any C4bp-mediated cofactor activity at all, in accordance with the weak binding of C4bp.
From the physiological point of view, the most obvious implication of
C4bp binding to cells is an increased complement-regulatory activity on
the cell surfaces. As C4bp is a regulator of the classical pathway C3
convertase, which acts early in the cascade, it can effectively control
most of the complement activation events, including opsonization,
cytolysis, C5a-mediated chemotaxis, and release of the anaphylatoxins
C3a, C5a, and C4a. C4bp on the cell surface could also interfere with
the local blood coagulation homeostasis, as the C4bp -chain
inactivates the anticoagulant protein S by a high affinity interaction.
However, the physiological function of C4bp binding to protein S in the
coagulation cascade is not known. As the cell lines used in this study
are malignant, it is tempting to propose that C4bp binding is a
complement-regulatory mechanism of some cancer cells. The tumor cells
may use this property to increase their resistance of humoral and
phagocytic immune responses. Ovarian adenocarcinomas, in particular,
seem to use this mechanism, as the SK-OV-3 and Caov-3 cells were the
strongest binders of C4bp when compared with other tumor cell lines.
The SW626 cell line has been obtained from an ovarian tumor, but it has
been suggested that it may actually have originated as a metastasis
from a colon carcinoma (30). PA-1 is an ovarian
teratocarcinoma cell line originating from an entirely different type
of cells. In contrast, this phenomenon may occur with certain normal
human cells as well. In this case, it would be interesting to
characterize the natural receptors for C4bp and their distribution and
physiological functions in tissues.
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Acknowledgments |
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-chain were raised by Dr. Björn
Dahlbäck (Lund University, Malmö, Sweden). The possibility
to use these mAbs in the present study is greatly appreciated. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Seppo Meri, Haartman Institute, Department of Bacteriology and Immunology, University of Helsinki, P.O. Box 21, 00014 Helsinki, Finland. E-mail address: seppo.meri{at}helsinki.fi
3 Abbreviations used in this paper: DAF, decay-accelerating factor; C4bp, C4b-binding protein; CCP, complement control protein; fH, factor H; fI, factor I; GVBS, 0.1% gelatin, Veronal-buffered saline; 125I-C4b, 125I-labeled C4b; 125I-C4bp, 125I-labeled C4bp; MCP, membrane cofactor protein.
Received for publication March 13, 2001. Accepted for publication May 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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polypeptide chain of the human complement component C4b-binding protein. J. Exp. Med. 173:1073.-chain is crucial for C4b binding and factor I cofactor function. J. Biol. Chem. 274:19237.M2 (CD11b/CD18). J. Immunol. 160:4057.1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway. Proc. Natl. Acad. Sci. USA 75:1971.1H control and generation of restriction on neuraminidase-treated cells. Proc. Natl. Acad. Sci. USA 75:2416.,6-diphenyl glycoluril (Iodogen). Anal. Biochem. 117:136.[Medline]
-chain is crucial for C4b binding and factor I-cofactor function. Biochem. J. 323:469.
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