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*
Department of Pathology, Case Western Reserve University, Cleveland, OH 44106;
Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM 87131; and
Department of Medicine, Washington University, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A structural feature that is unique to DAF among human regulators of complement activation proteins is that it contains a posttranslationally added GPI membrane anchoring structure (6). Proteins that are anchored in this way can be extracted from cells and incorporated into the surface membranes of other cells so as to confer their functions on the target cells, a procedure that has been termed "protein engineering" or cell surface "painting" (reviewed in 7). Previously, we used this methodology to identify the CCPs of DAF that provide for its regulation of the classical and alternative pathway C3 convertases (8). In the present study, we constructed a recombinant MCP variant that contains a GPI anchor and employed this technology for the purpose of studying how DAF and MCP interrelate functionally on the cell surface.
Our aims were to study whether DAF and MCP work in a cooperative fashion in preventing C3b deposition, and, if so, how they influence each other’s function. For this purpose, we used rabbit erythrocytes (Erab) and studied alternative pathway activation using purified components. Erab are well-suited for such studies as it has been shown that rabbit DAF has no effect on the human alternative pathway convertase (9, 10), Erab do not express MCP (11), and the cells classically function as potent "activators" of the human alternative pathway. Using this system, we found that DAF and MCP synergize each other’s actions in preventing alternative pathway-mediated C3b deposition. We further found that the complexing of Bb, the factor B activation fragment that generates the C3 convertase, with cell-associated C3b inhibits the ability of MCP to catalyze the cleavage of C3b and, that by dissociating Bb from the alternative pathway C3 convertase, DAF restores MCP’s activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isotonic Veronal-buffered saline (DGVB2+) consisted of 2.5 mM Veronal, 73.7 mM NaCl, pH 7.5, 0.1% gelatin, 2.5% dextrose, and 1 mM MgCl2/0.15 mM CaCl2. Isoionic Veronal buffer (GVB2+) contained 145 mM NaCl and lacked dextrose. DGVB2+/Ni contained 1 mM NiCl2 in place of MgCl2.
Factor I was purchased from Quidel (San Diego, CA). C3, factor B, factor D, and factor H were prepared as previously described (8, 12). C3 was radiolabeled with 125I employing Iodogen (Pierce, Rockfield, IL) and was biotinylated with NHS-Biotin (Pierce) as previously described (13).
Preparation of GPI-anchored MCP protein
The cDNA encoding GPI-anchored MCP was prepared by cutting MCP-BC1 cDNA (GenBank accession no. X59405) cloned into the EcoR1 site of pSG5 vector at MCP’s membrane proximal Fok1 site and pSG5’s EcoR1 site. A PCR-amplified sequence encoding the 30 aa of DAF’s C-terminal signal peptide flanked by Fok1 and EcoR1 sites then was ligated into the Fok1- and EcoR1-digested product.
For the preparation of transfectants, 10 µg of DNA was preincubated for 30 min at 25°C with 15 µl of lipofectin reagent (Life Technologies, Gaithersburg, MD) suspended in 1 ml of Opti-MEM (Life Technologies). The mixture then was added to Chinese hamster ovary (CHO)K1 cells grown to 60% confluency on 100-mm culture plates and the plates incubated for 4 h at 37°C. The cells subsequently were incubated for 18 h in complete medium, after which methionine sulfoximine (25 µM) was added and selection carried out over the next 4–6 wk.
Surviving colonies were isolated with cloning cylinders and tested for expression of recombinant MCP by staining with anti-MCP mAb GB24 (10 µg/ml) followed by FITC-labeled goat anti-murine F(ab')2 (Sigma, St. Louis, MO) and analyzing the stained cells in a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Colonies expressing the highest MCP levels were further expanded. To verify GPI-anchoring of the recombinant MCP, MCP-positive cells were incubated for 30 min at 37°C with 10 U/ml of recombinant Bacillus thurigensis phosphatidylinositol-specific phospholipase C (PI-PLC) (kindly provided by Dr. T. Rosenberry, (Mayo Foundation, Jacksonville, FL) or with buffer alone. Cells then were compared for MCP surface expression by flow cytometry following staining as described above. CHO-K1 cells expressing native MCP transmembrane protein (with the BC oligosaccharide region and type 1 cytoplasmic tail (BC1)) were prepared as described (14).
For isolation of GPI-anchored MCP protein, transfectants were grown to 80–95% confluency on 100-mm plates. The medium was replaced with 1 ml PBS containing 60 mM N-octyl-ß-D-glucopyrannoside (Sigma) and 30 µl of protease inhibitor mix (15), and the plates were incubated for 15 min on ice. Solubilized extracts and cell residues then were scraped, incubated on ice for an additional 5 min, and centrifuged for 5 min at 1000 x g. The supernatant was collected, again centrifuged, and the resulting supernatant aliquoted and stored at -70°C. The concentration of MCP in the extract was determined by two-site immunoradiometric assay (IRMA) (see below).
For purification of GPI-anchored MCP protein, extracts were rotated overnight at 4°C with cyanogen bromide-Sepharose beads coupled to anti-MCP mAb TRA-2-10 (50 µl beads per 1 µg of protein). Beads were pelleted and washed twice with 1 ml of 0.1% CHAPS, 0.5 M NaCl, and 1.0 M Tris, pH 7.4. Beads then were eluted five times (3 min each time) with 0.1% CHAPS, 0.05 M triethylamine, 50 mM Tris, pH 11.2, and the eluate was immediately neutralized with a 1:20 volume of 0.1% CHAPS and glycine-saturated 0.5 M Tris, pH 6.0. The affinity-purified products were dialyzed against PBS, aliquoted, and stored at -70°C.
Isolation of DAF proteins
Human erythrocyte DAF was obtained as described (4). Recombinant native DAF protein, derived from CHO cells transfected with full-length DAF cDNA, was purified as previously described (13). For flow cytometric analyses of surface DAF expression and for DAF protein purification, in all cases anti-DAF mAb IA10 and IA10-Sepharose beads were used as previously described (6, 8).
Quantitation of MCP and DAF proteins
MCP concentrations were quantitated using a two-site IRMA similar to that developed for DAF (16). The wells of 96-well plates were coated at 4°C for 18 h with anti-MCP mAb GB24 (10 µg/ml in PBS containing 0.02% sodium azide). Wells then were washed three times with the same buffer containing 1% BSA maintaining the final wash solution on the plate for 1 h at 20°C to allow for blocking. Samples were added in triplicate in the same buffer containing 0.05% Tween 20 in sequential 3-fold dilutions. Plates then were incubated overnight at 4°C. Wells were washed again with the same buffer after which [125I]-labeled anti-MCP mAb TRA-2-10 (25 µl/well, 2 x 105 cpm) was added and the plates further incubated at room temperature for 2 h. Wells were washed and cut from the plates, and cpm were counted. The concentration of MCP of each sample was calculated by comparing the cpm of each well with that of MCP-BC1 standards of known concentrations (14).
DAF protein concentrations were quantitated by the DAF IRMA in the same fashion using anti-DAF mAb IA10 for capture, [125I]-labeled anti-DAF mAb IIH6 for detection of bound protein, and human erythrocyte DAF of known concentration as standards (16).
Western blot analyses
Proteins were electrophoresed on 7.5% SDS-polyacrylamide gel under nonreducing conditions, transferred onto 0.45-µm Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) for 1 h at 100 mV in a Transblot apparatus (Bio-Rad, Hercules, CA), and blocked for 18 h at 4°C with 5% BSA. Blocked membranes then were incubated with 10 µg/ml anti-MCP GB24 mAb for 2 h at 4°C followed by incubation with HRP-conjugated sheep anti-mouse mAb for 1 h at room temperature. After washing, the blots were developed using an electrochemoluminescence (ECL) Western blotting reagents kit (Amersham, Chicago, IL).
Cytotoxicity assays
CHO K-1 parental cells or CHO cell transfectants were grown to 70% confluency on 100-mm culture plates. Cells were removed with 4 ml Versene (Life Technologies) and washed three times with PBS. A total of 1 x 107 cells then were suspended in 1 ml PBS containing 500 µCi 51Cr, and the cell suspensions were incubated for 2 h at 37°C with occasional shaking. After washing three times with GVB-E, 5 x 105 labeled cells were incubated for 15 min at 4°C with rabbit anti-hamster lymphocyte serum (1:2) (Sigma) in 200 µl of GVB-E. Cells were washed three more times with GVB2+, resuspended to 105 cells/ml in GVB2+, and 100-µl aliquots of the cell suspension added to the wells of 96-well V-bottom plates. Then serial dilutions of normal human serum were added in 100-µl volumes in triplicate wells for each cell type. A volume of 100 µl of 1% Triton X-100 and buffer alone were included as controls for 100% release and for spontaneous release, respectively. The percent specific release was calculated from the formula: % specific release = [(measured release – spontaneous release)/(100% release – spontaneous release)].
C3 deposition and cleavage studies
For uptake and cleavage studies, C3b was deposited onto Erab using purified alternative pathway components. A convertase was formed by first incubating 600 µg/ml C3, 400 µg/ml B, and 10 µg/ml D for 5 min at room temperature. The resulting convertase complexes then were added to 8 x 108 Erab and 240 µg C3 in GVB/Ni2+ buffer, and the mixture was incubated for 20 min at 37°C. The resulting ErabC3b cells were washed, resuspended to 108 cells/ml in GVB-E, and stored on ice.
For C3 deposition studies, MCP and DAF were incorporated for 45 min at
30°C as previously described (8, 17, 18). Briefly,
107 ErabC3b were incubated with MCP-GPI or
detergent buffer control to generate cells with or without incorporated
MCP-GPI. Washed cells then were resuspended in 100 µl of
DGVB2+ containing 55 µg/ml B and 4 µg/ml D, and the
mixtures were incubated for 10 min at 37°C. The cells bearing
then were quickly pelleted, resuspended in
100 µl DGVB2+ containing 400 µg/ml C3, and incubated
for another 10 min at 37°C. The resulting Erab bearing
large amounts of C3b were incubated with DAF or detergent-containing
buffer alone for 45 min at 30°C to prepare cells with and without
incorporated DAF. The amplification procedure then was repeated a
second time in which 40 µg/ml [125I]-C3 was added
together with 400 µg/ml C3. In some tubes, factor I (3.4 µg/ml)
together with factors B and D were added to the cell suspension.
For fluid-phase C3b cleavage assays, C3b (Advanced Research Technologies, San Diego, CA) was biotinylated by incubating it with NHS-LC-biotin (1 mg/ml) for 30 min at room temperature, and the product was extensively dialyzed. The biotinylated C3b then was incubated with varying concentrations of MCP and factor I or buffer alone for 1 h at 37°C. Then 15 µl of each reaction mixture was mixed with reducing buffer, boiled, and loaded onto 10% SDS-PAGE gels. The electrophoresed proteins were transferred to nitrocellulose, the blots were incubated with streptavidin-conjugated HRP, and the C3b products revealed using the enhanced ECL Western blotting reagents kit.
For cell-bound C3 cleavage assays, ErabC3b cells were incubated with MCP-GPI or detergent/buffer control. Additional C3b then was deposited onto the ErabC3b and ErabC3b (MCP) as described above, except that biotinylated C3 (40 µg/ml) was included during the second (final) round of C3 amplification. The resulting cells were washed three times in DGVB2+ and incubated at 30°C for 45 min with DAF or detergent/buffer control. The cells then were washed and resuspended in 100 µl of DGVB2+ containing 3.4 µg/ml factor I. In some studies, factors B and D were added together with factor I in the same concentrations used for amplification. Cells were washed, pelleted, and lysed by the addition of 1.5 ml H2O for 15 min at 4°C. Following lysis, erythrocyte stroma were collected by centrifugation at 10,000 x g for 30 min at 4°C, the pellet resuspended in reducing buffer and the sample electrophoresed on 7.5% SDS-PAGE gels. Separated proteins were blotted onto polyvinylidene difluoride membranes, and, following blocking, the membranes were incubated for 1.5 h at room temperature with HRP-conjugated streptavidin and developed using the ECL kit.
C3b cleavage was quantified by using the Kodak digital science
electrophoresis documentation and analysis system 120 (Rochester, NY).
Bands corresponding to the bound 
', ß, and ' cleavage fragments
were analyzed for net intensity after correcting each gel lane (for
loading variations) by standardizing to the net intensity of the ß
fragment. Percent cleavage was calculated by dividing the net intensity
of the ' cleavage fragment by the sum of the net intensities of the
bound ' and ' cleavage product. Percent restoration of cleavage
was calculated by subtracting the net intensity of the cleavage
fragment in the presence of B and D from the net intensities of the
lanes containing MCP-GPI in the absence or presence of DAF. The
corrected net intensity of the ' cleavage product then was divided
by the corrected net intensity of the ' cleavage fragment in the
presence of MCP-GPI alone.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preliminary to conducting interactive studies of DAF and MCP,
recombinant MCP protein was examined to verify that it was
appropriately expressed with a GPI anchor and that it retained the full
function of native MCP. As shown in Fig. 1
, following incubation with PI-PLC, the
CHO cell transfectant expressing the recombinant protein exhibited
markedly reduced MCP expression as compared to K562 cells expressing
native MCP or either cell type treated with buffer alone.
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, when tested in cytotoxicity
assays, cells expressing equivalent copy numbers (see Fig. 1
) of the
recombinant GPI-anchored protein were protected from
complement-mediated lysis as efficiently as cells expressing the
transmembrane form of the protein.
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). When analyzed by Western
blotting, the extracted MCP-GPI displayed an apparent
Mr of 55 kDa (Fig. 3A). The
recombinant protein bound to C3b-coated plates and C3b-Sepharose beads
with an affinity comparable to that of the extracted native protein
MCP-BC1 (data not shown). Also, when mixed with factor I (Fig. 3B), it promoted cleavage of fluid-phase C3b with efficiency
equivalent to that of extracted native (BC1) transmembrane MCP
protein.
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As described previously, we used Erab and purified
alternative pathway components to determine whether and how MCP and DAF
cooperate in regulating C3 convertase activity. In initial studies, the
activity of each protein individually following its incorporation was
assessed. As shown in Fig. 4A
(lanes 5 and 6), when incorporated into
ErabC3b (after the first amplification step), DAF inhibited
the further deposition of C3b on the cell surface in a dose-dependent
manner. Similarly, when MCP-GPI was incorporated (prior to the
amplification step) (lanes 1–4), and the cells
resuspended in factor I, it also displayed a dose-dependent inhibitory
effect on C3b deposition. Interestingly, in the absence of factor I,
incorporated MCP-GPI had no effect on the amount of C3b deposition (see
Discussion).
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B,
MCP-GPI alone on the cell surface conferred minimal inhibition of C3b
deposition (5.8%), as did a range of limiting DAF concentrations
(0–7.3% inhibition). In contrast, inhibition increased dramatically
when both proteins were incorporated into the cells (up to 43.3%).
With the fixed limiting dose of MCP (
20 incorporated
molecules/cell), inhibition was again dependent on the concentration of
the DAF added.
Next, the converse experiment was performed in which cells were
incubated with a range of limiting MCP-GPI concentrations followed by a
fixed limiting dose of DAF (45 incorporated molecules/cell) (Fig. 4C). Once again, inhibition dramatically increased in the
presence of both proteins (up to 64.1%) as compared to each protein
separately (from 0 to 8.2%). As above, either in the presence or
absence of DAF, MCP-GPI inhibited C3b deposition in a dose-dependent
fashion.
DAF is required for MCP’s function in the presence of excess factors B and D
Previous studies of cell-associated MCP function have relied
principally on cytotoxicity assays of transfectants in whole serum
(2, 19, 20). As an initial approach for directly assaying
MCP’s function on the cell surface, biotinylated C3b was deposited
during the last amplification step (see Materials and
Methods) on ErabC3b that contained incorporated
MCP-GPI. As shown in Fig. 5A,
upon incubation of the resulting cells with factor I, incorporated
MCP-GPI cleaved the cell-bound C3b to iC3b (as evident by the
appearance of the characteristic 43-kDa ' fragment of iC3b. The
cleavage did not occur in the absence of factor I or MCP (not shown).
In pretitrations, a mAb against factor H exhibited the ability to
completely block H cofactor activity (data not shown). Control studies
in which this mAb was included in the reaction mixture excluded the
possibility that the cleavage was mediated by contaminating factor H.
Additionally, no cleavage was seen in the absence of the MCP-GPI. The
factor I-mediated cleavage of cell-bound C3b was dependent on the
amount of MCP-GPI incorporated into the cells (Fig. 5B).
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C, the addition of B and D and consequent
assembly of C3b into C3bBb markedly reduced the amount of
cell-bound C3b cleavage. Further studies demonstrated that this
inhibition of cleavage was dependent on the amount of B added together
with factors D and I (data not shown).
Finally, the effect of the presence of DAF on the B- and D-mediated
inhibition of MCP function was assessed. As shown in Fig. 5D, when DAF was incorporated into the cells, the inhibition
of MCP-GPI/factor I-mediated cleavage of cell-bound C3b to iC3b in the
presence of factors B and D was reversed. The restoration of cleavage
was dependent on the amount of DAF incubated with the cells (Fig. 5E). A similar extent of cleavage restoration was seen at
higher DAF levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It was previously reported by Lublin and Coyne (21) that interchanging the cell attachment mechanisms of DAF and MCP between GPI and conventional transmembrane anchors does not alter either protein’s regulatory efficiency in CHO cell cytotoxicity assays. These studies indicated that neither differences in surface mobility conferred by the two anchoring mechanisms nor localization of GPI-anchored proteins in nucleated cell "rafts" markedly affects either protein’s regulatory function. Here we confirmed that an MCP molecule that is attached to the cell surface with a GPI anchor functions with an efficiency essentially equivalent to that of the native MCP molecule. Studies with isolated MCP-GPI protein showed that the replacement of the transmembrane anchoring mechanism with a GPI anchor had no effect on the protein’s ability to bind to C3b-coated plates or C3b-Sepharose beads when compared to the extracted native protein. In addition, the change of the C-terminal anchor did not affect the protein’s ability to catalyze the factor I-mediated cleavage of fluid-phase C3b. Based on the above, it is unlikely that replacement of MCP’s anchor with a GPI in the present study had a significant influence on the results that were obtained. The use of erythrocytes rather than nucleated cells for the assays precluded any involvement of "rafts."
The initial functional system that we used was designed to study the
ability of DAF and MCP either individually or together to inhibit the
deposition of C3b on the cell surface. Recent studies by others
(20) found that MCP does not affect the initial deposition
of C3b resulting from the "tickover" of C3 but rather that it works
to prevent the amplification of C3b deposition by inhibiting the
alternative pathway C3 convertase C3bBb. Previous studies
have shown that the same is true for DAF (4) because DAF
works to accelerate the decay of already formed convertases (22, 23). We found that the presence of this small amount of C3b had
little to no effect on the amount of MCP-GPI that incorporated into the
cells. However, if MCP-GPI was incubated with cells after C3b
amplification, a much larger amount of MCP associated with the cells
compared to control cells (730 vs 230 molecules of MCP per cell)
presumably due to the binding of MCP-GPI to cell-bound C3b. In our
system, following the incorporation of MCP-GPI into Erab,
C3b deposition next was amplified on the cell surface for one or more
rounds and then DAF was incorporated into the cells (as earlier
incorporation did not allow sufficient C3b deposition). A subsequent
round of amplification in the presence of factor I constituted the
final readout step for analysis. Because DAF was not incorporated until
after amplification, MCP exhibited an apparent greater inhibitory
effect when the proteins were studied individually in the C3b uptake
assays (Fig. 4A).
Although DAF and MCP each have been studied extensively, no investigations have focused on whether the two proteins interact in providing optimal protection of self cells from autologous complement despite the fact that nearly all cells express both proteins. Using the above experimental system with incorporated MCP-GPI and DAF in Erab, we demonstrated that the two regulators work synergistically on the cell surface in preventing alternative pathway-mediated C3b deposition. The magnitude of the inhibition of C3b uptake in the presence of the two proteins compared to each protein individually was striking. At higher concentrations of the proteins, this cooperative inhibition reached 43–64% as compared to 0–8% for each protein when the proteins were incorporated individually at the same concentrations.
In previous studies using soluble MCP added to C3b-bearing cells (24) in which it was found that (in the absence of factor I) the uptake of the soluble (fluid-phase) molecule was enhanced by C3b, it was hypothesized that MCP by itself may function to stabilize alternative pathway C3 convertase complexes. However, the data presented here clearly show that when MCP is in the cell membrane and factor I is absent, C3b uptake is not affected. In contrast, in the presence of factor I, a large decrease in the amount of C3b deposited on the cell surface is observed.
To our knowledge, the data presented here provide the first direct demonstration in well-defined erythrocyte intermediates that when present on the same surface, MCP functions to catalyze the cleavage of cell-bound C3b. A recent study, using Abs directed against iC3b in conjunction with CHO cell transfectants incubated with factor I, obtained similar results regarding MCP cofactor activity (25). A number of earlier studies strongly suggested this using fluid-phase C3b cleavage assays and cytotoxicity protection assays (5, 14, 26). However, several factors complicated these previously established systems. In the course of our work using some of these other systems, we found that MCP-mediated cleavage of cell-bound C3b is not easily observed when using serum depleted of C5 or subsequent components. Because a low dilution of serum (1:2 to 1:8) must be used, high background cleavage is observed, presumably due to serum factor H, which masks the effect of MCP. To eliminate background cleavage, purified complement components must be used. Some previous systems (26) have used purified components in fluid-phase cleavage assays, but such methods do not directly relate to the cell surface, and low ionic strength buffer (20 mM NaCl) is needed. Although, as mentioned, a GPI-anchored form of MCP was previously generated, this protein was used in transfected cell lines with whole serum containing factor H. By isolating and purifying MCP-GPI, we were able to incorporate the protein into erythrocyte (i.e., Erab) intermediates that could be easily used for cell-associated complement studies using purified components.
In our second experimental system designed to assess MCP’s function
directly, i.e., cofactor activity, we added biotinylated C3 in the
final C3 amplification step because this probe labels the sequential
43/41-kDa fragment of the C3b ' chain that is generated by the
cleavage reaction. We found that in the presence of factor I, MCP
promoted the formation of this band. We further found that the ability
of incorporated MCP-GPI to catalyze this cleavage was inhibited when B
and D were added at the same time as factor I. Our finding in the C3b
uptake studies that MCP alone was able to inhibit C3b deposition (Fig. 4A) is not inconsistent with this because not all bound C3b
molecules are involved in the formation of C3 convertases and because
much higher concentrations of MCP were used in those studies. Moreover,
of possibly greater relevance, the studies were done in the absence of
properdin and the spontaneous decay rate of non-properdin-stabilized
C3bBb complexes is faster than the incubation times
used.
Incorporation of DAF into the ErabC3b intermediates in the presence of B and D showed that the presence of DAF on the cell surface restored MCP-catalyzed cleavage of cell-bound C3b. Presumably, this is due to its ability to accelerate the decay of Bb from C3b and render the bound C3b molecules susceptible to MCP-dependent factor I-mediated cleavage. In our experimental system, complete restoration of C3b cleavage was not seen at the concentrations of DAF we added. This could be due to the fact that insufficient amounts of DAF were added or to the inability of DAF to act on nonactivated convertases (C3bB) (23). Because B associates with C3b prior to D-mediated cleavage to Bb and Ba, it is likely that some C3b molecules are bound to factor B zymogen, preventing DAF-mediated dissociation.
As found in the C3b deposition studies, within the range of
concentrations studied the maximal effect on restoration of C3b
cleavage in the presence of B and D was observed at a ratio of DAF to
MCP of 3:1. However, because saturation was not achieved, the precise
ratio of the two proteins providing for optimal synergism remains to be
determined. A possible mechanism of this synergism is proposed in Fig. 6. Initially, C3b, deposited by natural
"tickover," is bound by factor B. The binding of both DAF
(23, 27) and MCP is prevented. B is then cleaved by
D to form the alternative pathway C3 convertase,
C3bBb, which then prevents MCP from associating with C3b
and factor I cleaving it to iC3b. When DAF is present on the target
cell surface, it acts to decay Bb from C3b, thereby rendering C3b
susceptible to MCP-supported factor I-mediated cleavage. Because C3b
complexed with B (C3bB) is not affected by DAF, such C3b molecules are
consequently protected from MCP and factor I-mediated cleavage.
However, this complex cannot amplify C3b deposition. By this
formulation, DAF would incompletely restore MCP/factor I activity
(i.e., act on C3bBb but not C3bB) and, at the same time,
synergistically inhibit further C3b deposition.
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A growing interest in xenotransplants (cross-species transplants) has brought attention to ways of circumventing the hyperacute graft rejection by the recipient, which is in large part complement mediated. In this regard, transgenic animals are being produced that express one or more complement regulatory proteins. With respect to DAF and MCP, our findings suggest that both proteins must be present together and that a DAF to MCP ratio of 2:1 to 3:1 confers a high level of synergism.
Finally, numerous studies (30, 31, 32, 33) have shown that many tumor cells express relatively high levels of both proteins. Our findings further suggest that the above-defined synergism would enhance their ability to evade endogenous humoral immune responses as well as responses to exogenously administered complement-activating antitumor Abs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. Edward Medof, Institute of Pathology, Room 301, Case Western Reserve University School of Medicine, 2085 Adelbert Road, Cleveland, OH 44106.
3 Abbreviations used in this paper: DAF, decay-accelerating factor; MCP, membrane cofactor protein; CCPs, complement control protein repeats; S/T, serine/threonine; RT, reverse transcription; Erab, rabbit erythrocyte; UTR, untranslated region; CHO, Chinese hamster ovary; PI-PLC, phosphatidylinositol-specific phospholipase C; IRMA, immunoradiometric assay; ECL, electrochemoluminescence.
Received for publication March 27, 2000. Accepted for publication July 18, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 S. Uhrinova, F. Lin, G. Ball, K. Bromek, D. Uhrin, M. E. Medof, and P. N. Barlow Solution structure of a functionally active fragment of decay-accelerating factor PNAS, April 15, 2003; 100(8): 4718 - 4723. [Abstract] [Full Text] [PDF]
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