From the University of Wales College of Medicine,
Virus Receptor and Immune Evasion Group, Department of Medical
Biochemistry, Heath Park, Cardiff CF14 4XX, United Kingdom, the
¶ Division of Virology, Institute of Biomedical and Life Sciences,
University of Glasgow, Church Street, Glasgow G11 5JR, United Kingdom,
and the
Department of Clinical Chemistry, Lund University,
University Hospital Malmö, Malmö S-205 02 Sweden
Received for publication, November 13, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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Kaposi's sarcoma-associated herpesvirus (KSHV)
is closely associated with Kaposi's sarcoma and certain B-cell
lymphomas. The fourth open reading frame of the KSHV genome encodes a
protein (KSHV complement control protein (KCP, previously termed ORF4)) predicted to have complement-regulating activity. Here, we show that
soluble KCP strongly enhanced the decay of classical C3-convertase but
not the alternative pathway C3-convertase, when compared with the host
complement regulators: factor H, C4b-binding protein, and
decay-accelerating factor. The equilibrium affinity constant (KD) of KCP for C3b and C4b was determined by
surface plasmon resonance analysis to range between 0.47-10
µM and 0.025-6.1 µM, respectively,
depending on NaCl concentration and cation presence. Soluble and
cell-associated KCP acted as a cofactor for factor I (FI)-mediated
cleavage of both C4b and C3b and induced the cleavage products C4d and
iC3b, respectively. In the presence of KCP, FI further cleaved iC3b to
C3d, which has never been described before as complement receptor 1 only mediates the production of C3dg by FI. KCP would enhance virus
pathogenesis through evading complement attack, opsonization, and
anaphylaxis but may also aid in targeting KSHV to one of its host
reservoirs since C3d is a ligand for complement receptor 2 on
B-cells.
Kaposi's sarcoma-associated herpesvirus
(KSHV)1 is the likely
etiologic agent of Kaposi's sarcoma and is the most recently identified member of the human Herpesviridae family (1, 2). KSHV is also associated with the B-cell tumors body cavity-based primary effusion lymphomas and the plasma cell variant of multicentric Castleman's Disease (for reviews, see Refs. 3 and 4). KSHV belongs to
the rhadinovirus genus of the Gammaherpesvirinae subfamily, the prototype of which is Herpesvirus saimiri (HVS). The long unique
region of the KSHV genome comprises 140.5 kb and contains over 80 open
reading frames (ORFs) (5). Several of the ORFs encode host cell
homologues (e.g. viral cyclin D, viral interleukin-8 G protein-coupled receptor, and a bcl-2 homologue) with the potential to regulate the cell cycle and the immune response thereby contributing to the virulence and the pathogenesis of KSHV (5, 6).
The fourth open reading frame, ORF4, was initially speculated to have
complement regulatory abilities based on its homology to human
complement regulators decay-accelerating factor (DAF) and membrane
cofactor and to previously described virus-encoded complement
inhibitors (5). The KSHV ORF4 gene is predicted to encode a KCP protein
of 550 amino acids (data base reference SPTREMBL:O40912), and
the first 280 amino acids are predicted to encode four complement
control protein (CCP) domains. CCP domains are defined by a consensus
sequence of ~60 amino acids containing four invariant cysteine
residues that form disulfide links, which results in the CCP forming a
globular domain with a hydrophobic core enclosed by During our initial characterization of the KCP mRNA isolated from
primary effusion lymphoma cell lines, we found that expression of the
KCP cDNA in CHO cells significantly reduced C3b deposition on
antibody-sensitized CHO cells following incubation with whole human
serum; however, the mechanism of complement regulation was not
elucidated (9). Here we describe the mechanism of KCP complement regulation. We examined the ability of KCP to enhance the decay of
C3-convertases of both the alternative and classical pathways of
complement. These activities were compared with those of the host
complement regulators, factor H (FH), C4-binding protein (C4BP), and
DAF. The ability of KCP to act as a factor I (FI) cofactor for the
cleavage and inactivation of both C3b and C4b was also examined in the
soluble phase and on the surface of cells. Importantly, we show that
KCP has FI cofactor activity previously undescribed as it promotes FI
production of the C3d fragment.
Proteins--
Human C4BP (12), C1 (13), C2 (14), C4 (15), FH
(16), and FI (17) were purified from plasma as described previously. C3, C3b, C4b, factors B, D, and properdin were purchased from Advanced
Research Technologies. C1 and C2 were functionally pure (i.e. they were devoid of other complement factors), while
C4BP, FH, FI, C4, C3, C4b, and C3b were at least 95% pure as
determined by Coomassie staining of proteins separated by SDS-PAGE. All
proteins were stored in aliquots at Expression and Purification of KCP--
Soluble recombinant Fc
fusion proteins were constructed based on the naturally occurring
isoforms of KCP (described in Ref. 9) using the same strategy and
vectors as previously described to construct DAF-Fc (19). Cloning and
characterization of full-length KCP cDNA are described elsewhere
(9). Briefly, PCR primers were designed to truncate the recombinant KCP
isoforms immediately following the CCP domains (short:
5'-AGCTGCTGTATGGGTGTCTTCA-3'), prior to the Ser/Thr-rich region
(medium; 5'-TGGCTGGGATGTAGTTTTCTCAT-3'), or prior to the transmembrane
region (long: 5'-AATGGGAGGGAGTGTTGGTTCT-3'). All of these primers were
designed with a NotI restriction site to allow the in-frame
addition of the human IgG1-Fc C terminus when used for PCR in
combination with the common forward primer (F:
5'-GCGCTCTAGAGCTAGCATGGCCTTTTTAAGACAAAC-3'), followed by cloning into the previously described expression vector pDR2 C3b/C4b-degradation Assay--
Various concentrations
of putative or control cofactor proteins were mixed with 750 nM C3b (or 250 nM C4b), 60 nM FI,
and trace amounts of 125I-labeled C3b (or
125I-labeled C4b) in 50 µl of 50 mM Tris-HCl,
pH 7.4, supplemented with 150 mM NaCl. For examination of
cell-associated KCP cofactor activity the unlabelled C3b/C4b were not
included in the incubation. The samples were incubated for 1.5-4 h for
soluble proteins or overnight for cells at 37 °C, and the reaction
was terminated by the addition of SDS/PAGE sample buffer with reducing
agent. The samples were then incubated at 95 °C for 3 min before
protein separation on 10-15% gradient polyacrylamide gels and
visualization by autoradiography using Phosphorimaging analysis
(Molecular Dynamics).
Inhibition of Classical Pathway C3-convertase--
Sheep
erythrocytes were washed twice with DGVB2+ (2.5 mM veronal buffer, pH 7.3, 72 mM NaCl, 140 mM glucose, 0.1% gelatin, 1 mM
MgCl2, and 0.15 mM CaCl2),
suspended at a concentration of 109 cells/ml, and incubated
for 20 min at 37 °C with an equal volume of amboceptor (Roche
Molecular Biochemicals; diluted 1:3000 in DGVB2+) to make
EA cells. EAC1 cells were made by washing the EA cells twice
with ice-cold DGVB2+ and resuspending at 109
cells/ml; then C1 was added to 1010 cells drop-wise to a
final concentration of 5 µg/ml, and the mixture incubated with
agitation for 20 min at 30 °C. EAC1 cells were washed twice with
ice-cold buffer and incubated with agitation for 20 min at 30 °C
with 1 µg/ml C4. The resultant EAC14 cells were incubated in
DGVB2+ containing C2 (~5 µg/ml) for 5 min to allow
formation of C3-convertase. The cells were then placed on ice for 1 min, centrifuged, and resuspended in prewarmed DGVB2+. An
equal volume of these EAC142 cells was added to a range of prediluted
inhibitors and allowed to incubate at 30 °C with constant shaking
for 5 min. 100-ml aliquots of each sample were removed and added to 100 µl of guinea-pig serum-diluted 1:50 in 40 mM EDTA-GVB,
and the resultant lysis was determined following incubation at 37 °C
for 60 min. The amount of released hemoglobin was directly proportional
to the residual C3-convertase activity remaining on the EA142 cells and
was measured at 405 nm in the supernatant after pelleting the unlysed
cells by centrifugation. Soluble recombinant DAF-Fc or purified C4BP
were used as positive controls in these experiments, and all inhibitors
were compared with the amount of lysis observed in the absence of added inhibitors.
Inhibition of the Alternative Pathway C3-convertase--
Ten ml
of EAC14 (1.5 × 109/ml), prepared as described above,
were incubated with 0.25 µg/ml C2 and 50 µg/ml C3 for 30 min at 30 °C with agitation. The cells were washed once in 10 mM EDTA-GVB, resuspended in the same buffer, and incubated
for 2h at 37 °C to allow dissociation of C1 and C2 from the cells.
The resultant EAC43 were washed twice in EDTA-GVB, then twice in
Mg2+-EGTA buffer (2.5 mM veronal buffer,
pH 7.3, 70 mM NaCl, 140 mM glucose, 0.1%
gelatin, 7 mM MgCl2, and 10 mM
EGTA), and then resuspended at 2 × 109 cell/ml. EAC43
were then incubated with properdin (0.21 µg/ml), FD (21 ng/ml), FB
(10 ng/ml), and tested inhibitor or Mg2+-EGTA (final volume
150 µl). The mixtures were kept shaking at 30 °C and 100-µl
aliquots removed after 30 min and added to 100 µl of guinea pig serum
diluted 1:30 in GVB-EDTA. After 1 h at 37 °C, the samples were
centrifuged, and the amount of erythrocyte lysis was determined
spectrophotometrically as above. Soluble recombinant DAF-Fc and FH
served as positive controls in these experiments, and all inhibitors
were compared with the amount of lysis observed in the absence of added inhibitors.
N-terminal Protein Sequencing--
C3b was treated with FI in
the presence of the short form of KCP as described in the degradation
assay section. The proteins containing in total 7.5 µg of C3 were
separated by SDS/PAGE on 10-15% gradient gel and transferred by
semi-dry electroblotting onto polyvinylidene difluoride membrane. The
membrane was stained with Coomassie and the appropriate bands were
excised and subjected to N-terminal sequencing in an automated
sequenator (494 Protein Sequencer ProCise; Applied Biosystems).
Surface Plasmon Resonance (Biacore)--
The interaction between
C3b/C4b and KCPs was analyzed using surface plasmon resonance (Biacore
2000; Biacore). Each of four flow cells on a CM5 sensor chip were
activated with 20 µl of 0.2 M 1-ethyl-3-(3
dimethylaminopropyl) carbodiimide with 0.05 M
N-hydroxy-sulfosuccinimide at a flow rate of 5 µl/min, and
then the short form of recombinant KCP (0.01 mg/ml in 10 mM
sodium-acetate buffer, pH 4.5) was injected over flow cell 2 to reach
2000 resonance units. Medium and long forms of KCP were injected over
flow cells 3 and 4, respectively, to also reach 2000 resonance units.
In all cases unreacted groups were blocked with 20 µl of 1 M ethanolamine, pH 8.5. A negative control was prepared by
activating and subsequently blocking the surface of flow cell 1.
The association kinetics were studied for a range of C3b and C4b
concentrations (methylamine inactivation of C3 and C4 was used to
convert native C3 and C4 to C3b and C4b conformation, respectively
(20)) using the standard flow buffer (10 mM Hepes-KOH, pH
7.4 supplemented with 150 mM NaCl, 0.005% Tween 20). In
some experiments, 75 mM NaCl was used, and occasionally
standard flow buffer was supplemented with physiological concentrations
of calcium and zinc (2.5 mM CaCl2, 20 µM ZnCl2). Protein solutions were injected for 300 s to achieve saturation during the association phase at a
constant flow rate of 30 µl/min. The sample was injected first over
the negative control surface and then over immobilized KCP flow cells
and analyzed for a dissociation phase of 200 s at the same flow
rate. Signals were normalized by subtracting the nonspecific signal
measured by control flow cell 1. Between each different concentration
of C4b or C3b tested the flow cell surfaces were regenerated with a
30-µl injection of 2 M NaCl to remove bound ligands. All
sensograms were analyzed using the BiaEvaluation 3.0 software (Biacore)
to calculate equilibrium affinity constants.
Flow Cytometry--
Deposition of C3b and various fragments
thereof was measured by flow cytometry using polyclonal antibodies. C3b
was deposited in the absence of sensitizing antibody, on the surface of
sheep erythrocytes (EC3) after three rounds of incubation with C3, FB, FD, and nickel ions as previously described (16). The cells were then
incubated overnight at 37 °C with FI alone or in combination with
FH, the short form of KCP, or a source of CR1 (human erythrocyte ghosts) in DGVB2+. Aliquots of each of these treated EC3
cells were then incubated with a panel of rabbit polyclonal antibodies
directed against C3d (Dakopatts), C3c (two different antibodies from
Dakopatts), or whole C3 molecule (generated in house against whole
purified C3). EC3 cells were washed once in flow cytometry medium (FCM; phosphate-buffered saline, 15 mM EDTA, 30 mM
NaN3, 1% bovine serum albumin) and resuspended to a
concentration of 106 cells/ml. Primary antibodies (purified
IgG fractions) were diluted 1/100 in FCM and allowed to bind for 30 min
at 4 °C. Unbound antibody was removed by three washes in FCM prior
to incubation with secondary fluorescein isothiocyanate-conjugated goat
anti-rabbit immunoglobulin antibody (Dakopatts). After three final
washes in FCM the cells were analyzed on a flow cytometer (FACSCalibur,
BD Biosciences). All treated EC3 cells were compared with EC3 cells
that were incubated overnight in DGVB2+ only as a negative
control. All analyses were performed in triplicate, and the experiment
was performed twice.
Expression and Purification of Three Splice Variants of
KCP--
Three soluble recombinant Fc fusion forms of KCP were
constructed based on the naturally occurring transcript variants
observed in KSHV-infected cells (9). The shortest construct is composed of only the N-terminal CCP domains fused with the hinge and C terminus
of human IgG1 Fc region. The medium and longest recombinant fusion
proteins were truncated prior to the Ser/Thr-rich region or prior to
the transmembrane regions, respectively. Fig.
1 shows the molecular mass of all three
purified KCP-Fc forms compared with human DAF-Fc separated by SDS-PAGE
under reducing conditions. However, it should be noted that Fc fusion
proteins appear as IgG molecules with the Fab portion replaced by KCP,
and purified KCP-Fc proteins are therefore dimeric. Additionally,
because the longest KCP form retained both of the mRNA splice donor
sites and the single splice acceptor site, the resultant fusion protein existed as a mixture of full-length, medium, and short Fc fusion proteins.
KCP Accelerates Decay of the Classical Pathway
C3-convertase--
The decay of classical pathway C3-convertase was
measured in the presence of soluble KCP (Fig.
2). Putative or known inhibitory proteins
were added after the formation of the classical C3-convertase on the
surface of sheep erythrocytes, and decay acceleration was measured
after 5 min. All three forms of KCP-Fc accelerated the decay of
C3-convertase equivalently and were ~10-fold less active than the
positive control DAF-Fc (Fig. 2A). Monomeric KCP forms were
released from KCP-Fc with papain and found to be about 5-fold less
active than C4BP at enhancing the decay (Fig. 2B). Dimeric KCP-Fc was found to be equally as effective as monomeric KCP, indicating that the Fc fusion proteins did not cause steric hindrance of the active site, nor did the close proximity of two active sites
increase the decay-accelerating activity. No decay of classical pathway
C3-convertase was observed when dimeric CAR-Fc (Coxackie adenovirus
receptor) or monomeric CAR were tested, confirming that the
decay-accelerating activity measured was specific to KCP (Fig. 2).
KCP Shows Limited Inhibition of the Alternative Pathway
C3-convertase--
The decay of the alternative pathway C3-convertase
was also assessed by hemolytic assay. The alternative C3-convertase was assembled on the surface of sheep erythrocytes coincident with the
addition of putative and known inhibitors, and decay acceleration was
measured after 30 min. All three forms of KCP-Fc accelerated the decay
equivalently, but they were 1000-fold less efficient compared with
positive control DAF-Fc (Fig.
3A). Monomeric KCP proteins
were also tested after being released by papain cleavage, but the
decay-accelerating activity was not increased compared with dimeric Fc
fusion proteins, and activity was found to be 1000-fold lower than the
positive control FH (Fig. 3B). While KCP had poor
alternative pathway decay-accelerating activity, it was still much
greater than the negative control CAR-Fc and monomeric CAR, which had
no significant decay-accelerating activity (Fig. 3).
KCP Acts as FI Cofactor in Cleavage of C4b--
Cofactor activity
of KCP was assessed in a C4b-degradation assay in the presence of FI
(Fig. 4, left panel). FI
degradation of radiolabeled C4b was assessed in the presence or absence
of putative or control cofactors under physiological saline conditions. All three forms of KCP-Fc were found to catalyze FI cleavage of the C4b
KCP Displays Novel FI Cofactor Activity in Cleavage of
C3b--
All three forms of KCP-Fc were also tested for FI cofactor
cleavage of C3b (C3b degradation products depicted in Fig. 4,
right panel) as detailed above for C4b studies. FH and all
three forms of KCP-Fc, but not DAF-Fc, were capable of inducing FI
cleavage of the 104-kDa C3b
The ability of KCP to degrade cell-bound C3b was also measured by flow
cytometry analysis. Sheep erythrocytes were coated with C3b in the
absence of sensitizing antibody (EC3 cells) through three consecutive
rounds of incubation with C3, FB, FD, and properdin under alternative
pathway activating conditions. EC3 cells were then incubated overnight
with FI alone or in the presence of various cofactor proteins, and the
degree of C3b cleavage attained was measured by flow cytometry using a
panel of C3d- and C3c-specific antibodies (Table I). The same
amount of cell-surface C3d was detected irrespective of enzyme or
cofactor added. However, C3c epitopes
were almost entirely removed from cells incubated with FI in the
presence of CR1 and KCP but not FH. The amount of C3c remained the same
after overnight treatment with either FI alone or with buffer alone,
indicating that no significant nonspecific cleavage of C3b occurred
(Table I). The same trend in specific decreased fluorescence for cells
incubated with KCP or CR1 was observed with two separate polyclonal
antibodies against C3c as well as a polyclonal antibody against intact
C3.
Binding of C3b and C4b to KCP Proteins Assessed by Surface Plasmon
Resonance (Biacore)--
The affinity of binding between the KCP
proteins and C3b/C4b was studied with a Biacore 2000. KCP proteins were
immobilized on the surface of a CM5 chip using amino coupling. The
three different constructs occupied different flow cells of the chip.
C3b and C4b were then injected for sufficient time to reach saturation (Fig. 7A), and then
KD was calculated from a binding curve showing
response at equilibrium (Req) plotted against concentration (steady state affinity model; Fig. 7B). Binding of C3b/C4b
to all three forms of KCP was evaluated both in dimeric form as well as
papain-released monomeric form (Table
II). Buffers used contained either 75 or
150 mM NaCl, and 2.5 mM CaCl2, and
in some buffers 20 µM ZnCl2 was included
(Table II). The reason for testing these two ions is that our
preliminary homology-based three-dimensional model of CCP domains of
KCP predicts binding sites for both calcium and zinc. The results
obtained clearly show that all three KCP species are capable of binding
C3b and C4b. In most conditions tested the affinity of KCPs for C4b was
at least 10-fold higher than the affinity for C3b. Conversion of
dimeric Fc fusion protein to monomers was necessary to accurately
measure binding to C3b and C4b under physiological salt (150 mM) conditions, possibly due to the removal of steric
hindrances. The addition of physiological levels of zinc (20 µM) increased the binding of KCP to C4b 10-fold under all
conditions but was less efficient at increasing KCP binding to C3b.
Addition of calcium to buffers did not affect the affinity constants
(not shown).
Virus-encoded proteins with homologies to host complement
regulators have been previously described in two families:
Herpesviridae and Poxviridae. Vaccinia complement
control protein (VCP) was the first described and is the most
completely characterized (21-26). Other poxviruses that retain
homologues to VCP in their genome include variola virus (small pox),
cowpox, and monkeypox, the latter missing the fourth CCP domain and
perhaps not functional (27-30). With the exception of Herpes Simplex
virus type 1, which encodes glycoprotein C, a complement regulator that
does not have homology to any known inhibitor, all herpesviruses that
encode complement regulators belong to the rhadinovirus genus. Such
members include HVS, M Although the functional complement-regulating ability of all the above
listed proteins has been studied, there are no reports testing
decay-accelerating activity separately from FI cofactor activity as we
have detailed here for KSHV. We used a FI-deficient purified protein
system, and we showed that KCP has true decay-accelerating activity for
the classical pathway C3-convertase but is poor at enhancing the decay
of alternative pathway C3-convertase. The ability of KCP to decay
classical C3-convertase is 10-fold lower than for DAF-Fc but only
5-fold less than that observed for C4BP. However, it is important to
note that C4BP is a heptameter with several equivalent binding sites
and therefore activity of the single active site in KCP is comparable
to that of C4BP. Moreover, the local concentration of KCP in
vivo produced from KSHV-infected cells undergoing lytic
replication would be high at the site of complement activation
(i.e. the virally infected cell) and therefore would be far
more effective than soluble host complement regulators anyway. Since
KCP seems to be fairly specific for the regulation of the classical
pathway as its ability to decay alternative C3-convertase was not very
strong (1000-fold lower than DAF-Fc and FH), it suggests that the
alternative pathway may not play a significant role in clearance of the
virus in vivo. This is in contrast to the homologous viral
complement regulator encoded by M We determined that KCP has FI cofactor activity for the cleavage of C4b
and C3b. FI cofactor activity has not been tested for any of the other
rhadinoviruses, but VCP and the variola virus-encoded complement
regulator SPICE have both been shown to act as FI cofactors in the
cleavage of C4b and C3b (23, 24, 29). Interestingly, SPICE but not VCP
has been suggested to mediate FI cleavage to the C3dg fragment,
previously only described for CR1 (29, 34). However, SPICE and KCP
cofactor activities are distinctly different in that the final FI
degradation product for C3b in the presence of KCP was C3d not C3dg as
was reported for SPICE (Fig. 6 and Ref. 29), which we confirmed through
N-terminal sequence of the isolated degradation product. FI has never
been shown to cleave at this site before, even in the presence of CR1,
and the C3d fragment has only been reported as a cleavage product of
trypsin, plasmin, or elastase (35). The most common FI cleavage sites in C3b are the two Arg-Ser sequences that result in the release of C3f;
however, in the presence of CR1, FI cleaves iC3b at an Arg-Glu sequence
and under non-physiological conditions has been reported to cleave iC3b
at a Lys-Glu sequence (36). It is therefore surprising that under
physiological saline conditions we observed a FI cleavage of C3b at a
Glu-His sequence in the presence of KCP. The mechanism by which this
occurs is under further investigation. It is important to point out
that KCP is the only viral protein that has been shown to degrade
cell-associated C3b to C3d, proving that this activity is not an
artifact of soluble phase experiments (Table I).
We measured the binding affinity of KCP to C4b and C3b (Table II) using
Biacore. These data indicate that all three KCP species are capable of
binding C3b and C4b but that the affinity of KCP for C4b is at least
10-fold higher than for C3b. The addition of physiological levels of
zinc increased the binding of KCP to C4b 10-fold under all conditions.
The higher binding affinity of KCP for C4b, relative to C3b binding,
also supports our findings that KCP is better at accelerating the decay
of the classical, compared with the alternative, C3-convertase. In
comparison to host-encoded complement regulators, we found the that the
affinity of KCP for C4b was 7-fold less than that measured for C4BP
(KD = 0.5 µM in 150 mM
NaCl and 5 mM EDTA), and for C3b was 20-fold less than FH
(KD = 0.4 µM in 150 mM
NaCl and 5 mM EDTA) under the same conditions (not shown).
The ability of KCP to catalyze the cleavage of C3b to C3d by FI is
unique among the virus and host complement regulators. It will be
important to study the FI cofactor activity for the other rhadinovirus
members HVS and M The importance of complement evasion by rhadinoviruses has recently
been underscored in a thorough pathological examination of M
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-strands (7, 8).
While the CCP domain is not exclusive to complement control proteins,
all but one of the C3-convertase-regulating proteins identified to
date, either mammalian or viral, are composed of CCPs. Analyses of KCP
mRNA expressed in B-cells isolated from primary effusion lymphoma
patients revealed three major transcript variants (one unspliced
full-length form and two alternatively spliced variants), all
containing the N-terminal four CCP domains but with different amounts
of internal coding sequence removed by alternative splicing (9). All
three transcripts also encode the transmembrane region indicating that all forms are membrane-associated. The HVS-encoded complement regulator
is also alternatively spliced, but the transmembrane region is lost in
the single alternatively spliced form due to a frameshift (10). The
complement regulator homologue encoded by murine gammaherpesvirus 68 (M
HV68) has also been reported to be expressed as cell-bound and
soluble forms (11); however, no alternate splicing of the M
HV68
mRNA that could yield the latter form was observed by the authors,
and how the soluble protein arises has yet to be resolved.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C. Protein concentrations
were determined by measuring absorbance at 280 nm. C3b and C4b were labeled with 125I using the chloramine T method (18), and
the specific activity was determined to be 0.4-0.5 MBq/µg of
protein. Human erythrocyte ghosts were used as a source of CR1 and were
made through repeated washing (until all red color was removed) in
ice-cold phosphate buffer (7.5 mM phosphate, 1 mM EGTA, 0.2 M phenylmethylsulfonyl fluoride)
by resuspension and centrifugation at maximum speed in a benchtop
microfuge (4 °C) followed by discarding the supernatant.
EF1
(19). The
resultant plasmids were transfected into CHO cells (European culture
collection) using LipofectAMINE (Invitrogen). Cells were propagated in
RPMI 1640 cell medium containing 10% fetal calf serum, and stable
transfectants were created by propagating cells in medium containing
400 µg/ml of Hygromycin B (Invitrogen) for 2 weeks. Cell supernatants
were collected, and the Fc fusion proteins isolated and purified as
previously described using protein A-Sepharose (Amersham Biosciences)
(19). The resulting three KCP-Fc fusion proteins of different sizes
resemble IgG molecules with the Fab portion replaced with KCP proteins.
These proteins were released by incubation with papain-coupled agarose
(Sigma) at 37 °C for 1 h (the optimal ratio between amount of
the protein and papain was determined by titration). Papain-agarose was
then removed by centrifugation, and the free Fc and uncleaved Fc fusion
protein removed from the supernatant by binding to a protein
A-Sepharose column. The purity of all proteins was confirmed by silver
staining following separation by SDS-PAGE, and the protein
concentration was determined by measuring absorbance at 280 nm. Cell
surface expression of all three KCP fusion proteins was accomplished by exchanging in-frame the human Fc portion of the recombinant proteins with the cDNA encoding the signal for GPI anchoring of human DAF (see Ref. 9). Uniform expression was confirmed by flow cytometry using
monoclonal anti-KCP antibodies raised in-house.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of KCP proteins by SDS/PAGE.
Purified DAF-Fc and the three recombinant forms of KCP-Fc (~2
µg/well) were separated by SDS/PAGE on 7.5% gel under reducing
conditions and then visualized by silver staining. The molecular mass
markers are shown to the left.
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Fig. 2.
Acceleration of decay of classical
C3-convertase for Fc fusion proteins (A) or released
monomers (B). EAC142 were incubated in
DGVB2+ alone or with varying concentrations of inhibitors
for 5 min. The remaining active C3-convertases were measured by
hemolysis after addition of guinea pig serum diluted in EDTA-containing
buffer. Data are given as mean ± S.D. of quadruplicates.
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Fig. 3.
Acceleration of decay of alternative
C3-convertase for Fc fusion proteins (A) or released
monomers (B). Increasing concentrations of
inhibitors were added to EAC43 together with properdin, FD, and FB. The
remaining active C3-convertases were measured by measuring released
hemoglobin after the addition of guinea pig serum diluted in
EDTA-containing buffer. Data are given as mean ± S.D. of
quadruplicates.
-chain from 85 kDa to the major fragment of 46 kDa, consistent with
the cleavage of C4b to C4d (Fig. 5).
Cleavage of C4b was not seen if C4b was incubated with KCP alone or FI
alone, confirming co-operative activity. Further, no C4b cleavage was
observed following incubation with DAF-Fc and FI (not shown),
indicating the specificity for the KCP portion of the KCP-Fc molecule.
All three forms were roughly equivalent in their ability to act as
FI-cofactors, and 50% of the total C4b was converted to C4d by FI when
KCP was present at 1.85 µM, 2.7 µM, and
1.55 µM, respectively, for short, medium, and long KCP-Fc
forms compared with C4BP, which had a 50% total cofactor activity at
0.2 µM (not shown). However, this quantitative difference
does not take into account that C4BP has seven active sites compared
with the two active sites of KCP-Fc. Stably transfected CHO cells
expressing GPI-anchored KCP short, medium, or long forms or unmodified
full-length KCP, were also able to act as FI cofactors for the cleavage
of C4b to C4d, while control CHO cells were unable to support FI
activity (Fig. 5A). Hence, KCP was capable of FI cofactor
activity in both cell-bound and soluble forms.
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Fig. 4.
Diagrammatic depiction of the FI cleavage
sites for C3b and C4b in the presence of various cofactors.
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[in a new window]
Fig. 5.
C4b-degradation assay for CHO cells
expressing GPI-anchored monomeric forms of short (S),
medium (M), or long (L) and CHO cells
expressing naturally occurring transmembrane form (TM)
(A) or forms of KCP-Fc (S,
M, L) were examined
(B). Positive control source of CR1 were human
erythrocyte ghosts (A) or C4BP (B). Samples
containing 125I-labeled C4b, FI, and indicated cofactors
were incubated at 37 °C for 2 h. They were then mixed with a
sample buffer containing reducing agent and heated, and the proteins
were separated by 10-15% gradient SDS/PAGE. Radioactive C4b fragments
were then visualized by autoradiography.
-chain releasing a product of 64 kDa as
well as a 40-38-kDa doublet consistent with the formation of iC3b
(Fig. 6). Moreover, all three KCP-Fc
forms, but not FH, induced further cleavage of iC3b to a 30-kDa
degradation product that was smaller than the 35-kDa C3dg degradation
product produced by FI in the presence of CR1 (Fig. 6A).
N-terminal sequencing of the 30-kDa species yielded the sequence
HLIVTPSGCGEQNMIGMTPT. This sequence confirmed that the first 47 amino
acid residues of C3dg were missing, which is consistent with the
molecular mass and sequence of C3d. Fifty percent of the C3b was
degraded by FI to smaller fragments when KCP-Fc was present at
concentrations of 1.9 µM, 2.15 µM, and 1.25 µM, respectively, for short, medium, and long isoforms. This level of activity was calculated relative to FH, which catalyzed 50% FI cleavage of total C3b to the 64- and 40-38-kDa cleavage products (as FH did not induce production of C3dg) when present at a
concentration of 50 nM. No cleavage was observed in the
absence of FI (Fig. 6B), and incubation of C3b with KCP
alone did not result in any cleavage (not shown). All the
cell-associated forms of KCP, including the naturally occurring form,
were also found to be as effective as soluble KCP for the cleavage of
C3b to iC3b and C3dg, while control CHO cells did not act as FI
cofactors (Fig. 6C).
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Fig. 6.
C3b-degradation assay for short form of
KCP-Fc and CR1 in the presence and absence of FI under non reducing
conditions (A), soluble short
(S), medium (M), and long
(L) KCP-Fc forms under reducing conditions
(B), and CHO cells expressing naturally occurring
transmembrane (TM) or GPI-anchored S,
M, L forms of KCP
(C). Human erythrocyte ghosts were used as a
source of CR1 used as a positive control (A) or FH
(B), whereas untransfected CHO cells were included as a
negative control (C). Cofactors were incubated with
125I-labeled C3b and FI for 1.5 h at 37 °C.
Radioactive C3b fragments were visualized by autoradiography after
separation by 10-15% gradient SDS/PAGE.
Flow cytometry analysis of sheep erythrocyte-bound C3b degradation
products
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Fig. 7.
Surface plasmon resonance analysis.
A, purified C4met was injected at various concentrations
over a CM5-chip with short, medium, and long forms of KCP immobilized
in separate flow cells. Identical samples were injected over a control
flow cell, and nonspecific binding was subtracted. The amount of C4met
associated with KCP was measured in arbitrary resonance units.
B, the response obtained for each concentration of C4met at
equilibrium (Req) was plotted against concentration, which
allowed for estimation of KD.
Summary of surface plasmon resonance analysis (equilibrium dissociation
constants)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
HV68, and KSHV. The genomic organization has
been conserved between the different rhadinoviruses so that the gene encoding the complement regulator is designated as ORF4 (5, 10,
31-33).
HV68, which has been reported to
inhibit both alternative and classical pathways of whole mouse and
human serum (11).
HV68, as these have not yet been investigated.
There may be an evolutionary reason for KSHV or KSHV-infected cells to
promote the production of C3d: the receptor for C3d is CR2, expressed
predominantly by B-cells, which has been identified as one of the KSHV
viral reservoirs (37, 38). Thus KSHV may have evolved a mechanism that
not only allows evasion of clearance of virions and virus-infected
cells but may be using the complement system to target the virus to
primary sites of pathogenesis during natural infection as B-cell
infection by KSHV is a central aspect of primary effusion lymphoma and
Castleman's disease (reviewed in Ref. 3 and 4). However, KCP is only expressed in infected cells during lytic replication in B cells (9).
While this expression profile may facilitate KSHV transmission from
infected cells to uninfected B-cells, whether KCP facilitates infection
by extracellular virions through expression on the envelope is
currently under investigation.
HV68
infection compared with a recombinant virus lacking the complement
regulating homologue (39). Deletion of ORF4 from M
HV68 resulted in a
dramatic decrease in virulence during acute central nervous system
infection of immunocompetent mice and in mortality in persistently
infected immunocompromised mice, and this attenuation was not seen in
C3-deficient mice. Furthermore, C3 played a role in regulating latency
of the virus, but the M
HV68 ORF4 could not counteract this effect
(39). This also emphasizes the importance of KCP inhibition of the
classical pathway C3-convertase and degradation of cell-bound C3b and
C4b that we have detailed here. Extended to in vivo
infections, these complement-regulating activities would facilitate
KSHV replication and enhance virus pathogenesis through a combination
of blocking virion and/or infected cell opsonization, blocking
complement-mediated virolysis, and inhibiting anaphylaxis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Kevin Marchbank (University of Wales College of Medicine, Cardiff, UK) for guidance and discussion on C3dg studies, Profs. Patrick Moore and Yuan Chang (University of Pittsburgh Cancer Institute, Pittsburgh, PA) for valuable provision of resources and discussion, Dr. Bruno Villoutreix (University of Paris) for homology-based modeling, and Prof. B. Paul Morgan (University of Wales College of Medicine) and Prof. Björn Dahlbäck (Lund University, Malmö, Sweden) for continuous generous support.
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FOOTNOTES |
---|
* This study was supported by grants from the Swedish Research Council (to A. B.), Kock's Trust, Österlunds Trust, Crafoord Trust, Royal Physiographic Society in Lund, Groschinsky's Trust, King Gustav V's 80th Anniversary Foundation, Zoega's Trust, and University Hospital in Malmö. This study was also supported by the Wellcome Trust Foundation, Cancer Research UK (to O. B. S. and D. J. B.) Grant C7934 and the Association for International Cancer Research Grant 01-242 (to D. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Wellcome Trust Career Development Fellow.
** To whom correspondence should be addressed. Tel.: 46-40-33-82-33; Fax: 46-40-33-70-44; E-mail: Anna.Blom@klkemi.mas.lu.se.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211579200
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ABBREVIATIONS |
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The abbreviations used are: KSHV, Kaposi's sarcoma-associated virus; HVS, Herpesvirus saimiri; ORF, open reading frame; DAF, decay accelerating factor; CCP, complement control protein; CHO, Chinese hamster ovary; FB, factor B; FD, factor D; FH, factor H; FI, factor I; C4BP, C4-binding protein; GPI, glycosylphosphatidyl inositol; KCP, KSHV complement control protein; FCM, flow cytometry medium; VCP, vaccinia complement control protein; CAR, Coxackie adenovirus receptor; CR1, complement receptor 1.
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