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*
Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, and
Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and
National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune, India
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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 role for CR2 has been invoked in B cell activation, the generation of immunologic memory, Ig class switching, and B cell tolerance (1, 2, 3, 4, 6, 7, 8, 9, 10, 11). Binding of CR2 to its natural ligands (C3d or iC3b) attached to an Ag, immune complex, or pathogen as a consequence of complement activation, triggers the immunological responses of the receptor (8, 12). Coupling of an Ag to two or three copies of C3d has been shown to elicit a 1,000- or 10,000-fold enhanced T-dependent B cell response (13, 14). It is believed that this enhancement of the humoral response is the result of enhanced retention of Ag by follicular dendritic cells in germinal centers and of enhanced recruitment of the CR2/CD19/CD81 coreceptor into the B cell-Ag receptor complex (8, 12). In addition to iC3b and C3d, the EBV surface glycoprotein gp350/220 also binds to CR2, and mediates EBV infection of CR2-expressing cells (15, 16, 17). It is noteworthy that several cellular responses with similar characteristics of the C3d-CR2 interaction have been observed upon gp350/220 binding to CR2 (18, 19, 20). Specifically, gp350/220 has been shown to induce B cell proliferation (19), and when coupled to an Ag (monoclonal anti-human IgD Ab), it was found to enhance membrane Ig-mediated B cell stimulation in vitro (20). The similarities between the binding of gp350 and the C3 fragments to CR2 extend even further. The binding site for iC3b, C3d, and gp350 has been located in the N-terminal SCR 1 and 2 domains of the receptor (21, 22). Furthermore, the viral protein competes with C3d and iC3b for interaction with CR2.
Based on mAb competitions, peptide mapping, and site-directed mutagenesis studies, it has been determined that the CR2 binding site on gp350/220 lies within residues 1–470 of the viral glycoprotein. Specifically, a mAb that binds to this region (72 A1) inhibits gp350/220 binding to CR2 and viral entry into host cells (15, 23). Furthermore, a peptide that binds to CR2 has been identified within this fragment of the molecule (aa 21–30, EDPGFFNVEI) (15), and deletion of aa 28 and 29 in this region (VE) abolishes the binding of gp350/220 to CR2 (23).
Whether CR2 binds to C3 at a single or multiple sites is
controversial. Chemical fragmentation and peptide mapping have
identified a region on C3d, aa 1199–1210 (mature C3 numbering) to
which CR2 binds (24). A polymeric synthetic peptide
K1187-A1214
(KFLTTAKDKNRWEDPFKQLYNVEATSKYA) binds to CR2 on Raji cells
(24) and stimulates cell proliferation (18, 25, 26). Furthermore, when a peptide (equivalent to residues
1195–1210 of C3) was coupled to
anti-idiotype Ab, it induced a strong idiotype and Ag-specific
response in mice (27). This peptide is homologous in
sequence to the CR2-binding region on gp350/220. The observation that
synthetic peptides representing aa 295–306 (
-chain) and 744–755
(
-chain) showed similarity to the above peptide of C3 that binds to
CR2 (25) suggested that additional residues in C3 might
participate in the interaction with CR2. These peptides were identified
based on their sequence homology with the C3d residues 1201–1214, and
the binding was corroborated by the ability of peptides and C3c
immobilized to microspheres to bind to Raji cells through CR2
(25).
Kalli and coworkers (28) have shown that soluble C3d and iC3b inhibit soluble CR2 binding to iC3b-coated erythrocytes to a similar extent. The authors thus concluded that the interaction between CR2 and C3 is delimited to the C3d region. In view of this conclusion and in an effort to map the CR2 binding sites on C3, mutagenic scan studies have been performed in which erythrocytes coated with mutated iC3b were tested for their ability to bind to soluble CR2 and induce the rosetting of CR2-bearing Raji cells. In these studies, mutagenesis of residues 1199–1210 (EDPFKQLYNVEA) was shown to reduce the binding of iC3b to CR2 by only 20% (29). The recent availability of the x-ray crystallographic structure of C3d has indicated the presence of a negatively charged pocket on the concave surface of the molecule (30). The authors of this x-ray crystallographic study proposed this pocket as a candidate site for CR2 interaction. Replacement of two clusters of negatively charged C3d residues (E371008/E391010, and E1601131/D1631134/E166 1137, C3d numbering, mature C3 numbering) present on the opposite side of the acidic pocket led to severe inhibition of the interaction (31).
Because the results of the localization of the C3 binding sites for CR2 are apparently contradictory, it is not clear whether the interaction between CR2 and C3 is mediated only through the C3d region or other regions in C3 are involved in the interaction. In addition, it is not clear whether C3d interacts with CR2 through single or multiple sites and whether differences in experimental settings may affect these interactions. In the present study, we have analyzed the kinetics of the interaction of CR2 with C3d and iC3b in real time using surface plasmon resonance (SPR) and have asked whether these interactions are similar in nature. Because gp350 also serves as a ligand for CR2, we have analyzed its interaction with CR2 and have compared this interaction with those of CR2 with C3d and iC3b. Our results indicate that the binding of CR2 to viral gp350 follows a simple 1:1 binding model, whereas its binding to the C3 fragments is more complex. We observed direct binding of CR2 to a synthetic peptide with a sequence comprising C3 amino acids K1187-A1214, suggesting that this region in C3d is involved in its interaction with the receptor. We have also detected kinetic differences between the binding of iC3b and C3d to CR2. Finally, we observed direct binding of CR2 to the C3c fragment, which suggests that the regions of iC3b and C3d involved in their interaction with CR2, in addition to common contact sites, may involve regions that differ from each other.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we have expressed a truncated form of EBV
gp350/220 (gp350 (470t)), the ectodomain of CR2 (soluble CR2 containing
SCR 1–15), and soluble CR2 in fusion with biotinylation tag (CR2-BCCP)
using the baculovirus system. The cloning procedure for the expression
of soluble CR2 in the baculovirus system has been described elsewhere
(32). This clone was a gift from Michael Holers
(Department of Medicine and Immunology, University of Colorado Health
Science Center, Denver, CO). The EBV gp350 (470t) and CR2-BCCP
expression constructs were cloned as follows: to obtain gp350, the
gp350 (470t) cDNA was amplified by the PCR from the pGEM-gp350 clone
(33) using the primers 5'-TCGG GA TCCA ATG
GAG GCA GCC TTG ATT GTG TGT CAG TA-3' (forward) and 5'-CCT GCGGCCGC CTA
ATG GTG ATG GTG ATG GTG GGA T GAT ACA GTG GGG CCT GT-3' (reverse),
which added six C-terminal His codons and a stop codon. The His codons
were added to provide a binding site for the nickel-nitro acetic acid
(Ni-NTA)-agarose resin (Qiagen, Chatsworth, CA) used in the
purification of the expressed protein. The PCR-amplified product was
cloned into the pVT-Bac vector (34), which contains the
honeybee mellitin signal sequence. CR2-BCCP is composed of a CR2
molecule (SCR 1–15) fused at its C terminus to residues 70–156 of the
biotin carboxyl carrier protein (BCCP) from Escherichia coli
(35). The construction of this clone involved several
steps. First, total DNA was extracted from an overnight culture of
E. coli DH5 cells. The cDNA encoding the BCCP protein was
then amplified by PCR using the primers 5'-AAC TGA GCT CAT GGA AGC GCC
AGC AGC A-3' (forward) and 5'-ATT AGC TAG CCT ACT CGA TGA CGA CCA GCG
G-3' (reverse) and cloned into the pVT-Bac vector (pVT-Bac-BCCP). The
CR2 cDNA was then PCR-amplified from the pGEM-CR2 clone
(32) with primers 5'-AAT TCC CGG GAT GGG CGC CGC GGG CCT
G-3' and 5'-CAC GAG CTC TGA ACG GGA TCT GCA AAC-3' and cloned into the
pVT-Bac-BCCP construct, in-frame with the BCCP protein.
The resulting gp350 (470t) and CR2-BCCP constructs were recombined into baculovirus (Autographa californica nuclear polyhedrosis virus) by cotransfection with Baculogold DNA (PharMingen, San Diego, CA). Recombinant plaques were isolated and amplified, and the resulting culture supernatants were screened for protein expression by SDS-PAGE and Western blotting. Baculovirus recombinants expressing the protein of interest were subjected to two additional rounds of plaque purification. For production of larger quantities of protein, Spodoptera frugiperda Sf9 cells (Life Technologies, Gaithersburg, MD) grown in suspension cultures were infected at a multiplicity of infection of 4. Forty-eight to 72 h after infection the supernatants were cleared by centrifugation, concentrated, and then dialyzed against PBS.
The baculovirus-expressed recombinant proteins were purified by using Ni-NTA agarose or mAb HB5 affinity columns. The His-tagged protein gp350 (470t) was affinity-purified over an Ni-NTA-agar column. The bound protein was eluted with increasing concentrations of imidazole (0.01–0.25 M) in 0.02 M phosphate buffer (pH 7.5) with 0.5 M NaCl. The other two recombinant proteins (CR2 and CR2-BCCP) were affinity-purified over a Sepharose column covalently coupled to mAb HB5 specific for CR2 (American Type Culture Collection, Manassas, VA). Soluble CR2 was further purified over a protein G-Sepharose column to remove traces of HB5 Ab that might have leaked from the column. The eluates from all purifications were dialyzed against PBS and concentrated using the Amicon (Beverly, MA) ultrafiltration system.
The biotin holoenzyme synthetase protein, BirA, catalyzes the incorporation of biotin into BCCP. This protein was expressed in E. coli using a clone that was provided by Dr. D. Beckett (Department of Chemistry and Biochemistry, University of Maryland, College Park, MD) (36). The expressed protein was affinity-purified over an Ni-NTA-agar column by eluting with increasing concentrations of imidazole (0.01–0.25 M) in 0.02 M phosphate buffer (pH 7.5) with 0.5 M NaCl. The eluted protein was dialyzed overnight into a buffer solution containing 200 mM KCl with 50 mM Tris and 5% glycerol (pH 7.5).
Serum proteins
C3 (37) and factors I (38) and H (39) were purified from human serum as previously described. To obtain C3b or C3c and C3d, C3 was cleaved at 37°C with 1% w/w trypsin for 10 min, or 5% w/w trypsin for 1 h, respectively (40). The reactions were stopped by adding a 3-fold (w/w) excess of soybean trypsin inhibitor over the amount of trypsin used. To obtain iC3b, 500 µg of C3b was incubated with 150 µg of factor H and 64 µg of factor I for 4 h at 37°C. C3b, C3d, C3c, and iC3b were purified on a Mono Q column and gel filtration (Pharmacia, Piscataway, NJ) as previously described (41, 42).
Peptide synthesis, purification, and characterization
The peptide with the C3 sequence K1187-A1214 (Biotin-K F L T T A K D L N R W E D P G K Q L Y N V E A T S Y A) corresponding to C3 residues 1187–1214 (24) and a control peptide (Biotin-G S G S K P F P A P Q T P G R L Q P A P V I P S A P A A P) were synthesized in an Applied Biosystems (Foster City, CA) peptide synthesizer (model 431A) using F-moc amide resin (4-(2',4'-dimethoxyphenyl-F-moc-aminomethyl)-phenoxy resin). The side chain-protecting groups were Asp (otBu), Arg (Pmc), Thr (tBu), Gln (Trt), and Trp (Boc). Biotin (1 mmol) was dissolved in equal volumes of DMSO and N-methylpyrrolidone, placed in an amino acid cartridge, and activated according to Applied Biosystems User Bulletin no. 35 before carrying on the peptide synthesis. The peptides were cleaved from the resin by incubation for 3 h at 22°C with a solvent mixture containing 5% phenol, 5% thioanisole, 5% water, 2.5% ethanedithiol, and 82.5% trifluoroacetic acid (TFA). The reaction mixture was filtered through a fritted funnel, precipitated with cold ether, dissolved in 50% acetonitrile containing 0.1% TFA, and lyophilized. The crude peptides obtained after cleavage were dissolved in 10% acetonitrile containing 0.1% TFA and purified using a reversed-phase C-18 column (Waters, Milford, MA). The purity of all peptides was monitored by analytical chromatography on a reversed-phase C-18 column and by laser desorption mass spectrometry (43).
Protein sequencing and mass spectrometry
To obtain the NH2-terminal sequence of gp350 (470t), the protein was subjected to electrophoresis and electroblotted onto a Problot membrane (PerkinElmer-Applied Biosystems, Foster City, CA). After the bands were excised from the membrane, automated Edman degradation was performed with the PerkinElmer-Applied Biosystems model 473A Protein Sequencer equipped with on-line phenylthiohydantoin-amino acid analysis, using programmed chemistry cycles and HPLC operation programs provided by the manufacturer. For mass spectrometric analysis, gp350 (470t) and soluble CR2 were desalted using a matrix para-crystalline method previously described (43), and thereafter subjected to matrix-assisted laser desorption ionization-mass spectrometry analysis using a Micromass (Beverly, MA) TofSpec 2E time-of-flight mass spectrometer (1.0 m flight tube) equipped with a nitrogen laser (337 nm).
Site-specific biotinylation of CR2-BCCP and C3
To incorporate biotin at a single residue on CR2-BCCP and C3, the following strategies were used: 1) CR2-BCCP labeling was achieved in the presence of the BirA protein, which catalyzed the reaction; and 2) the labeling of C3 and its fragments was performed using the sulfhydryl-reactive EZ-Link polyethylene oxide (PEO)-maleimide-activated biotin (Pierce, Rockford, IL).
For labeling, CR2-BCCP was first dialyzed using 40 mM Tris-Cl, 5.5 mM MgCl2, and 100 mM KCl (pH 8) and then 31 µM of the protein was incubated with 365 µM of the BirA protein, 24 µg of d-biotin (Sigma, St. Louis, MO), and 20 mM ATP (Sigma) for 1 h at 37°C. Excess unincorporated biotin was removed from the reaction mixture by PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) gel filtration chromatography and extensive dialysis against PBS. The specificity of this reaction was determined by using soluble CR2 as a control.
Human C3 and C3 fragments were biotinylated as follows. The thioester bond of native C3 was hydrolyzed by incubating 7.8 mg of C3 (43 µM) with an equal volume of a 0.2 M solution of methylamine (pH 7.3) overnight at 37°C (44, 45, 46). The buffer was then exchanged into a solution consisting of 0.1 M NaH2PO4 and 5 mM EDTA (pH 6.3) by using a PD-10 column (Amersham Pharmacia Biotech). EZ-Link PEO-maleimide-activated biotin (Pierce) was dissolved in PBS at a concentration of 10 mM and then added to the C3 protein suspension at a 5:1 molar ratio. The reaction mixture was incubated for 30 min at room temperature, and then the sample was desalted and exchanged into PBS twice over a PD-10 column to remove the free biotin. The C3 fragments C3b, iC3b, and C3d were obtained by limited tryptic cleavage of the same stock of biotinylated C3 protein as described above. Monitoring of the biotinylation reactions, cleavages, and purifications was performed with SDS-PAGE and Western blotting.
Western blot assays
Proteins were separated on SDS-PAGE and electrotransferred to a nitrocellulose membrane. Nonspecific binding to the membrane was prevented by incubation in blocking buffer (PBS containing 10% milk). For CR2 detection, the membranes were incubated with a monoclonal anti-CR2 Ab (HB5, 2 µg/ml); gp350 (470t) was detected with a 1/5000 dilution of an anti-His tag (C-terminal) mAb (Invitrogen, Carlsbad, CA) or a 1/500 dilution of a polyclonal anti-EBV Ab (provided by Dr. Michael Holers). Bound mAbs were detected with a peroxidase-labeled goat-anti mouse IgG Ab (Bio-Rad, Richmond, CA), and bound polyclonal Abs were detected with a peroxidase-labeled mouse anti-rabbit Ab (Bio-Rad). The incorporation of biotin was verified by reactivity with peroxidase-labeled streptavidin (Life Technologies). All of these incubation steps were performed for 1 h at 22°C, and the membranes were washed with PBS containing Tween 20 (0.05%) between each incubation step. The proteins were detected using the ECL kit (Amersham Pharmacia Biotech).
SPR measurements
The kinetics of CR2 binding to its ligands C3d, iC3b, gp350, C31187–1214 peptide, and C3c were determined on the SPR-based biosensor BIAcore X (BIAcore, Piscataway, NJ). All the experiments were performed at 25°C in phosphate buffer containing 0.05% Tween 20 (pH 7.4), either at physiologic ionic strength (150 mM NaCl), or in 75 mM NaCl. To orient C3d, iC3b, CR2, and C31187–1214 peptide onto the chip and thus obtain a homogeneous ligand surface, the proteins were biotinylated at a specific residue as previously described and then immobilized on a streptavidin chip (Sensor Chip SA; BIAcore). C3c was immobilized on a CM5 chip by using amine-coupling chemistry (Sensor Chip SA). One hundred forty resonance units (RU) of CR2, 250 RU of C3d, 350 RU of iC3b, 2800 RU of C3c, or 500 RU of C31187–1214 peptide were immobilized on flow cell two (Fc2). Flow cell one (Fc1) was used as a blank control, except for the binding experiments involving the C31187–1214 peptide, in which a biotinylated control peptide was immobilized on Fc1. Binding was measured at 50 µl/min to avoid mass transport effects. At this flow the initial on-rate was maximum. Flow was allowed to occur for several seconds to establish a baseline, and then various concentrations of analyte were injected and the association was followed for 120 s. At that time, sample was replaced with running buffer, and the dissociation of the complex was monitored for 240 s. The sensor chip was regenerated with brief pulses of 0.2 M sodium carbonate (pH 9.5). The activity of the immobilized protein was monitored by observing the binding response of a single concentration of analyte throughout the experiment. Biosensor data for the control Fc1 were subtracted from those obtained for the immobilized proteins. Each binding experiment was performed at least twice, and the responses were averaged. Several buffer injections were also performed, averaged, and subtracted from Fc2 signal. BIAevaluation 3.0 (BIAcore) software was used to analyze the binding data, using global fitting. Linear analysis of the binding data was obtained as previously described (46, 47, 48) by plotting dRU/dt vs RU, where RU is the relative response of the biosensor at time t. The apparent equilibrium dissociation constant (KD) was calculated from the equation KD = koff/kon.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze the interaction of CR2 with its ligands, we expressed
both the receptor and its ligand gp350 (470t) using the baculovirus
system. Because previous studies had shown that EBV gp350/220, when
truncated at residue 470, can inhibit EBV binding to CR2
(23), we chose to express this gp350 (470t) construct for
use in the interaction analysis. A C-terminal His tag was added to this
protein to aid in its purification. gp350 (470t) does not have a
well-defined signal peptide. To verify whether its putative signal
peptide had been processed, we sequenced its N terminus. Edman
degradation yielded the sequence DPMEAALIV, with DP being the amino
acids added in the cloning process and MEAALIV the putative signal
peptide. Thus, its putative signal peptide had not undergone cleavage
upon secretion of the protein. The molecular size of gp350 (470t) as
predicted from the amino acid sequence of the protein was 50 kDa.
However, analysis by SDS-PAGE and Western blot revealed a band of
65–70 kDa (Fig. 1
). Mass spectrometric
analysis showed a single homogeneous species and indicated that the
gp350 (470t) had a total mass of 72 kDa (Fig. 1A). Because
this protein contains 18 potential N-glycosylation sites, we conclude
that glycosylation contributed 20 kDa to the total size of the
molecule. This conclusion is consistent with previous studies of the
gp350 protein, which have indicated that it is heavily glycosylated
(23, 49, 50).
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B) which does not differ substantially from the size
deduced from its amino acid composition (111 kDa). It is possible that
associated carbohydrates added 5 kDa of mass because CR2 contains
eleven putative sites of N-glycosylation. The recombinant CR2-BCCP was
analyzed by SDS-PAGE and Western blotting. Purified CR2-BCCP migrated
as a single band, as visualized by its reactivity with mAb HB5
(Fig. 1). Generation and characterization of site-specific biotinylated proteins
To measure the affinity and binding kinetics of the interaction
between CR2 and its ligands, we used SPR technology. In such studies,
immobilization of the ligand is generally achieved by the use of
amine-coupling chemistry. However, this approach produces heterogeneous
surface ligands and precludes the measurement of
homogeneous binding kinetics. To overcome this problem, we designed an
enzymatic and a chemical strategy to incorporate biotin at a specific
residue on CR2 and C3, respectively (Fig. 2). This strategy provided a means of
orienting these proteins on a streptavidin sensor chip surface in a way
mimicking their physiological orientation.
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, only
CR2-BCCP reacted with peroxidase-labeled streptavidin, indicating that
only the BCCP-tagged protein had incorporated biotin.
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). Biotin
incorporation was detected mainly in the fragments containing
Cys988 (i.e., the 68-kDa fragment of iC3b
and C3d). Kinetic analysis of the interaction of CR2 with iC3b, C3d, and gp350
In the present study, we have examined the interaction of CR2 with its ligands by SPR technology using a BIAcore X biosensor, and we addressed the following: 1) whether C3d and iC3b have different affinities for soluble and surface-bound CR2; and 2) whether the affinities of CR2 for fluid-phase and surface-attached C3d/iC3b (e.g., when C3d/iC3b are attached to complement-activating particles) can vary as a result of differential exposure of interacting residues. Thus we tested these possibilities by measuring the binding of iC3b and gp350 to CR2 immobilized on the sensor chip as well as the interaction of soluble CR2 with iC3b and C3d immobilized on the sensor chip.
When we examined the binding of gp350 (470t) to CR2 immobilized on the
sensor chip, we found that the binding reaction was dose-dependent and
saturable. Analysis of the binding data by global fitting
analysis (BIAevaluation software 3.0) showed a close fit to 1:1
Langmuir binding model (A + B 
AB; Fig. 4A). A random distribution of
residuals and a 
2 value for this interaction
of 1.8 indicated that this model describes well the experimental
data.
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2 values were <2, the values obtained for the
maximum response (Rmax) were lower
than the predicted values, and even lower than the observed response
values. Eliminating the refractive index (Ri)
parameter that ascribes a bulk to the interaction resulted in a
2 value >120. Linear transformation of the
binding data showed nonlinear plots (Fig. 4, B–D),
indicating that the binding follows complex models.
Data from our laboratory and others have indicated that two regions of
CR2 (52, 53) and more than one region of C3d/iC3b
(24, 25, 29, 31, 54) may be involved in C3d/iC3b-CR2
interactions. Thus, it is possible that two regions of C3d/iC3b may
interact with two regions of CR2. To test this possibility, we tried to
fit our data to a bivalent analyte model: AA + B AAB; AAB + B
AABB (Fig. 4). These data include binding of CR2 to immobilized iC3b
and C3d and binding of iC3b to immobilized CR2. The latter experiment
was performed in the presence of 75 mM NaCl in the binding buffer,
which allowed us to observe better binding. For these analyses, the
residual plot for the fitted data and the 2
values ranged from 1 to 3 and from 1.5 to 2.1, respectively, and the
residuals were randomly distributed around the fit. Besides, the
Rmax values were close to the
predicted maximum response. Other models, such as a conformational
change model, did not describe the data obtained. A two-step binding
model describes well the binding data where two or more binding steps
are affecting the binding equilibrium between CR2 and C3d or between
CR2 and iC3b. The overlaid data, with the result of the global fitting
analysis for a bivalent model for iC3b binding to immobilized CR2, are
shown in Fig. 4B, and those for the binding of soluble CR2
to immobilized C3d or iC3b are shown in Fig. 4, C and
D, respectively. The kinetic constants for the interaction
of CR2 with its ligands that we obtained from the global fitting
analysis are summarized in Table I.
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In light of our observation that C3d interacts with CR2 in a
complex manner and the controversy regarding the involvement of the C3
region comprising aa 1187–1214 in CR2 binding, we analyzed the binding
of CR2 to a synthetic peptide carrying this amino acid sequence
(24). The C31187–1214 peptide
(24) was biotinylated on its N terminus and immobilized on
the Fc2 of a streptavidin sensor chip. As control we used an
irrelevant biotinylated peptide, which was immobilized on Fc1. The
experiment was performed in the presence of 75 mM NaCl in the running
buffer to allow for better detection. Binding of CR2 to the
C31187–1214 peptide was dose-dependent (Fig. 5). Furthermore, the interaction was
inhibited by soluble C3d, but not by a control protein (BSA) (data not
shown). Although global fitting of the data was not possible, these
data indicate that the C3 region, 1187–1214, binds to CR2.
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Recent availability of the x-ray structure of human C3d (30) and mutation analysis of charged residues of C3d (31) have highlighted the importance of ionic interactions in C3d-CR2 interactions. To understand the influence of ionic contacts on the kinetics of CR2-ligand interactions, we have studied these interactions at various salt concentrations using SPR technology.
In the present study we measured the binding of CR2 to its ligands at
physiologic (150 mM) and half-physiologic (75 mM) ionic strength
buffers. In all the interactions analyzed in this study, decreasing the
amount of NaCl in the binding buffer lowered by 
4- or 5-fold the
quantity of C3 fragments or gp350, respectively, that was required to
obtain a similar response in PBS (data not shown). Therefore, a
decrease in the salt concentration apparently facilitates the binding
of CR2 to its ligands, by increasing the affinity of these
interactions.
We then attempted to fit these binding data to a kinetic model. The
binding of gp350 to immobilized CR2 and that of CR2 to immobilized C3d
and iC3b fit well with a 1:1 and a bivalent interaction model,
respectively (data not shown). Therefore, these data suggest that the
mechanism of binding of CR2 to C3d, iC3b, and gp350 is not affected by
the ionic strength of the buffer. The
KD values obtained from these analyses
are presented in Table I.
To better understand the significance of these values for the binding
of CR2 to C3d, we have overlaid the sensograms for the interaction of
soluble CR2 with C3d in the presence of either 75 or 150 mM NaCl. As
shown in Fig. 6, the total binding
of 475 nM CR2 was increased by lowering the salt concentration. We then
split the total binding curves into the components of a bivalent model.
This strategy allowed us to analyze the parameter
T50, which is the time in the
association phase at which the encounter and final complexes are
equimolar, as defined by Lipschultz et al. (55).
T50 at 75 mM NaCl was 40 s
whereas at 150 mM NaCl T50 was greater
than the contact time. Thus, it appears that the initial
charge-dependent binding is critical in the formation of stable CR2-C3d
or CR2-iC3b complexes.
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The existence of kinetic differences between the binding of C3d
and iC3b to CR2 led us to test the hypothesis that additional binding
sites for CR2 may exist on C3c. C3c was immobilized on a sensor chip by
amine-coupling chemistry, and soluble CR2 was allowed to interact with
it. The experiment was performed under half ionic strength (75 mM NaCl)
buffer conditions to allow better detection. Binding of CR2 to C3c was
dose-dependent (Fig. 7A) and was inhibited by the presence
of low molar excess of soluble C3c but not C3d (data not shown). The
presence of traces of C3d on the C3c sample was examined with the
reactivity of an anti-C3d monoclonal Ab (C3-19) in a Western blot
assay (data not shown). The detection limit of this assay was 0.8 pmol
of C3d, which represents <3% of the amount of C3c loaded on the gel.
Thus, the C3c sample contained <3% of C3d. To further eliminate any
possibility of presence of C3d, the binding response of two
anti-C3d mAbs (mAb 130, mAb C3-19) to the sensor chip
containing immobilized C3c was similar to that of a control Ab (mAb
72A1), and significantly lower that that of two anti-C3c Abs (C3-9,
133-H11) (Fig. 7B). All these data argue strongly against
the possibility that the observed CR2 binding was due to the presence
of traces of C3d in the C3c sample.
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To assess whether protein orientation had any effect on the
kinetics of CR2-iC3b complex formation, we compared the binding of
soluble CR2 to iC3b and vice versa, in the presence of 75 mM NaCl (Fig. 8). The initial association and
dissociation phases occurred more rapidly when soluble CR2 bound to
iC3b immobilized on the chip (Fig. 8A) than in the converse
situation (Fig. 8B). This observation suggests that, indeed,
experimental design may affect the interaction between CR2 and its
ligands as a function of time.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an attempt to mimic the actual in vivo orientation of these proteins, we have biotinylated them at key residues, i.e, the Cys988 residue that participates in the thioester bond formation in C3 (45, 56), and the C-terminal end of CR2. The immobilization of biotinylated C3d/iC3b on a streptavidin surface (i.e., the BIAcore chip) was intended to simulate the deposition of C3 onto activating particles. Likewise, immobilization of a CR2 molecule containing biotin at its C terminus mimicked anchoring on the cell surface. Orientation of proteins in this way provided an advantage over the random amine-coupling method generally used in SPR-based assays, because it produced a homogeneous ligand surface that facilitated the measurement of homogeneous binding constants.
To study the interaction of CR2 with EBV gp350, we expressed a
truncated form of gp350/220, consisting of residues 1–470. This
truncated molecule had previously been shown to inhibit EBV binding to
CR2 (23). The SPR data obtained for this CR2-gp350 (470t)
interaction showed a close fit to 1:1 Langmuir binding model (Fig. 4).
The apparent KD value obtained for this
interaction was 45 nM (Table I). Affinities for the interaction of
gp350/220 with CR2 have been previously determined by equilibrium
binding in two different experimental settings. Tanner and coworkers
(23) examined the ability of gp350/220 to inhibit the
binding of 125I-labeled gp350/220 to
CR2-expressing cells (Raji), and Moore and coworkers (57)
studied the binding of soluble CR2 to gp350/220 by measuring the
changes in mobility of the CR2-gp350/220 complexes in the
ultracentrifuge as a function of ligand concentration. The
KD values obtained by the two groups
were 12 and 3.2 nM, respectively. The differences observed between
these studies could reflect different affinities of CR2 for gp350/220
when present on the cell surface or in solution. In our case, the
differences in KD values observed
between the present study (CR2 anchored to the sensor chip;
KD = 45 nM) and the previous study
(23) (CR2 present on cell surface;
KD = 12 nM) could be due to truncation
of the protein used in our study. We used a protein truncated at
residue 470, whereas the previous studies used the entire molecule.
Therefore, these data suggest that other regions in gp350 may be
directly involved or influencing its binding to CR2.
Our analysis of the binding between CR2 and its natural ligands C3d and
iC3b revealed that these interactions do not follow a simple 1:1
binding model (Fig. 4). We eliminated surface heterogeneity by
implementing the directed orientation of the immobilized ligands.
Additionally, the fact that the gp350 binding data fit a 1:1 model both
argue strongly against the influence of experimental artifacts in these
data. Linear transformation of the association and dissociation data
showed nonlinear plots (Fig. 4). These nonlinear binding curves could
reflect multiple binding sites with different affinities,
cooperativity, or more complex models. Global fitting analysis of the
binding data suggested that the interaction between CR2 and C3d/iC3b
follows a bivalent model (Fig. 4). It is possible that two regions of
the C3d/iC3b molecule actually interact with two regions of the CR2
molecule; in this case the stoichiometry of the complex would be 1:1,
which is in accordance with the previous findings of Moore and
coworkers (57).
Several lines of evidence support a multiple site interaction model for
C3d/iC3b binding to CR2. First, it has been shown that two different
regions within SCR 1 and 2 of CR2 are involved in its interaction with
C3 (22, 53). Furthermore, data from our laboratory and
others have indicated that at least two regions of human C3d are
involved in its interaction with CR2 (24, 29, 31, 54).
However, involvement of the region in C3 comprising aa 1199–1210 in
binding to CR2 has been disputed (29, 31). To reassess
this interaction using SPR technology, we performed a direct binding
assay. We observed that CR2 binds to a synthetic peptide with the C3
sequence 1187K F L T T A K D L N R W E D P G K Q
L Y N V E A T S Y A1214 (24) (Fig. 5). The binding data could not be fit to any kinetic model, but it
allowed us to observe that the dissociation of CR2 from
C31187–1214 appeared to occur much faster than
that from C3d (in the same buffer conditions). This suggests that other
regions in C3d may be involved in its interaction with the receptor,
possibly stabilizing the complex. In fact, when we attempted to
competitively inhibit the binding of soluble CR2 to immobilized
C3d with the C31187–1214 peptide, no inhibition
of the CR2-C3d interaction could be observed (data not shown),
suggesting that other regions on C3d also interact with the receptor.
Alternatively, correct orientation of the peptide may be necessary for
it to bind to CR2.
The data obtained in this study (Fig. 6)
and in previous studies (31, 57) clearly indicate that the
binding of C3d to CR2 is highly dependent on ionic interactions. SPR
data obtained in the present study indicate the involvement of two
components in the C3d-CR2 interaction (Fig. 4, right panel).
To elucidate the influence of ionic interactions on these components we
obtained T50 values (the time required
to achieve an equimolar distribution of initial and final complexes)
for binding of C3d to CR2 at physiologic and half-physiologic ionic
strengths. We observed that lowering the amount of salt reduced the
time required for the onset of secondary contacts in the interaction
(Fig. 6). This finding suggests that some or all of the charged cluster
of amino acids on C3d (E37, E39, E160, D163, and E166) that are known
to influence CR2 binding when absent (31) may participate
in the second component of the interaction. In the absence of these
residues, binding of CR2 to C3d may have such fast association and
dissociation rates that it may not be detected in assays that do not
involve chemical cross-linking or SPR. In fact, our SPR data revealed
that dissociation of CR2 from the C31187–1214
peptide occurs much faster than its dissociation from C3d (Figs. 5 and 6). Alternatively, the charged cluster of residues in C3d may
cooperatively contribute to the interaction.
A careful analysis of our SPR data indicates that the interactions of
C3d and iC3b with CR2 are not identical. Although both interactions
seemed to follow a complex model, there appear to be significant
differences between the binding of soluble CR2 to immobilized C3d and
iC3b. For example, the on-rates of both the first and second components
of C3d and iC3b differed from each other by >2-fold (Table I). Our
results indicate that the interactions of C3d and iC3b with CR2,
although similar, appear to involve some notable differences. This may
be due to the participation of the CR2-interacting iC3b residues
located outside the C3d region (25). We observed that CR2
interacted with C3c when the latter was immobilized in a sensor chip
(Fig. 7). This result points toward the presence of CR2-interacting
residues on the C3c fragment that may explain the differences observed
between C3d and iC3b binding to CR2. Interestingly, this interaction
could be inhibited by soluble C3c (data not shown), which suggests that
the CR2 binding residues in C3c are exposed when in soluble C3c, i.e.,
immobilization of C3c did not significantly alter the conformation of
these residues. High amounts (2800 RU) of C3c were immobilized in the
chip in a random orientation, two factors that may induce surface
heterogeneity. Therefore, we did not attempt to fit the CR2-C3c binding
data to any kinetic model.
In addition to the presence of contact sites on C3c, other factors may contribute to differential binding of iC3b and C3d to CR2. Several residues in the C3d region of iC3b may be less accessible to CR2 as compared with those of C3d. Evidence suggesting that such steric hindrance may exist was presented in a study that used mAb 130 (58, 59). This Ab, which recognizes a neoantigenic determinant that is expressed when C3b is cleaved to iC3b and inhibits CR2 binding (24), was found to bind C3d better than iC3b (59).
Although it is clear from our data that C3d/iC3b-CR2 interactions do
not follow simple 1:1 interactions and that the data show two
components (Fig. 4, right panel) and fit well with a
bivalent interaction model (Fig. 4, left panel), the
complexity of the model precludes us from concluding that these
are bivalent interactions, because increasing the complexity of a model
increases the potential for multiple local minima in the
2 function that decrease the stability of the
fitting procedure. However, we have shown in this study that multiple
regions in C3 interact directly with the receptor.
It is also conceivable that CR2 has different affinities for soluble or
surface-bound C3 fragments (i.e., those bound to the activating
particle at their thioester site). In this study we have examined the
effect of ligand orientation on the binding kinetics of the iC3b-CR2
interaction. We observed differences in the on- and off-rates of iC3b,
depending on whether it was oriented on the surface or present in
solution. Complex formation and dissociation was initially faster when
iC3b was immobilized than when it was presented in soluble form.
Immobilization of iC3b through Cys988, which is
involved in the thioester bond formation, may render the
CR2-interacting residues more accessible. Similarly, differential
expression of various epitopes on fluid-phase vs surface-bound C3 has
already been proposed. Nilsson and coworkers have found that the
reactivity of several mAbs recognizing the C3 -chain differ when the
C3 fragments are in fluid phase or surface-bound (60).
In summary, we have used SPR technology to gain insight into the interaction between CR2 and its ligands. We have found that in contrast to gp350 (470t), C3d and iC3b appear to bind to CR2 in a complex manner.
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Acknowledgments |
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Footnotes |
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2 This work was in partial fulfillment of a PhD thesis (M.R.S.), which will be submitted to the Department of Cell Biology, Physiology, and Immunology, Universitat Autònoma de Barcelona, Spain.
3 Address correspondence and reprint requests to Dr. John D. Lambris, Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, 401 Stellar-Chance Laboratories, University of Pennsylvania, Philadelphia, PA 19104. E-mail address: lambris{at}mail.med.upenn.edu
4 Abbreviations used in this paper: CR, complement receptor; SCR, short consensus repeat; BCCP, biotin carboxyl carrier protein; Ni-NTA, nickel-nitro acetic acid; TFA, trifluoroacetic acid; PEO, polyethylene oxide; RU, resonance units; SPR, surface plasmon resonance; Fc1, flow cell one; Fc2, flow cell two; BirA, biotin holoenzyme synthetase.
Received for publication February 2, 2001. Accepted for publication May 25, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1H globulin. J. Exp. Med. 152:1625.2-macroglobulin, C3 and C4. FEBS Lett. 308:280.[Medline]
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