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Departments of Medicine and of Immunology, Allergy, and Infectious Disease, University of New South Wales, St. George Hospital, Sydney, New South Wales, Australia
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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60 amino acid residues in length
and contains four conserved cysteine residues, which are disulfide
linked in the order Cys1–3, Cys2–4. While the
first four domains are typical examples of this CCP superfamily, the
fifth domain in ß2GPI is aberrant, containing an
additional disulfide bond and a long C-terminal tail. It is now widely accepted that ß2GPI is an absolute requirement for the binding of "antiphospholipid" (aPL) Abs purified from patients with autoimmune disease when assayed using anionic phospholipid ELISAs (3, 4, 5). These autoantibodies are of considerable clinical importance because of their association with arterial and venous thrombosis, recurrent fetal loss, and thrombocytopenia (6). The interaction of autoantibodies with ß2GPI may be important in relation to the pathogenesis of thrombosis in vivo. ß2GPI is known to bind to negatively charged surfaces as well as to activated platelets and to act as an inhibitor of the intrinsic blood coagulation pathway in vitro (7).
Although there has been considerable controversy as to the exact nature of the antigenic epitope to which aPL Abs bind, it has become clear that ß2GPI is the most common and best-characterized antigenic target (8, 9, 10). aPL autoantibodies preferentially bind ß2GPI that has been immobilized on anionic phospholipid membranes or certain synthetic surfaces (11, 12, 13). Binding in the fluid phase is very weak, even when high concentrations of ß2GPI are used (12). A number of hypotheses have been put forward to explain this pattern of reactivity. It has been proposed that the binding of ß2GPI to aPL induces a conformational change in ß2GPI, thus exposing a cryptic epitope on ß2GPI for the autoantibodies to bind (11). Alternatively, it has been suggested that binding of ß2GPI to anionic phospholipid increases the local concentration of ß2GPI, thus promoting an increase in the intrinic affinity and binding of the autoantibodies to ß2GPI (12).
To distinguish between these possibilities, we generated two mutants, using a site-directed mutagenesis technique. The first mutant, F307*, lacks an aberrant C-terminal tail by stop codon introduction. This exposes Cys288, allowing for spontaneous dimer formation. Although the second mutant, F307*/C288A, also lacks an aberrant C-terminal tail, the Cys288 residue is replaced by an Ala residue, so that dimerization does not occur as easily as it does with the first mutant. Expression and characterization of these mutants in insect cells have allowed us to define, for the first time, the most likely explanation for the binding reactivity of autoantibodies to ß2GPI occurring in patients with the antiphospholipid syndrome (APS).
The present data demonstrate that affinity-purified anti-ß2GPI Abs from the sera of autoimmune patients bind to a mutant of ß2GPI that dimerizes with a higher affinity than to wild-type or a similar mutant that does not dimerize as readily. Our results provide direct evidence, for the first time, that anti-ß2GPI Abs bind to native ß2GPI and that the binding is dependent upon density of Ag and bivalency and not on neo-epitope formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Escherichia coli strains used for this study were JM109 (endA1, recA1, gyrA96, thi, hsdR17, relA1, supE44, D (lac-proAB), (F', traD36, ProAB, LacIZ D M15)) and ES1301 mutants (lacZ53, mutS201::Tn5, thyA36, rha-5, metB1, deoC, IN (rrnD-rrn-E)). Plasmid dsDNA was isolated using the Wizard Maxipreps DNA Purification System (Promega, Madison, WI).
Cells and virus
Spodoptera frugiperda (Sf21) insect cells were maintained in serum-free medium, Sf-900II (Life Technologies, Gaithersburg, MD). A BacPAK 6 viral DNA (Clontech), which was modified from AcMNPV DNA, was digested with Bsu361 to remove part of an essential viral gene, forcing recombination with cotransfected transfer vectors. Nearly 100% of the virus recovered contained the target gene.
Construction of an expression vector for mutants of ß2GPI
The construction of the wild-type ß2GPI expression vector has been previously described (14). Site-directed mutagenesis was conducted using an oligonucleotide-directed kit, Altered Sites in vitro mutagenesis system (Promega). The cDNA for the F307* mutant was constructed from the wild-type ß2GPI template using the mutagenic oligonucleotide 5'-GTC CCC AAA TGC TAG AAG GAA CA-3'. For the F307*/C288A mutant, two site-directed mutations were introduced simultaneously, using two separate mutagenic oligonucleotides: 5'-GTC CCC AAA TGC TAG AAG GAA CA-3' and 5'-AAG GAA AAG AAG GCT AGC TAT ACA GA-3'. The mutation in each primer is underlined. Following digestion with XbaI and PstI, the mutant recombinant fragments were released from pALTER vector and subcloned to pBacPAK 9 transfer vector, replacing the wild-type sequence in this region. The mutants were identified by dideoxy sequencing from double-stranded, alkali-denatured templates, utilizing the T7 sequencing kit (Pharmacia, Uppsala, Sweden) with an internal primer.
Generation and purification of recombinant viruses
Sf21 cells (2 x 106) were cotransfected with 100 ng of BacPAK 6 viral DNA and 500 ng of the expression vector by the lipofectin method in a 60-cm2 tissue culture dish. A pure clone of a recombinant virus was obtained by diluting the cotransfection supernatant (collecting 4 days after the infection) containing progeny viruses and performing a plaque assay to produce individual plaques.
Expression of ß2GPI mutants in Sf21 cells
Sf21 cells (5 x 106) grown in a monolayer were
infected by recombinant virus with a multiplicity of infection of 10
in 100-cm2 tissue culture dishes. The infected cells
were cultured in 10 ml of serum-free medium, Sf-900II, for 3 to 5 days
at 27°C. Ten microliters of culture supernatant was collected for
electrophoresis and subjected to SDS-PAGE and Western blot.
SDS-PAGE and Western blot analysis
SDS-PAGE and Western blot was performed as previously described (15).
Preparation of recombinant wild-type and mutants of ß2GPI
Recombinant wild-type and mutants of ß2GPI were purified from the culture supernatant of ß2GPI-transfected Sf21 cells by affinity chromatography using a polyclonal anti-ß2GPI Ab (15, 16). Culture supernatant was diluted with 10 mM sodium phosphate (PBS), pH 7.4, containing 150 mM NaCl, and was applied to the anti-ß2GPI affinity column. Bound protein was eluted with 0.1 M glycine-HCL, pH 2.5. The eluted fractions were immediately neutralized with 2 M Tris, pH 8.0, and the eluted protein was concentrated with a Centricon 10 ultrafilter (Amicon, Beverly, MA). The concentrated eluants were dialyzed against PBS. The purity of the sample was assessed by SDS-PAGE.
Binding of anti-ß2GPI Abs to wild-type and mutants of ß2GPI in an anti-ß2GPI ELISA
An anti-ß2GPI ELISA in the absence of phospholipid was performed as described by Wang et al. (17). Wells of 96-well microtiter polystyrene plates (plain and irradiated) were coated with wild-type (50 µl) and mutants of ß2GPI or haptoglobin as a control (10 µg/ml in carbonate buffer, pH 9.6) overnight at 4°C. The plates were then washed with PBS and blocked with 1% milk/0.3% gelatin-PBS for 1 h at room temperature. After three washings, purified anti-ß2GPI Abs were added to wells and incubated at room temperature for 3 h. The plates were washed three times with PBS and then incubated with alkaline phosphatase-conjugated goat anti-human IgG (Sigma, St. Louis, MO; 1:1000 in 1% BSA-PBS) for 90 min. After three washes, a chromogenic substrate (50 µl of p-nitrophenylphosphate (1 mg/ml) in 10% diethanolamine buffer, pH 9.8; Sigma) was added to each well, and OD was read at 405 nm in an ELISA reader (Titertek Multiskan MCC). The irradiated microtiter plates were Linbro Titertek plates that had received a dose of 10 kGy; irradiation was performed at the Australian Nuclear Science and Technology Organization (Sydney, Australia).
The anti-ß2GPI Abs used in this assay were purified by sequential cardiolipin (CL) affinity (18) and cation exchange (3) chromatography from serum obtained from five patients with APS.
The OD values are expressed as net binding of Abs to mutants and wild-type ß2GPI after subtraction of binding of Abs to the control protein (haptoglobin).
Effect of different concentrations of wild-type and mutants of ß2GPI in an anti-ß2GPI ELISA
A fixed amount of purified anti-ß2GPI Ab was tested for its reactivity to different concentrations of wild-type or mutants F307* and F307*/C288A of ß2GPI in an anti-ß2GPI ELISA. Wild-type and mutants F307* and F307*/C288A were coated on an irradiated plate (10 kGy) at 0.5, 1, 2, 4, 8, and 16 µg/ml in carbonate buffer, pH 9.6, overnight at 4°C. The plates were then processed as described above for the anti-ß2GPI Ab ELISA.
Effect of fluid phase wild-type or mutants of ß2GPI on the binding of anti-ß2GPI Abs to solid phase ß2GPI
To measure the inhibition of binding of anti-ß2GPI Abs to solid phase ß2GPI by fluid phase wild-type and mutants of ß2GPI, Abs were first diluted to a dilution previously found in preliminary experiments to be midway along the linear portion of the standard curve. Diluted Abs were then preincubated for 3 h at room temperature with an equal volume of buffer or increasing concentrations of wild-type or mutants of ß2GPI (6.25, 12.5, 25, 50, 100, and 200 µg/ml). The amount of free Ab in the Ab inhibitor mixtures was then measured in the anti-ß2 GPI ELISA using irradiated plates, and the average Ab affinity was calculated according to the method of Friguet et al. (19). Haptoglobin (Sigma) was used as a control Ag, since it is in the same protein superfamily as ß2GPI.
The inhibitory activity was expressed as the percentage of inhibition compared with that of the uninhibited control (Abs plus buffer).
Preparation of F(ab')2 and Fab' fragments of an anti-ß2GPI Ab
F(ab')2 fragments were obtained from one purified anti-ß2GPI Ab digested with pepsin: 1 ml of a purified anti-ß2GPI Ab (0.68 mg) was incubated in sodium acetate buffer for 5 h in a shaking water bath at 37°C with 250 µl of immobilized pepsin (Sigma). The reaction was stopped by adding 15 mM Tris-HCI, pH 7.5, and the immobilized pepsin gel was separated from the digest by centrifugation at 1000 x g for 5 min. Intact IgG and Fc' fragments were removed by protein A affinity chromatography. Purity of the F(ab')2 fragments was monitored by SDS-PAGE under nonreducing conditions. Fab' fragments were obtained from the purified anti-ß2GPI Ab by digestion with papain (Sigma) using a similar method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using the Altered Sites in vitro mutagenesis system, two mutants
were generated, F307* mutant (from Phe307 to stop codon),
F307*/C288A mutant (from Phe307 to stop codon and from
Cys288 to Ala288; Fig. 1
). Each was expressed at a
concentration of 10 µg/ml of medium in insect cells as previously
described (14).
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The recombinant ß2GPI secreted into the serum-free
culture medium was purified from culture supernatants by
anti-ß2GPI affinity chromatography as previously
described (14). The recovery of eluted ß2GPI from the
affinity column was 50%. Purified recombinant ß2GPIs
were subjected to SDS-PAGE and analyzed by staining with Coomassie
brilliant blue R-250 (Fig. 2A).
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). However, mutant F307* in
addition to a prominent band of 37 kDa, also had a significant band at
74 kDa consistent with this being a dimer, which was confirmed using a
specific rabbit anti-ß2GPI Ab by western blot (Fig. 2B). Binding of anti-ß2GPI Abs to wild-type ß2GPI and mutants of ß2GPI in an anti-ß2GPI ELISA
The binding of purified anti-ß2GPI Abs (from
five different autoimmune patients) to wild-type, mutant F307*, and
mutant F307*/C288A were tested by using normal and irradiated
polystyrene microtiter plates. In the anti-ß2GPI
ELISA, using irradiated plates, the mean OD for mutant F307* binding by
anti-ß2GPI Abs was 1.259 (range, 0.743–1.524) vs
0.742 (range, 0.333–1.113) for wild-type and 0.609 (range,
0.279–1.019) for mutant F307*/C288A. As shown in Figure 3A, in the presence of 3
µg/ml of purified anti-ß2GPI Ab, the amount of
binding observed was 36 to 65% higher with mutant F307* than with
wild-type or mutant F307*/C288A and was negligible with the control
protein haptoglobin. In the anti-ß2GPI ELISA, using
normal plates, the anti-ß2GPI Abs bound mutant F307*;
the mean OD for mutant F307* binding of anti-ß2GPI
Abs was 0.584 (range, 0.26–1.386) (Fig. 3B). The relative
amount of wild-type or mutants of ß2GPI bound to the
plates was assessed with a rabbit polyclonal
anti-ß2GPI Ab, which confirmed that equal amounts of
wild-type, mutant F307*, and F307*/C288A were coated onto the
microtiter wells.
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We next investigated the dose-dependent binding with a fixed
amount of Ab with increasing concentrations of wild-type and mutants of
ß2GPI. As shown in Figure 4, in the presence of 3 µg/ml of
purified anti-ß2GPI Ab, binding increased, with
increasing concentrations of mutant F307*, in a dose-dependent manner.
Wild-type, native, and mutant F307*/C288A exhibited a dose-dependent
increase in binding activity that was 50% of that obtained with
mutant F307*. The binding to mutant F307* did not reach a plateau at
the concentrations used, in contrast to the results obtained with
wild-type and mutant F307*/C288A. This may be due to the fact that
mutant F307* is a mixture of monomers and dimers. The relative amount
of ß2GPI bound to the plates was assessed using a rabbit
polyclonal anti-ß2GPI Ab, which confirmed that equal
amounts of wild-type, native, and mutants F307* and F307*/C288A were
coated on the microtiter wells (data not shown).
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Inhibition experiments using fluid phase preparations of
ß2GPI in an anti-ß2GPI system were
performed to confirm the specificity of the purified
anti-ß2GPI Abs (n = 5) for wild-type
and mutants of ß2GPI and to assess the affinity of the
autoantibody preparations for wild-type, mutant F307*, and mutant
F307*/C288A of ß2GPI. Direct binding assays with serial
dilutions of the Abs in preliminary experiments were performed to
determine the optimal concentration of Abs for the inhibition assays. A
concentration of 6 µg/ml of each Ab was found to be optimal (results
not shown). Fluid phase mutant F307* inhibited
anti-ß2GPI Ab binding to wild-type
ß2GPI immobilized on the microtiter plates significantly
better than that obtained with wild-type ß2GPI or mutant
F307*/C288A. The mean inhibitory concentration of mutant F307* for 50%
inhibition (IC50) was 28.2 µg/ml (range, 16–42 µg/ml),
which is an underestimate because of the presence of monomers of the
mutant. The IC50 for wild-type and mutant F307*/C288A
ß2GPI was estimated to be >200 µg/ml, as inhibition
did not reach the 50% level for some of the Abs. Table I summarizes the OD data used in the
inhibition experiments to calculate the Kd for
each preparation. As mutant F307* was a mixture of dimers and monomers,
the best estimate of its m.w. to calculate the
Kd was based on the assumption that 30% of
the molecule was dimerized (from the intensity of Coomassie brilliant
blue staining). The method of Friguet et al. (19) was used to measure
Ab affinity for each ß2GPI preparation by deriving the
dissociation constant, Kd, for each Ab in the
fluid phase competitive inhibition anti-ß2GPI ELISA.
Table II summarizes the calculated
Kd for each Ab preparation for wild-type and the
two mutants of ß2GPI. No inhibition of patient Abs was
observed with similar concentrations of haptoglobin in parallel
experiments (data not shown).
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To assess whether the binding of purified
anti-ß2GPI Abs to mutant F307* of ß2GPI
required Ab bivalency, F(ab')2 (bivalent) and Fab'
(monovalent) fragments were prepared from one affinity-purified
anti-ß2GPI Ab. As shown in Figure 5, no binding of monovalent Fab'
fragments to mutant F307* was demonstrated, while significant
dose-dependent binding of bivalent F(ab')2 fragments was
observed. No binding of either F(ab')2 or Fab' was detected
to haptoglobin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs to ß2GPI have now been extensively studied using a phospholipid-free ELISA system. Matsuura et al. (11) reported that aCL Abs can bind directly to ß2GPI coated on irradiated wells but not on untreated wells. The extent of aCL binding was dependent on the radiation dose applied to the surface, and aCL Abs binding to ß2GPI adsorbed to these wells correlated well with that of ß2GPI complexed to negatively charged phospholipid. The binding in this study was inhibited by preincubation with beads coated with CL and ß2GPI, but not ß2GPI, or by CL-coated beads alone (11). These data have been interpreted as supporting the theory that aCL Abs bind a cryptic epitope on ß2GPI, which is exposed only after binding to negatively charged phospholipid or an appropriately treated surface (11). In contrast, we have affinity purified anti-ß2GPI Abs with solid phase ß2GPI in the absence of phospholipid from patients with APS and demonstrated that these Abs bind directly to ß2GPI. This confirms that these Abs bind native ß2GPI and not a neo-epitope on ß2GPI following its interaction with phospholipid or irradiated polystyrene surfaces (15). We have also shown that both fluid phase ß2GPI and synthetic peptides from the fifth domain of ß2GPI inhibit, in a dose-dependent manner, the binding of anti-ß2GPI Abs to ß2GPI, providing further support for this notion (17).
Roubey et al. (12) have shown that these Abs bind to ß2GPI on irradiated polystyrene plates and are intrinsically low affinity Abs that require increased density of ß2GPI to bind in vitro (12). Fab' fragments showed significantly less binding than whole IgG or F(ab')2 to ß2GPI, suggesting a critical role for Ab bivalency (12). Further support for this notion is provided by a recent report that concluded that human polyclonal anti-ß2GPI Abs are mainly monoreactive autoantibodies that bind an epitope on native ß2GPI, which is preserved on ß2GPI purified from mouse, guinea pig, bovine, and human sera (13). In addition, the demonstration that autoantibody preparations from APS patients can bind ß2GPI on Western blotting in the absence of any phospholipid provides further evidence that the epitope for these autoantibodies is on the native ß2GPI molecule (14).
It has been proposed that autoantibodies to ß2GPI are
intrinsically of low affinity, and fluid phase inhibition experiments
using native ß2GPI have calculated the
Kd for anti-ß2GPI
autoantibodies to be 10-5 M. Since the minimum
Kd required for Abs to bind in an ELISA
system is 10-6 M, this presumably accounts for the absence
of binding of these Abs when ß2GPI is coated onto
nontreated ELISA plates. It has been proposed that clustering of
ß2GPI on ELISA plates, either by coating with CL or by
irradiation, allows bivalent binding to occur and thus increases
affinity. Our results with polyclonal anti-ß2GPI
autoantibodies using the mutant F307* (which spontaneously dimerizes)
coated on untreated ELISA plates demonstrate significant binding when
compared with wild-type and mutant F307*/C288A. These data provide
strong evidence that the antigenic target for these autoantibodies is
not a neo-epitope on ß2GPI. Binding of Abs to mutant
F307* of ß2GPI was abolished when Fab' fragments were
used, as compared with F(ab')2. This would argue for the
importance of bivalent binding of these autoantibodies in the ELISA
system. However, we have examined the Abs from only five patients, and
it could well be that if we examined a much larger population sample
that there would be Abs that recognize neo-epitopes on
ß2GPI. In addition, we cannot exclude the possibility,
although it is unlikely, that a neo-epitope identical to that which
occurs when ß2GPI interacts with CL has been
created by the mutation in F307* and not in F307*/C288A.
In the present study, we have demonstrated that purified
anti-ß2GPI Abs bind to mutant F307* much better than
wild-type and mutant F307*/C288A. The most likely explanation for these
observations is that mutant F307* forms dimers (Fig. 2). When the same
amount of mutant F307*, F307*/C288A, and wild-type ß2GPI
are coated on the plates, the clustering of mutant F307* is much more
concentrated than wild-type or mutant F307*/C288A. Clustering of
immobilized Ag in dimers allows an increase in affinity of the
anti-ß2GPI Abs and bivalent binding to occur (Fig. 6). The calculated affinity of the
dimerized ß2GPI mutant would actually be greater, as the
preparation used contained monomers as well (Fig. 2). The increased Ag
density required for such bivalent binding of
anti-ß2GPI autoantibodies is achieved when mutant
F307* is coated on both normal and irradiated microtiter plates. This
evidence suggests that the binding of anti-ß2GPI Abs
from patients with APS bind native ß2GPI and not
neo-epitopes formed when ß2GPI binds to a suitably
charged surface.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steven A Krilis, Department of Immunology, Allergy and Infectious Disease, St. George Hospital, South Street, Kogarah, 2217, New South Wales, Australia. E-mail address:
3 Abbreviations used in this paper: ß2GPI, ß2-glycoprotein I; aCL, anticardiolipin; CL, cardiolipin; aPL, antiphospholipid; Sf21, Spodoptera frugiperda; APS, antiphospholipid syndrome; CCP, complement control protein; IC50, mean inhibitory concentration of mutant F307* for 50% inhibition.
Received for publication January 13, 1998. Accepted for publication April 15, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 B. C. H. Lutters, J. C. M. Meijers, R. H. W. M. Derksen, J. Arnout, and P. G. de Groot Dimers of beta 2-Glycoprotein I Mimic the in Vitro Effects of beta 2-Glycoprotein I-Anti-beta 2-glycoprotein I Antibody Complexes J. Biol. Chem., January 26, 2001; 276(5): 3060 - 3067. [Abstract] [Full Text] [PDF]
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J. Guerin, Y. Sheng, S. Reddel, G. M. Iverson, M. G. Chapman, and S. A Krilis Heparin Inhibits the Binding of beta 2-glycoprotein I to Phospholipids and Promotes the Plasmin-mediated Inactivation of This Blood Protein. ELUCIDATION OF THE CONSEQUENCES OF THE TWO BIOLOGICAL EVENTS IN PATIENTS WITH THE ANTI-PHOSPHOLIPID SYNDROME J. Biol. Chem., January 18, 2002; 277(4): 2644 - 2649. [Abstract] [Full Text] [PDF]
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), recombinant wild-type ß2GPI
(•), mutant F307* (
), mutant F307*/C288A (
). Results are
expressed as the mean (± SE) of duplicates.
