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2-Glycoprotein I to Determine Fine Antigenic Specificity of Antiphospholipid Autoantibodies1
* La Jolla Pharmaceutical Co., San Diego, CA 92121;
Departments of Medicine and Immunology and Infectious Disease, St. George Hospital, University of New South Wales, Sydney, Australia; and
Laboratory of Molecular Biophysics, Rockefeller University, New York, NY 10012
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-glycoprotein I
(2GPI) appear to be a critical feature of the
antiphospholipid syndrome (APS). As determined using domain deletion
mutants, human autoantibodies bind to the first of five domains present
in 2GPI. In this study the fine detail of the domain I
epitope has been examined using 10 selected mutants of whole
2GPI containing single point mutations in the first
domain. The binding to 2GPI was significantly affected
by a number of single point mutations in domain I, particularly by
mutations in the region of aa 40–43. Molecular modeling predicted
these mutations to affect the surface shape and electrostatic charge of
a facet of domain I. Mutation K19E also had an effect, albeit one less
severe and involving fewer patients. Similar results were obtained in
two different laboratories using affinity-purified
anti-2GPI in a competitive inhibition ELISA and with
whole serum in a direct binding ELISA. This study confirms that
anti-2GPI autoantibodies bind to domain I, and that
the charged surface patch defined by residues 40–43 contributes to a
dominant target epitope. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-glycoprotein I
(2GPI)4
are recognized to be strongly associated with the antiphospholipid
syndrome (APS) (1, 2, 3). Abs detected by the anticardiolipin
(aCL) assay from patients with clinical features of APS generally bind
to 2GPI, but samples from patients with other
causes of a positive aCL assay, such as a variety of infections, do not
(4). Likewise, lupus anticoagulant activity in APS patient
plasma is often due to anti-2GPI Abs
(1). Knowing the location of an epitope for pathogenic Abs
may permit the design of therapeutic agents able to act as toleragens
for the specific B cells producing the autoantibodies (5).
This would provide a far more specific approach to treatment than the
anticoagulation or general immunosuppression currently used in
APS.
2GPI is a member of the short consensus repeat
protein family, which is characterized by repeating domains of 
60 aa
with two disulfide bridges. 2GPI has five
domains; the amino-terminal first through fourth domains are typical,
and the fifth domain contains an extra 20-aa C-terminal loop ending
in an extra terminal disulfide bond and a lysine-rich region that
functions as the major anionic phospholipid binding site (6, 7). The recent structure of 2GPI, as
determined by x-ray crystallography, shows that it has a J shape
(8, 9). This suggests that the base of the J shape
containing domain 5 provides the membrane binding interface. The
remaining domains thus extend progressively away from the phospholipid
surface. In this situation domain I would have the greatest exposure
and accessibility (8, 9). No specific function has been
ascribed to domains I–IV.
The identification of which region(s) of 2GPI
is bound by autoantibodies has been the subject of some debate
(10, 11, 12, 13, 14). Wang et al. (10) tested several
human mAbs generated from patients with APS that bound to cardiolipin
in the presence of 2GPI. Several short
peptides containing sequences from domain V of
2GPI enabled these mAbs to bind to cardiolipin
or inhibited binding of mAb to 2GPI. However,
polyclonal autoantibodies from APS patients did not bind these
peptides. It was suggested that these mAbs bound epitopes on domain V.
Arvieux et al. (15) identified patients who had
anti-2GPI Abs that were aCL assay negative
and inhibited the binding of 2GPI to
cardiolipin, suggesting binding to the cardiolipin binding site on
domain V of 2GPI. Yang et al.
(13) have recently described direct binding of
autoantibodies from patients with systemic lupus erythematosus to a
recombinant domain V-GST fusion protein. However, contrary to the
studies of Arvieux et al. (15), these patient samples were
also aCL assay positive.
Several studies have used recombinant mutants of
2GPI with one or more domains deleted
(11, 12, 16). Igarashi et al. (16) found that
several mouse and human monoclonal 2GPI
autoantibodies bound to wild-type 2GPI,
domains I–IV (i.e., the deletion mutant containing only domains I–IV)
and, to a lesser degree, to domains I–III, but not mutants without
domain I. This was interpreted to suggest that domain IV may play a
critical role in exposure of the cryptic epitope. Using these
mutants and purified anti-2GPI from four
APS patients, George et al. (12) found binding to domains
I–IV, but not domains I–III or domains II–V. Three mouse mAbs, two
of which were reported to bind to domain IV, were used in competitive
inhibition assays. Binding of one of the two anti-domain IV mAbs
was inhibited by three of the four patient
anti-2GPI preparations. These findings
were interpreted as demonstrating that domain IV contains the epitope
site for human anti-2GPI
(12). Using a single point and a triple point domain IV
mutant, Koike et al. (17) found significantly decreased
binding of anti-2GPI to both mutants in an
ELISA, again suggesting domain IV as the site of the epitope(s).
Other studies using domain-deleted mutants of
2GPI reached different conclusions. Using
anti-2GPI purified from 11 patients with
APS, significant competitive inhibition of binding to the wild type
could be demonstrated using all mutants containing domain I, most
importantly including domain I alone, but there was little if any
inhibition using mutants without domain I (11). In a
direct binding ELISA study in which the wild-type and domain-deleted
mutants were coated onto irradiated polystyrene plates, sera from 21
patients with anti-2GPI Abs were also
shown to bind to full-length 2GPI and to the
domain I–IV mutant at the same level. In only three was there any
degree of binding to domains II–V (14). These studies
suggested that domain I, not domain IV or V, is the dominant epitope
site on 2GPI for
anti-2GPI autoantibodies from the majority
of APS patients.
This present study used recombinant wild-type
2GPI plus variants with single point mutations
in domain I in both direct binding and competitive inhibition ELISAs.
This was done firstly to attempt to better localize the epitope, and
secondly to address the possibility that the deletion of entire domains
in the previous studies resulted in conformational changes elsewhere in
the protein, which could potentially affect autoantibody binding and/or
the coating characteristics on ELISA plates (11, 14).
Using these domain I point mutants we have examined the binding of
anti-2GPI autoantibodies in a wide range
of ELISA conditions, with two separate groups of
anti-2GPI patient samples. In one
laboratory (La Jolla Pharmaceutical Co.) the domain I point mutants
were examined for their ability to competitively inhibit the binding of
affinity-purified anti-2GPI autoantibodies
from binding to wild-type 2GPI. The
competition assays were performed using both irradiated and Maxisorp
microwell plates (Nalge Nunc International, Roskilde, Denmark). In the
other laboratory (St. George Hospital) a separate panel of serum
samples with anti-2GPI autoantibodies was
examined for binding to wild-type and domain I single point mutant
2GPI coated directly onto irradiated
plates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2GPI
autoantibodies
Autoantibodies binding 2GPI were
purified from 15 patients with APS. No patient selection criteria were
applied other than the availability of sufficient volume and titer to
yield an adequate amount of affinity-purified Ab for the inhibition
studies. Anti-2GPI was affinity-purified by
sequential fractionation with cardiolipin-containing liposomes, then
protein A- agarose beads, as previously described (11).
Patient selection for the direct binding
anti-2GPI ELISA
Serum samples were screened with an in-house and a commercial
anti-2GPI ELISA (INOVA Diagnostics, San
Diego, CA). Samples scoring above normal with the in-house assay and
>40 U in the commercial assay (normal is <20 U) were selected for
further study.
Anti-2GPI competitive inhibition ELISA
MaxiSorp microplates (Nunc) and irradiated microplates (10 kGy;
ICN, Costa Mesa, CA) were coated overnight at 4°C with 50 µl of
full-length recombinant 2GPI at 10 µg/ml in
0.1 M carbonate, pH 9.5, then washed three times with 0.15 M PBS, pH
7.2, and blocked for 1 h at room temperature with 75 µl of 2%
nonfat dried milk (NFDM) and PBS. Each Ab preparation was titrated to
determine the concentration required to produce 80% maximum
binding, typically between 2.5 and 10 µg/ml. Test inhibitors
(wild-type or domain I point mutants) were diluted to the required
concentration in 2% NFDM, or 2% NFDM alone was added to coated wells.
Affinity-purified anti-2GPI Ab was diluted
in 2% NFDM, and 25 µl of a constant concentration was added to the
wells. The contents of the wells were mixed, and the plates were
incubated at 37°C for 1 h. The plates were washed three times
with PBS, then 50 µl of alkaline phosphatase-conjugated
anti-human IgG or anti-mouse IgG (
-chain specific; Zymed,
South San Francisco, CA) diluted in 2% NFDM was added and incubated at
37°C for 1 h. The plates were washed three times with PBS, 50
µl of alkaline phosphatase chromogenic substrate was added, and the
plates were incubated for 30 min at room temperature. The
A550 was measured in a microplate autoreader
(Bio-Tek Instruments, Winooski, VT). The percent inhibition was
determined as follows: [(mean A550 obtained
from the control wells without inhibitor less
A550 of background)
-(A550 obtained in the presence of inhibitor
less A550 of background)/(the mean
A550 obtained from the control wells without
inhibitor less A550 of background)] x 100.
Each inhibitor was tested at seven different concentrations, allowing
an inhibition curve to be constructed. This was then used to calculate
the amount of mutant or wild-type 2GPI
required to give 50% inhibition (IC50). The
IC50 of each mutant was expressed as a ratio with
the IC50 of wild-type
2GPI for each
anti-2GPI tested.
Anti-2GPI direct binding ELISA
Flat-bottom microtiter polystyrene plates (ICN Biomedical) were
used after gamma irradiation to 10 kGy (7). Preparations
of 2GPI were wild-type
2GPI and 10 mutants with a single point
mutation in domain I, as described above. The washing buffer was 1 M
NaCl, 20 mM sodium phosphate buffer adjusted to pH 7.2 plus 0.1% (v/v)
Tween 20, and the blocking buffer comprised wash buffer with the
addition of fatty acid-free 1% (w/v) BSA (Sigma-Aldrich, Sydney,
Australia). The final wash was with 0.15 M NaCl and 20 mm sodium
phosphate, pH 7.2, without Tween 20. Second Abs were alkaline
phosphatase-conjugated goat anti-mouse IgG,
anti-human IgG, or anti-human IgM (Sigma-Aldrich).
The colorimetricsubstrate was p-nitrophenyl
phosphate (Sigma-Aldrich; 1 mg/ml) in 1 M diethanolamine and 0.5 mM
MgCl2, pH 9.8, and reading was performed in a
TiterTek Mulitskan MCC instrument (Eflab, Espoo, Finland) at
A405. The wild-type and point mutant
2GPI preparations were coated at 10 µg/ml in
50 µl/well of 50 mM sodium carbonate/bicarbonate buffer, pH 9.6, at
4°C overnight, a concentration that ensured adequate Ag coating to
enable patient autoantibodies to bind as previously determined
(14). The wells were washed three times with 200 µl of
wash buffer, blocked with 200 µl of blocking buffer for 1 h at
4°C, and washed three times. Patient serum, diluted 1/100 in blocking
buffer, was added and incubated for 2 h at room temperature, then
wells were washed three times. Where mAbs were used these were at the
stated concentration diluted in blocking buffer. The appropriate
alkaline phosphatase-conjugated anti-Ig was diluted 1/1000 in
blocking buffer, and 50 µl/well was added and incubated for 2 h
at room temperature. The plates were washed three times with wash
buffer, then once with final wash buffer, before 50 µl/well of
prewarmed substrate was added and incubated in the dark at 37°C.
Patient samples were read 30 min after the addition of substrate. Assays were accepted if the positive standard control was within 10% of 1.5 absorption units (AU), and the normal serum control within the normal range. All samples were tested in parallel in duplicate wells. All samples tested against point mutants were also tested against the wild type at the same time. Assays with mAbs were read when the positive control OD was in the range 1.5–1.9 AU.
The direct binding ELISA was reproducible with an intraassay coefficient of variation of 5.3, and an interassay coefficient of variation of 2.6% for the positive standard and of 8.4% for all samples. Forty normal blood donors had a mean of 0.081 AU and an SD of 0.054 AU. The mean ± 5 SD (0.353 AU) was used as the cut-off for abnormality.
Anti-2GPI mAbs
mAbs 1, 10, 11, and 16 were obtained from immunized mice and
were used at 0.1, 0.2, 0.1, and 0.2 µg/ml, respectively. mAbs 1, 11,
and 16 bind domain I, while mAb 10 binds domain IV (18).
mAb FC1, used at 1 µg/ml, is an IgG that binds domain I and was
obtained from an autoimmune NZW x BXSB F1
mouse (19). FC3 is an isotype-matched
non-2GPI-binding control from the same mouse.
IgM mAbs EY2C9 (anti-2GPI) and TH1B9
(negative control; a gift from Dr. T. Koike) were used at 2 µg/ml and
were derived from an autoimmune patient as previously described
(20). mAb 4E7H10 was obtained from mice immunized with a
domain I-keyhole limpet hemocyanin conjugate.
2GPI point mutation models
Three-dimensional models of the first domain of
2GPI were calculated using a method of
comparative modeling by satisfaction of spatial restraints implemented
in the MODELLER program (21). Ten different models were
produced for each point mutation based on the x-ray structure of the
native 2GPI (Protein Data Bank code 1c1z
(8)). The final models of each point mutant were selected
based on the value of the MODELLER objective function
(21). To study changes in antigenicity caused by point
mutations in the 2GPI sequence, the surface
electrostatic potentials of the native structure and the mutant models
were calculated with the GRASP program (22) using the
atomic charges from the CHARMM22 force field (23).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To further examine the epitope(s) recognized by these
autoantibodies we made full-length recombinant
2GPI proteins with single point mutants in
domain I. First we determined which, if any, of the amino acid
positions in domain I of 2GPI are important
for autoantibody binding by screening filamentous phage-displayed
peptide libraries in which mutants of domain I were expressed as an
amino-terminal fusion with the pIII protein. The mutations were created
by error-prone PCR, such that an average of one mutation in domain I
was generated in each phage clone. A phage micropan assay was used to
screen individual phage for the ability to bind affinity-purified Abs
from patients with antiphospholipid syndrome. Using this approach we
rapidly identified phage with missense mutations covering approximately
half the 63 domain I residues. This allowed us to identify a number of
mutants that adversely affected Ab binding. Ten mutants, some of which
affected Ab binding and some of which did not, were chosen for further
study as follows: D8A, K19E, K33E, S38T, G40E, M42K, M42V, R43G, T50A,
and N56T, where the nomenclature of D8A signifies a change from D to A
at position 8. These were incorporated into full-length
2GPI mutant proteins produced in insect cell
cultures as previously described (11).
Direct binding anti-2GPI ELISA
mAbs were used to assess the ability of the point mutants to
coat the wells of gamma-irradiated microplate wells. Both mouse
anti-domain IV mAb 10 and mouse anti-domain I mAb 16 showed no
reduction in binding to the point mutants compared with wild type,
suggesting similar microplate coating for each mutant (Fig. 1
). The autoimmune mouse mAb FC1 showed a
20% increase in binding to T50A, but otherwise similar binding
compared with the wild type. The binding of the human mAb EY2C9 to
mutant T50A was negligible and was reduced by >50% to G40E, M42K, and
R43G. The results suggest that the antigenic epitope recognized by
EY2C9 is located on domain I.
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2GPI adsorbed onto gamma-irradiated microplate
wells. The patient samples showed a decreased binding to several point
mutants, in particular to those with mutations between G40E and R43G.
The results from five patients are shown graphically in Fig. 2, and the results for all 27 samples are
tabulated in Table I. A majority of
the samples (60%) had a >50% decrease in binding to R43G
compared with the wild type. Nine sera (33%) showed a >50% decrease
in binding to K19E. Two sera (no. 12 and 17) showed little to no
binding to the G40E, M42V, and R43G mutants while displaying very good
binding to the remaining seven single point mutants and the wild
type.
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Wild-type 2GPI and 10 different mutants
were tested for their ability to inhibit mouse mAbs 4E7H10 and 16 from
binding to wild-type 2GPI immobilized on ELISA
plates (both Nunc MaxiSorp and irradiated polystyrene). These mAbs were
chosen because they recognize conformational epitopes on domain I and
because they bind to different epitopes than the human autoantibodies
(data not shown). The results (Fig. 3)
show that on both types of ELISA plates all 10 mutants inhibited the
binding of both anti-2GPI mAbs to
wild-type 2GPI. The IC50
for each of the single point 2GPI mutants was
within a 10-fold range of the wild-type value. This suggests that the
single amino acid substitutions in domain I did not alter the overall
conformation of the domain, inasmuch as that part of the domain
recognized by these two mAbs remained unaffected.
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2GPI immobilized on
both Nunc and gamma-irradiated ELISA plates. The amount of each of the
mutants required to give 50% inhibition was divided by the amount of
wild type required to give 50% inhibition. This value, a fold
increase, was used as a quantitative measurement, so that thereactivity
of each mutant could be compared from one Ab sample to another. The
complete inhibition results from two of the Ab preparations are shown
graphically in Fig. 4, and the tabulated
results of all 15 of the autoantibody preparations tested are shown in
Table II. The data show that most of the
mutants were generally within 10-fold of the wild-type
2GPI concentration in their ability to achieve
50% inhibition. By contrast, the G40E and R43G mutant
2GPI proteins were much less effective in
inhibiting the autoantibodies than was wild-type
2GPI, and in most cases were unable to achieve
50% inhibition at concentrations up to 1000-fold greater than that
required for the wild type. The K19E mutation generally required
concentrations 10- to 500-fold greater than wild-type
2GPI to achieve 50% inhibition.
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, show results similar to those
obtained in the competitive inhibition assays.
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The amino acids R43 and G40 are exposed to solvent and located
close together on one face of 2GPI domain I.
Comparative modeling and electrostatic calculations of the G40E and
R43G mutants predict an alteration of the surface shape and
electrostatic charge of domain I. The disrupted region includes both a
protruding mobile loop and the adjacent concave surface (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2GPI
(11). In that report we used a competitive inhibition
ELISA employing a panel of whole domain deletions in recombinant
2GPI. It is possible that the deletion of
whole domains might have altered the conformation of the remaining
domains in such a way as to prevent the binding of autoantibodies,
thereby giving a false impression of their domain specificity. In this
study we employed only full-length
2GPI (all five domains), each having a
single amino acid substitution in domain I. These single point mutants
were compared with full-length wild-type 2GPI
for their ability to bind anti-2GPI
autoantibodies from APS patients.
mAbs were used in both direct binding and competitive inhibition assays
to assess whether the selected mutations in domain I altered the
conformation of the molecule. With one exception (APS patient-derived
mAb EY2C9), binding of mAbs was unaffected by single amino acid
substitutions. These results confirm that the overall conformation of
2GPI was not changed. The data further show
that the substituted amino acids are not critical components of the
epitopes recognized by the mouse mAbs. Importantly, the data show that
all the mutants were coated equally on the microplate wells in the
direct binding assays, as mouse mAbs FC1, 10, and 16 bound equivalently
to wild-type 2GPI and to each of the
mutants.
Contrary to this finding, binding of human mAb EY2C9 was affected by a number of mutations in domain I. There was essentially no binding to T50A and markedly decreased binding to the mutations at aa 40–43. Paradoxically, EY2C9 was previously reported to bind domain IV (17). The current study strongly supports localization of an epitope for EY2C9 on domain I.
Autoantibodies from patient sera were also affected by mutations in
domain I, particularly in the region of aa 40–43 (G40E, M42V, and
R43G). With some patient sera there was a reduction in direct binding
by 80–90% relative to wild-type 2GPI (Table I
). In the competitive inhibition studies the same mutants were
generally unable to achieve 50% inhibition at concentrations 1000-fold
higher than required with the wild type (Table II). These data clearly
show that domain I contains the dominant epitope(s) for
anti-2GPI associated with APS.
While there are some differences in the results from the two different assay methods, the overall pictures from both are similar. The main difference is that mutation K19E had a greater effect in the direct binding assays than in the competitive inhibition assays. However, autoantibody binding to K19E was diminished in both assays. It is unclear whether K19 is part of the same epitope as that affected by G40E and R43G for some Abs, if K19E affects other amino acids that are part of that epitope, or if K19E affects a second, distinct epitope.
The conformation of a region in a protein can be altered by a point
mutation to a degree that depends on the specific substitution and the
residues adjacent to that substitution (24). Modeling of
domain I predicts that the G40E and R43G mutations are surface exposed,
and they are likely to induce conformation and electrostatic changes
over a surface region of the domain, as shown in Fig. 5. The altered
area of the domain is therefore likely to contribute significantly to
the Ab-Ag binding either because G40 and R43 are themselves critical
residues of the epitope or because the substitutions at these points
affect the conformation of the nearby critical residues. The
autoantibodies and the mAbs do not bind to reduced and alkylated domain
1 (data not shown), further suggesting that the epitope(s) is
conformational and not linear.
A similar restriction of autoantibodies to a single immunodominant region has been shown for other autoantigens, such as thyroid peroxidase (25). Given that potential epitopes would be expected to be found over much of the protein (26), this restriction of autoantibody repertoire found in both our study and another suggests that generation of autoantibody-producing B cell clones is influenced by other factors in addition to the availability of epitopes.
In conclusion, this study has shown that single amino acid
substitutions in domain I of full-length 2GPI
disrupt autoantibody binding to 2GPI. This was
demonstrated with competitive inhibition and direct binding assays. The
competitive inhibition method used mutants in fluid phase to circumvent
difficulties with ensuring adequate Ag binding to the solid phase of
the microplate well. This study provides strong support for the
hypothesis that the epitopes for anti-2GPI
autoantibodies from patients with APS are located on domain I of
2GPI. Amino acids 40–43 form a
surface-exposed region that contributes to the immunodominant
epitope.
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Acknowledgments |
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Footnotes |
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2 G.M.I. and S.R. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. G. Michael Iverson, La Jolla Pharmaceutical Co., 6455 Nancy Ridge Drive, San Diego, CA 92121. E-mail address: mike.iverson{at}ljpc.com
4 Abbreviations used in this paper: 2GPI, 2-glycoprotein 1; aCL, anticardiolipin; APS, antiphospholipid syndrome; AU, absorption units; NFDM, nonfat dried milk.
Received for publication March 22, 2002. Accepted for publication October 8, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 T. Shi, G. M. Iverson, J. C. Qi, K. A. Cockerill, M. D. Linnik, P. Konecny, and S. A. Krilis {beta}2-Glycoprotein I binds factor XI and inhibits its activation by thrombin and factor XIIa: Loss of inhibition by clipped {beta}2-glycoprotein I PNAS, March 16, 2004; 101(11): 3939 - 3944. [Abstract] [Full Text] [PDF]
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