International Immunology

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International Immunology, Vol. 12, No. 8, 1183-1192, August 2000
© 2000 Japanese Society for Immunology

Proteolytic cleavage of ß₂-glycoprotein I: reduction of antigenicity and the structural relationship

Eiji Matsuura¹, Junko Inagaki¹, Hideki Kasahara², Daisuke Yamamoto³, Tatsuya Atsumi², Kazuko Kobayashi¹, Keiko Kaihara¹, Dandan Zhao^1,4, Kenji Ichikawa², Akito Tsutsumi², Tatsuji Yasuda¹, Douglas A. Triplett and Takao Koike²

¹ Department of Cell Chemistry, Institute of Cellular and Molecular Biology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
² Department of Medicine II, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
³ Biomedical Computation Center, Osaka Medical College, Takatsuki 569-8686, Japan
⁴ Department of Pathology, Ball Memorial Hospital, Muncie, IN 47303, USA

Correspondence to: E. Matsuura

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Binding of ß₂-glycoprotein I (ß₂-GPI)-dependent anticardiolipin antibodies (aCL) derived from antiphospholipid syndrome (APS) is significantly reduced in aCL ELISA due to loss of the phospholipid (PL) binding property of ß₂-GPI by plasmin treatment. In the present study, the treatment generated a nicked form of ß₂-GPI and resulted in loss of antigenicity for the autoantibodies detected in ELISA, using an ß₂-GPI directly adsorbed polyoxygenated carboxylated plate, the assay system of which was not related to PL binding. The nicked form bound to neither Cu²⁺-oxidized low-density lipoprotein (oxLDL) nor to ß₂-GPI-specific lipid ligands isolated from oxLDL, the result being a complete loss of subsequent binding of anti-ß₂-GPI autoantibodies. The conformational change in the nicked domain V was predicted from its intact structure determined by an X-ray analysis (implemented in Protein Data Bank: 1C1Z), molecular modeling and epitope mapping of a monoclonal anti-ß₂-GPI antibody, i.e. Cof-18, which recognizes the related structure. The analysis revealed that novel hydrophobic and electrostatic interactions appeared in domain V after the cleavage, thereby affecting the PL binding of ß₂-GPI. Such a conformational change may have important implications for exposure of cryptic epitopes located in the domains such as domain IV.

Keywords: antiphospholipid syndrome, anti-ß₂-glycoprotein I antibodies, epitope mapping, plasmin, structure

	Introduction

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Antiphospholipid syndrome (APS) is characterized by the predominant clinical features of venous and/or arterial thrombosis, recurrent pregnancy loss, and thrombocytopenia, accompanied by autoantibodies, i.e. antiphospholipid antibodies (aPL) (1–4). In 1990, three individual research groups, including ours, first reported that a 50 kDa plasma cofactor is required for cardiolipin (CL) binding of anticardiolipin antibodies (aCL) found in APS patients (5–7). In recent years, ß₂-glycoprotein I (ß₂-GPI), which binds to anionic phospholipids (PL), is thought to be the major antigen for aCL. We also reported that epitopes for such aCL are cryptic and that they appear in domain IV of ß₂-GPI only when ß₂-GPI interacts with lipid membranes containing anionic PL, such as CL and phosphatidylserine, or with a polyoxygenated polystyrene surface (8,9). Alternatively, it has been proposed that ß₂-GPI binding to anionic PL increases the local concentration of ß₂-GPI, thus promoting an increase in intrinsic affinity and antibody binding to ß₂-GPI (10,11).

ß₂-GPI is a plasma glycoprotein first described by Schulze et al. in 1961 (12). ß₂-GPI consists of a single polypeptide chain containing 326 amino acid residues and five potential N-glycosylation sites (13,14). The amino acid sequence is highly conserved (>80%) among the human, bovine and mouse counterparts (15). The ß₂-GPI chain is typically folded into five contiguous homologous `sushi' domains, which correspond to the short consensus repeat (SCR) of the complement control protein superfamily. Each domain has two disulfide bridges but domain V diverges from the SCR module as it has an additional disulfide bridge and a long C-terminal loop (14,16). The crystal structure of human ß₂-GPI was recently reported (17,18).

Domain V of ß₂-GPI has a potent PL binding region and a structure consisting of a particular region, K²⁸²NKEKK²⁸⁷, was identified using synthesized peptides and mutant proteins of ß₂-GPI (19–23). X-ray analysis on ß₂-GPI (17) showed that the PL binding is provided by a patch consisting of 14 residues of positively charged amino acids on the aberrant half of domain V and by a flexible loop of S³¹¹–K³¹⁷. Sequential bindings of ß₂-GPI to PL such as CL and of ß₂-GPI-dependent aCL are significantly reduced by cleavage of the peptide bond between K³¹⁷ and T³¹⁸ on the C-terminal loop (20,21). Either factor Xa or plasmin specifically cleaves the site (24,25).

ß₂-GPI affects multiple PL-dependent coagulation pathways but also lipoprotein metabolism (26), and clearance of `non-self' particles and senescent/apoptotic cells (27–29). Previous in vitro studies also suggested that ß₂-GPI mediates activation of platelets (30) and endothelial cells (31) by aPL. An early event in atherosclerosis is the accumulation of cholesterol-laden foam cells, which originate mainly from monocyte macrophage cells (32–34), by their uptake of chemically modified low-density lipoprotein (LDL), such as Cu²⁺-oxidized LDL (oxLDL) (35,36). We reported that ß₂-GPI directly binds to oxLDL, and that the complex of oxLDL and ß₂-GPI is subsequently recognized by anti-ß₂-GPI autoantibodies to be taken up by macrophages (37). Romero et al. reported that development of arterial thrombosis is associated with anti-ß₂-GPI antibodies but not with anti-oxLDL antibodies in systemic lupus erythematosus and the secondary APS (38).

In the present study, we found that ß₂-GPI treatment with plasmin reduced its antigenicity detected in antibody ELISA when we used a ß₂-GPI adsorbed polyoxygenated (carboxylated) plate or using an oxLDL-derived antigen-adsorbed plain plate. Further, these reductions were accompanied by a particular conformational change in domain V.

	Methods

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Microtiter plates
Plain (Sumilon S-type) and polyoxygenated (carboxylated, Sumilon C-type) polystyrene plates were obtained from Sumitomo Bakelite (Tokyo, Japan). Density of oxygen atoms covalently introduced onto their surfaces was analyzed as follows: the plain plate, C–O, 1.3 mol%; and the polyoxygenated plate, C–O, 10.5 mol%, C=O, 5.6 mol%, and O–C=O, 3.7 mol% (9). Another plate (Immulon IIHB) was obtained from Dynex Technologies (Chantilly, VA).

Sera and mAb
ß₂-GPI-dependent aCL⁺ sera were obtained from secondary APS patients with systemic lupus erythematosus. Two human monoclonal aCL, i.e. EY1C8 and EY2C9 (anti-ß₂-GPI antibodies), originated from an APS patient (39). Mouse monoclonal anti-human ß₂-GPI antibodies (Cof-18, -20, -21, -22 and -23) were prepared from splenocytes of BALB/c mice immunized with human ß₂-GPI in complete Freund's adjuvant (40,41).

Human ß₂-GPI and domain V
ß₂-GPI was purified from normal human plasma by sequential CL-affinity column, ion-exchange column and Protein A–Sepharose column chromatography (8). A recombinant domain V protein was expressed in methylotrophic yeast, Pichia pastoris and was purified (23).

Treatment with plasmin
ß₂-GPI (2 mg) and domain V (0.5 mg) were treated with human plasmin (Calbiochem-Novabiochem, La Jolla, CA) in 2 ml of PBS at 37°C for 3 h. The reaction was performed at a molar ratio of plasmin:substrate of 1:3.2 and was terminated by adding 3 mg of aprotinin. The nicked samples were subsequently purified by affinity chromatography, using anti-ß₂-GPI– and heparin–Sepharose columns and by an ion-exchange chromatography, using a Mono-Q column (Pharmacia-Biotech, Uppsala, Sweden).

SDS–PAGE and immunoblot
Reduced samples were subjected to 12 or 16.5% SDS–PAGE, according to Laemmli (42). Proteins were transferred onto a nitrocellulose sheet and the sheet was blocked by incubation with 10 mM Tris–HCl/150 mM NaCl, pH 7.4, containing 0.05% Tween (TBS/Tween) and 3% skim milk. Following incubation with anti-ß₂-GPI antibodies in TBS/Tween, the sheet was exposed to horseradish peroxidase (HRP)-labeled anti-mouse IgG antibodies and then to 4-chloro-1-naphthol with H₂O₂. Between each step, extensive washings were done using TBS/Tween.

Anti-ß₂-GPI ELISA
Anti-ß₂-GPI ELISA was performed as described (9). Briefly, ß₂-GPI or domain V was adsorbed on a plain (Sumilon S-type) or polyoxygenated (carboxylated, Sumilon C-type) plate by incubating at 10 µg/ml (50 µl/well). The plates were blocked with 3% gelatin and 100 µl/well of anti-ß₂-GPI mAb (1 µg/ml) or of 100-fold diluted serum samples were applied for 1 h. Antibody binding to ß₂-GPI was probed using HRP-labeled anti-mouse IgG antibodies or HRP-labeled anti-human IgG/IgM antibodies. o-Phenylenediamine solution containing H₂O₂ was applied. The color reaction was stopped by adding 2 N H₂SO₄. Optical density was measured at 492 nm. Between each step, we used PBS containing 0.05% Tween 20 (PBS/Tween) for extensive washings.

Competitive assay for ß₂-GPI binding to solid-phase CL
CL (2.5 µg in 50 µl of ethanol/well) was adsorbed by evaporation on a plain plate. The plate was blocked with 1% BSA and then biotinyl-ß₂-GPI was incubated with the indicated concentrations of mouse anti-ß₂-GPI mAb in the CL plates for 1 h. ß₂-GPI binding was detected using HRP-labeled avidin. Further steps were as described in `Anti-ß₂-GPI ELISA'.

Preparation of oxLDL- and ß₂-GPI-specific ligands
LDL, prepared by ultracentrifugation of normal human plasmas, was oxidized by incubation with CuSO₄, as described (37). To isolate ligands specific for ß₂-GPI from oxLDL preparation, lipid extraction (43), and silica-gel TLC (Kiesegel 60, Merck, Darmstadt, Germany; solvent (i): chloroform/methanol/H₂O/NH₄OH: 60/40/5/2.5 and solvent (ii): chloroform/methanol: 8/1) and reversed-phase HPLC on Sephasil peptide C18 (Pharmacia-Biotech, Uppsala, Sweden) (solvent: acetonitrile/ isopropanol: 30/70) were subsequently done. The ß₂-GPI-specific ligands were monitored by a ligand blot analysis using ß₂-GPI, anti-ß₂-GPI mAb (Cof-22 and EY2C9) and HRP-labeled anti-mouse IgG antibodies/anti-human IgM antibodies. These ligands were characterized as oxidative products of ketocholesteryl-linoleate (submitted).

Assay for ß₂-GPI binding to oxLDL
F(ab)'₂ of anti-apoB100 mAb, i.e. 1D2 mAb, was adsorbed on a microtiter plate (Immulon IIHB) by incubating with 5 µg/ml (50 µl/well). The plate was blocked with 1% skim milk, followed by incubating with 10 µg/ml of native LDL or Cu²⁺-oxLDL for 1 h. Then, ß₂-GPI (10 µg/ml), Cof-22 (1 µg/ml) and HRP-labeled anti-mouse IgG antibodies were subsequently incubated for each 1 h. Further steps were as described under methods for `Anti-ß₂-GPI ELISA'.

Assay for ß₂-GPI and antibody binding to ß₂-GPI-specific ligands
ß₂-GPI-specific ligands (1.25 µg in 50 µl of ethanol/well) were adsorbed by evaporation on a plain plate and the plate was then blocked with 1% BSA. For detection of ß₂-GPI binding, ß₂-GPI (15 µg/ml), anti-ß₂-GPI antibody (Cof-22, 1 µg/ml) and HRP-labeled anti-mouse IgG antibodies were subsequently incubated. For detection of antibody binding, intact or the nicked ß₂-GPI or domain V (15 µg/ml), anti-ß₂-GPI mAb (1 µg/ml) or 100-fold diluted serum samples, and HRP-labeled anti-human IgG or IgM antibodies were subsequently incubated. Further steps were as described under methods for `Anti-ß₂-GPI ELISA'.

Construction and optimization of the nicked domain V's conformation
A possible conformation of the nicked domain V was constructed from its intact structure determined by an X-ray analysis (implemented in Protein Data Bank: 1C1Z), based on molecular modeling. According to the high thermal factors of residues of S³¹¹–W³¹⁶, and the presence of a hydrophobic hole composed of F²⁷⁹, F²⁸⁰ and F³⁰⁷, we predicted a possibly hydrophobic interaction between a region F³¹⁵–W³¹⁶ and the hole in the nicked ß₂-GPI. An initial model was created by following two processes using Quanta 97 (Molecular Simulations, San Diego, CA): (i) cleavage of the peptide bond of K³¹⁷–T³¹⁸, and (ii) closing the distance in 5Å between the side chains of amino acids related to the interaction described above.

The initial model was then optimized by 2000 cycles of energy minimization by the CHARMm program (44), with hydrophilic hydrogen atoms and TIP3 water molecules (45) in 8Å radius around the protein, adopting the standard amino acid parameters in the CHARMm force field. Molecular dynamics simulation (5 ps) of the model was then performed with 0.002 ps time steps. Cut-off distance for non-bonded interactions was set to 15Å and the dielectric constant was 1.0 when calculating the electrostatic energy. A non-bonded pair list was updated every 10 steps. Finally, the model was again optimized by 2000 cycles of energy minimization. The nicked domain V consisted of 82 amino acid residues (811 protein atoms) with 585 water molecules and had a total energy of –1.64x10⁴ kcal/mol, and averaged root-mean-square forces 0.355. For purposes of comparison with the nicked model, optimization and dynamics simulation were also performed in the case of the original X-ray structure of intact domain V. The optimized structures of intact and the nicked structures had a total energy of –3.86 x10³ and –4.11x10³ kcal/mol, under conditions where water molecules were omitted respectively.

Epitope mapping
Epitope mapping of Cof-18 mAb was performed by using a combinatorial library of a 12mer random peptide fused with a minor coat protein (pIII) of the filamentous coliphage, M13 (PH.D-12 Kit, New England Biolabs, Beverly, MA). A plain plate was coated with Cof-18 mAb (100 µg/ml) and blocked with 5 mg/ml of BSA. Phages (2x10¹²/ ml) were incubated for 1 h and the bound phages were eluted with 0.2 M glycine–HCl (pH 2.2). After three rounds of the biopanning, the specific phages were cloned and sequenced.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Proteolytic cleavage of human ß₂-GPI
ß₂-GPI nicked at the site between K³¹⁷ and T³¹⁸ was generated by a limiting enzymatic cleavage with plasmin. Almost complete cleavage (>98%) occurred in both ß₂-GPI and domain V by treatment at 37°C for 3 h (Fig. 1A).

View larger version (50K): [in this window] [in a new window]   Fig. 1. SDS–PAGE and immunoblot of ß2-GPI and domain V. (A) Structures of ß2-GPI and domain V, treated with or without plasmin (T or NT respectively), were subjected to 16.5% SDS–PAGE under the reducing conditions. (B) Transferred proteins were stained with mouse anti-human ß2-GPI mAb and HRP-labeled anti-mouse IgG antibodies.

 
Features of anti-ß₂-GPI mAb
We previously established mouse anti-ß₂-GPI mAb (i.e. Cof-18, -20, -21, -22 and -23), and determined that their epitopes to locate in domains V, III, IV, III, and IV, respectively (40,41). Immunoblot analysis showed that four mAb, i.e. Cof-20, -21, -22 and -23, reacted with both intact and the nicked ß₂-GPI proteins but not with any domain V proteins (Fig. 1B), because their epitopes are present in domain III or IV. In contrast, Cof-18 mAb bound to intact ß₂-GPI and domain V but not to their nicked forms. Three antibodies, i.e. Cof-20, -21 and -22, revealed almost equal binding to intact and the nicked ß₂-GPIs when adsorbed on either a plain or a polyoxygenated plate (Fig. 2). In ELISA using ß₂-GPI-adsorbed plates, binding of Cof-18 (specific to domain V) and Cof-23 (to domain IV) was affected by treatment of ß₂-GPI with plasmin. In particular, Cof-18 binding to the nicked ß₂-GPI adsorbed on those plates was diminished.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Binding of mouse Cof mAb to intact or nicked ß2-GPI in anti-ß2-GPI ELISA. Cof mAb were incubated in wells of a ß2-GPI protein-adsorbed plain (A) or polyoxygenated (B) plate. Antibody binding was detected with HRP-labeled anti-mouse IgG antibodies. Open columns, native ß2-GPI; solid columns, nicked ß2-GPI.

 
As shown in Fig. 3, Cof-18 mAb inhibited the ß₂-GPI binding to solid-phase CL in a dose-dependent manner. Thus, Cof-18 mAb competitively affected PL binding of ß₂-GPI. The specificity of Cof-18 was confirmed using other irrelevant Cof mAb. In contrast, Cof-18 mAb inhibited the plasmin-mediated cleavage in domain V (Fig. 4). Other irrelevant Cof antibodies or an anti-ß₂-GPI autoantibody, EY2C9, did not affect the cleavage (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Inhibitory effect of the simultaneous addition of Cof mAb on ß2-GPI binding to solid-phase CL. Cof mAb were incubated at varying concentrations with biotinyl-ß2-GPI in a CL-adsorbed plain plate. ß2-GPI binding to solid-phase CL was detected with HRP-labeled avidin. •, Cof-18; , Cof-20; , Cof-21; , Cof-22; , Cof-23.

 

View larger version (39K): [in this window] [in a new window]   Fig. 4. Prevention of plasmin-mediated cleavage in domain V by Cof-18 mAb. ß2-GPI was incubated with plasmin and Cof-18 mAb (ß2-GPI:IgG 1:5, molar ratio) at 37°C for 3 h. Reaction mixtures were subjected to 12% SDS–PAGE under reducing conditions.

 
Features of autoimmune anti-ß₂-GPI antibodies
In ELISA using a polyoxygenated carboxylated plate, anti-ß₂-GPI mAb, EY1C8 and EY2C9, and antibodies in all tested anti-ß₂-GPI⁺ sera (n = 30) bound only to intact ß₂-GPI but not to the nicked one (Fig. 5). No antibodies in the APS sera bound to either forms of domain V.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Binding of anti-ß2-GPI autoantibodies to a ß2-GPI protein adsorbed on a polyoxygenated plate. Anti-ß2-GPI mAb (A) and anti-ß2-GPI antibody-positive serum samples (A and B) were incubated in an intact or nicked ß2-GPI (or domain V) adsorbed polyoxygenated plate. Antibody binding was detected with HRP-labeled anti-human IgG or IgM antibodies.

 
Specific binding of ß₂-GPI to oxLDL and to the ligand derived from oxLDL was completely lost by plasmin treatment (Fig. 6A–C). Subsequent binding of anti-ß₂-GPI antibodies derived from APS patients was diminished due to lack of the nicked ß₂-GPI binding to the solid-phase ligands (Fig. 6D).

View larger version (28K): [in this window] [in a new window]   Fig. 6. ß2-GPI and subsequent anti-ß2-GPI autoantibody binding to LDL and the ß2-GPI-specific ligand derived from oxLDL. Native LDL (A) or oxLDL (B) was incubated in an anti-apoB100 F(ab)'2-adsorbed plain plate. The ß2-GPI-specific ligand (C) was directly adsorbed on a plain plate. Intact or nicked ß2-GPI binding was detected with anti-ß2-GPI mAb (Cof-22) and HRP-labeled anti-mouse IgG antibodies. (D) Intact or nicked ß2-GPI or domain V antibodies and HRP-labeled anti-human IgG or IgM antibodies were subsequently incubated in a ß2-GPI-specific ligand-adsorbed plain plate to detect autoantibody binding.

 
Structure of the nicked domain V
We predicted the conformation of nicked domain V from the X-ray structure of the intact domain (Fig. 7). In intact domain V, a hydrophobic hole, consisting of F²⁷⁹, F²⁸⁰ and F³⁰⁷, was hidden by the C-terminal region, T³¹⁸–K³²⁴, and another hydrophobic region, F³¹⁵ and W³¹⁶, was exposed to the solvent (Fig. 8). The nicked structure was constructed by considering interactions between these two hydrophobic regions and optimized with the solvent phase. The nicked structure was more stable than the intact one (total energy –3.86x10³ kcal/mol for intact domain V and –4.11x 10³ kcal/mol for the nicked one).

View larger version (120K): [in this window] [in a new window]   Fig. 7. Structural representations of intact and the nicked domain V of human ß2-GPI. Ribbon representation with the secondary structure elements. The ß-sheets are shown in blue, K317 in green, T318 in yellow, and a disulfide bond between C288 and C326 in purple.

 

View larger version (100K): [in this window] [in a new window]   Fig. 8. Superimposed stereoviews of the partial structure of intact ß2-GPI from X-ray crystallography and its possible structure of the nicked form. The residues from E309 to C326 and C288 of the intact structure are indicated by a blue line, except for K317 (green), T318 (yellow), and the cleaved peptide bond between K317 and T318 (light blue). The residues from E309 to W316 and those from D319 to C326 in the nicked structure are indicated by red and orange lines respectively. Color schemes of K317, T318 and cleaved peptide of the nicked form are similar to those of the intact one. Hydrophobic residues of F279, F280 and F307 (white lines) are able to interact with F315 and W316 (red bold lines), and these residues are shown with transparent spacefills. Novel electrostatic interactions of K250–E309, K262–K317 (the C-terminal charge), E265–K317, R260–D319/D222 and T318 (the N-terminal charge)–D319/D222 are shown in the nicked structure. Panel (B) is a view of panel (A) rotated by 90° on the horizontal axis.

 
In the nicked structure, the side chains of F³¹⁵ and W³¹⁶ oriented to the hydrophobic hole, and those of D³¹⁹ and D³²², containing in the C-terminal loop (colored orange), were tightly fixed by electrostatic interaction with R²⁶⁰. Positive charges of K³¹⁷ and K²⁵⁰ were weak due to their interaction with E²⁶⁵ and E³⁰⁹ respectively. Positive charges of five residues, i.e., K²⁵⁰, R²⁶⁰, K²⁶², K²⁸⁴ and K³²⁴, on the patch (17) were neutralized by interactions with carboxyl groups of E³⁰⁹, D³¹⁹/D³²², K³¹⁷ (on the main chain), E²⁸⁵ and C³²⁶ (on the main chain) respectively. Further, the patch was dislocated by the relocation of D³¹⁹ and D³²².

Epitopic structure for Cof-18 mAb
As an approach to characterize the conformational change which occurred in domain V, we analyzed the epitopic structure recognized by Cof-18 mAb because the mAb recognized a structure related to both PL binding and plasmin-mediated cleavage. Phage clones carried the consensus sequence GMKLTSEQKAML and SDWRLMFESWIR (Table 1). The amino acid sequence SE*K, located downstream of the first consensus sequence, was closely related to the sequence SD*R located upstream of the second ones. The suggested corresponding amino acid residues from these two consensus sequences are shown in the intact structure (Fig. 9A). A cluster composed of these amino acid residues for the epitopic structure located across three discontinuous loops.

View this table: [in this window] [in a new window]   Table 1. Consensus amino acid sequences deduced from cloned phages and an epitope for Cof-18 mAb

 

View larger version (106K): [in this window] [in a new window]   Fig. 9. Epitopic structure of Cof-18 mAb. Space-filling models of intact domain V (A) and the nicked one (B) are shown.

 
We synthesized a 16mer peptide, i.e. pCof-18, which was basically designed from these two consensus sequences and several 12 mer scrambled control peptides. Binding of phage #1 to the solid-phase Cof-18 mAb was stoichiometrically evident by analysis using the IAsys optical biosensor (Fig. 10A). Phage binding to Cof-18 mAb was dose-dependently inhibited by the simultaneous addition of pCof-18 (Fig. 10B). The binding was not affected by any control peptides tested in the system. The inhibitory effect of ß₂-GPI on phage binding to Cof-18 antibody was observed in ELISA, using the solid-phase phage #1 (Fig. 10C). The structural analysis indicated that the cluster of amino acids which formed a Cof-18's epitope stretched after the cleavage (Fig. 9B).

View larger version (23K): [in this window] [in a new window]   Fig. 10. Analysis of interactions among the cloned phages, Cof-18 mAb, and the synthesized peptide, pCof-18. (A) Biotinyl-Cof-18 mAb was immobilized on the wall of a biotinyl-cuvette via streptavidin. Different concentrations of (1) 2.6x108, (2) 6.5x108, (3) 1.3x109, (4) 2.6x109 and (5) 5.2x109/ml of cloned phage #1 were added to the cuvette and phage binding was measured using an optical biosensor, IAsys. Plots of measured on-rate constant (kon) for concentrations of phage are also indicated. (B) Inhibitory effect of pCof-18 on phage #1 binding to solid-phase Cof-18 mAb was assayed by the IAsys. (C) Inhibitory effect of ß2-GPI on Cof-18 mAb binding to the solid-phase phage #1 was assayed using ELISA. Indicated concentrations of ß2-GPI and Cof-18 mAb were incubated in the phage #1-adsorbed plate (by previously incubating with 6x1011/well) for 1 h and antibody binding was then measured with HRP-labeled anti-mouse IgG antibodies.

 

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In 1994, we demonstrated that anti-ß₂-GPI autoantibodies bind to ß₂-GPI adsorbed on a polyoxygenated but not on a plain plate and also considered that those epitopes are probably cryptic (9). Later, it was shown that domain IV is dominantly involved in expression of such epitopes, determined using a series of deletion mutants of ß₂-GPI (40). So far as tested in the present study, anti-ß₂-GPI autoantibodies did not bind directly to any solid-phase domain V (Fig. 5). By epitope mapping and structural analysis, we identified fine structures of those epitopes located in domain IV (submitted). Thus, it is most likely that anti-ß₂-GPI autoantibodies recognize cryptic epitopes located in domain IV. In the present study, we showed that ß₂-GPI treatment with plasmin reduced expression of the epitopes and the reduction was accompanied by a particular conformational change in domain V.

It was reported that binding of aCL from sera of APS patients in ELISA using a CL-adsorbed plate was diminished due to the loss of PL binding by the plasmin-mediated cleavage of ß₂-GPI (20). Human and mouse anti-ß₂-GPI autoantibodies did not bind to the nicked ß₂-GPI in an anti-ß₂-GPI ELISA when we used a polyoxygenated carboxylated plate, on which antigen proteins were directly adsorbed but not so with any PL (Fig. 5). In contrast, ß₂-GPI binding of irrelevant mouse anti-human ß₂-GPI mAb was equal to its nicked form adsorbed either on a plain or polyoxygenated plate. These observations imply that proteolytic cleavage does not simply reduce PL binding but it also prevents exposure of the cryptic epitopes.

Conformational changes occurring with the cleavage were analyzed by constructing the nicked structure. A hydrophobic region, F³¹⁵–W³¹⁶, originating in the flexible C-terminal loop (17) possibly interacted with another hydrophobic hole consisting of F²⁷⁹, F²⁸⁰ and F³⁰⁷, the result being reduced positive charges on the patch and dislocation of the patch (Fig. 8). The epitope of Cof-18 mAb located in close proximity in the cleaved region and the positively charged patch was stretched in the nicked ß₂-GPI (Fig. 9). Thus, the structural analysis of the nicked domain V indicates a possible relationship between the plasmin-mediated cleavage and the loss of the PL binding.

Against an `elongated' crystal structure of ß₂-GPI, (17,18), we constructed an alternative model, in which three electrostatic interactions, i.e. D¹⁹³–K²⁴⁶, D²²²–K³⁰⁵ and E²²⁸–K³⁰⁸, were observed between domain IV and V (submitted). We also found in the study that binding of anti-ß₂-GPI autoantibodies was significantly reduced by replacement of these three acidic amino acids in domain IV by valines (V). These observations also supported the notion that the conformational change in domain V may affect exposure of the cryptic epitopes in domain IV.

In contrast, Iverson et al. (46) reported mainly detecting autoantibodies directed to an intact structure of domain I, in ELISA using deletion mutants proteins similar to those we had previously designed (40). A lysine-enriched patch for PL binding was found to locate on a surface of domain V (17). It is generally thought that ß₂-GPI is adsorbed on a CL-plain polystyrene plate, via both hydrophobic and electrostatic interactions to express antigenicity for aCL. However, their mutant proteins were stretched on the nickel chelate-coated plate via their extra C-terminal glycine (G)–histidine (H) six residues but neither via the PL binding region nor by hydrophobic protein adsorption. We are entertaining the notion that both the orientation of adsorbed ß₂-GPI and direct adsorption via the PL binding region might be essential for expression of such cryptic epitopes, even in anti-ß₂-GPI ELISA using a polyoxygenated plate.

Numerous in vitro studies showed that ß₂-GPI acts as a natural anti-coagulant protein and that autoimmune abnormalities accompanied by the induction of anti-ß₂-GPI autoantibodies may lead to a pathological stage, i.e. athero-thrombosis. We also reported that subsequent binding of ß₂-GPI and anti-ß₂-GPI autoantibodies directed to oxLDL enhanced oxLDL uptake by macrophages (37). Further, several in vivo studies attractively demonstrated pathogenesis of anti-ß₂-GPI antibodies, i.e. immunization with ß₂-GPI-induced autoantibodies in mice and rabbits (47), induced APS-like syndrome (48), and accelerated atherosclerosis in mice (49). The present study showed that ß₂-GPI binding and subsequent binding of anti-ß₂-GPI autoantibodies either to oxLDL or to the ß₂-GPI-specific ligands from oxLDL were lost by plasmin treatment (Fig. 5). It is likely that the plasmin-mediated cleavage has important implications for down-regulation of `autoimmune' athero-thrombosis in APS.

One possible hypothesis that has been advocated is that anti-ß₂-GPI autoantibodies probably prevented natural anticoagulant activity of ß₂-GPI that affects the PL-dependent coagulation cascades and it results in the thrombotic events developing. Brighton et al. first found that ß₂-GPI levels in plasma were significantly low in patients with thrombosis or disseminated intravascular coagulation syndrome (DIC) (50). Horbach et al. recently reported that plasma ß₂-GPI levels were lower in patients with DIC as compared with controls and nicked ß₂-GPI levels were contrarily high (51). They suggested that the reduction of ß₂-GPI levels and the generation of nicked ß₂-GPI occurred due to absorption to apoptotic cells and activation of fibrinolysis respectively. Kato et al. reported that the nicked form of ß₂-GPI was generated either by factor Xa or by plasmin, but the latter was much more effective (24,25), and that an increment of serum nicked ß₂-GPI was observed in APS patients (52). Thus, the cleavage of ß₂-GPI by the multiple cascades on blood coagulation or fibrinolysis, which leads its reduced antigenicity, may play a role in a natural self-defense against the autoimmunity to develop thrombosis in APS. The clinical significance of the nicked ß₂-GPI should be elucidated in future studies.

	Acknowledgments

 
This work was supported in part by a grant for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant from the Ministry of Health and Welfare of Japan. We thank Drs Y. Goto and H. Kato (Osaka, Japan) for providing the domain V protein, and M. Ohara for pertinent comments.

	Abbreviations

 

aCL anticardiolipin antibody

aPL antiphospholipid antibody

APS antiphospholipid syndrome

ß2-GPI ß2-glycoprotein I

CL cardiolipin

DIC disseminated intravascular coagulation syndrome

HRP horseradish peroxidase

LDL low-density lipoprotein

oxLDL oxidized LDL

PL phospholipid

SCR short consensus repeat

	Notes

 
Transmitting editor: K. Okumura

Received 9 March 2000, accepted 2 May 2000.

	References

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