- Email: [email protected]
A Stable, Multi-Subunit Complex of β2Glycoprotein I
Thrombosis Research 90 (1998) 131–137
REGULAR ARTICLE
A Stable, Multi-Subunit Complex of b2Glycoprotein I
Margaret Galazka1,3, Lynn B. Keil2,3, Joseph D. Kohles1, Jianmin Li1, Stephen P. Kelty1, Matthew Petersheim1 and Vincent A. DeBari2,3 1 Department of Chemistry, College of Arts and Sciences, and 2Department of Medicine, School of Graduate Medical Education, Seton Hall University, South Orange, New Jersey and 3 The Rheumatology Laboratory, St. Joseph’s Hospital and Medical Center, Paterson, New Jersey. (Received 24 September 1997 by Editor D.A. Triplett; revised/accepted 3 March 1998)
Abstract b2glycoprotein I (b2GPI) is a 54-kDa plasma protein which is recognized as an autoantigen for antibodies from patients with antiphospholipid syndrome (APS). SDS-PAGE (under reducing conditions) of b2GPI from three sources indicates that the 54-kDa b2GPI band is accompanied by a band corresponding to an 8-kDa protein. In the absence of detergent and reducing agents (native PAGE), b2GPI demonstrated a large complex (molecular mass ≈320 kDa) which is dissociable by boiling in 6–8 M urea, yielding several lower molecular mass bands, one of which corresponds to the 8-kDa protein observed in SDS-PAGE. Sera from five healthy adults demonstrated native b2GPI migration equivalent to the commercially purified protein. Atomic force microscopy (AFM) images of native b2GPI show aggregrates of particles each having a diameter of 30–35 nm. This is consistent with a globular unit the size of which would be substantially larger than that expected for a 54Abbreviations: AFM, atomic force microscopy; APS, antiphospholipid syndrome; b2GPI, b2glycoprotein I; b-me, b-mercaptoethanol; BSA, bovine serum albumin; HEPES, hydroxyethylpiperazine ethanesulfonate; PBS, phosphate-buffered saline; tricine, N-tris hydroxymethyl methylglycine; tris, tris hydroxymethylaminomethane. Corresponding author: Vincent A. DeBari, St. Joseph’s Hospital and Medical Center, 703 Main Street, Paterson, New Jersey 07503. Tel: (973) 754-3560; Fax: (973) 754-3555; E-mail: ,debariv@sjh mc.org..
kDa protein. These experiments suggest that the 54-kDa b2GPI monomer subunits exist as a multimeric complex with the 8-kDa protein. 1998 Elsevier Science Ltd. Key Words: b2glycoprotein I; Antiphospholipid syndrome; Phospholipids; Proteins; Electrophoresis; Atomic force microscopy
ince 1990, when it was discovered that b2 glycoprotein I (b2GPI) was a cofactor for anticardiolipin antibodies from patients with antiphospholipid syndrome (APS) [1,2], this protein has been the subject of intense investigation by clinical and molecular immunologists. In the intervening years, evidence has mounted that b2GPI is, itself, the target antigen for APS [3–6], presenting an epitope which is encrypted except when the protein is bound to anionic phospholipids or an analogous surface [3,7,8]. Alternatively, it has been proposed that the surface binding results in aggregate formation which may be required to achieve a suitable antigen density for high-affinity antibody binding [9,10]. The precise physiologic function of b2GPI has not yet been elucidated. b2GPI binds anionic phospholipids [11] and inhibits clotting via the intrinsic pathway [12]. It also binds platelets, presumably by a hydrophobic interaction with the steroid ring system [11], which results in the inhibition of prothrombinase activity [13]. Investigators have also proposed other mechanisms for the hypercoagula-
S
0049-3848/98 $19.00 1 .00 1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII S0049-3848(98)00047-4
132
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
ble state observed in APS patients, focusing on prothrombin and its activation system [14–16]. Recently Rand et al. [17] have provided evidence that the b2GPI–anti-b2GPI system may reduce annexin V levels on the surface of vascular endothelium, resulting in thrombosis. The primary structure of b2GPI was determined by Lozier et al. [18] and was found to be a single peptide chain of 326 amino acids, arranged as five short consensus repeats or “sushi” domains [19], relating it to the complement family of proteins. The fifth domain is critical for phospholipid binding and epitope presentation [20] and site-directed mutagenesis of b2GPI has confirmed that the sequence, Lys 282-Asn-Lys-Glu-Lys-Lys 287, is responsible for phospholipid binding [21]. The molecular mass of b2GPI based on its amino acid sequence is 40 kDa but SDS-PAGE of b2GPI preparations yields an estimated mass of slightly greater than 50 kDa due to extensive glycosylation at five sites on the second, third, and fourth domains [22]. We examined three commercial preparations of b2GPI by SDS-PAGE and observed a molecular mass of z54 kDa. This monomer may represent neither the circulating form of the protein nor the immunoreactive form which the protein assumes on a surface and which leads to its likely involvement in the pathogenesis of APS. Several recent reports [21,23] indicate that, under certain conditions, b2GPI may exist as a dimer. We examined the migration of b2GPI in a native PAGE system and report our observations, suggesting a quarternary structure for b2GPI.
1. Materials and Methods 1.1. Materials b2GPI was obtained from Crystal Chemical Corp. (Chicago, IL). This material and its preparation have been described previously [7] as have its immunological properties as they relate to human autoantibodies from APS patients [8]. Additionally, we obtained samples of b2GPI from Alexis Corp. (San Diego, CA) and from the Cappel Research Products Division of Organon Teknika Corp. (Durham, NC). Two of the three suppliers state a purity of >95% and an apparent molecular weight (by SDS-PAGE) of 48–55 kDa. The third
states that “one major band is present” with a molecular weight of 54 kDa. To compare the migration of purified b2GPI with the native serum protein, we studied sera from healthy adult subjects. These specimens had been stored in our serum bank at 2708C (618C). HEPES was obtained from Aldrich Chemical Corp. (Milwaukee, WI) and PBS (0.15 M NaCI, 0.010 M phosphate, pH 7.4) was from Sigma Chemical Corp. (St. Louis, MO). HEPES was used at 10 mM in 0.15 M NaCl (normal saline, from Baxter Healthcare Corp., Deerfield, IL). SDS, b-me, EDTA (disodium salt) and urea were obtained from Sigma Chemical Corp. Nitrocellulose membranes (0.45 mm pore size) were obtained from Schleicher & Schuell (Keene, NH). BSA (Cohn fraction V) was obtained from Boehringer Mannheim Corp. (Indiannapolis, IN). Rabbit antihuman b2GPI was from Cedarlane Laboratories Ltd. (Hornby, Ont., Canada). Horseradish peroxidase conjugated goat anti-rabbit IgG was obtained from Organon Teknika Corp. (West Chester, PA). Horseradish peroxidase conjugate substrate kit (a-chloronaphthol and H2O2) was obtained from Bio-Rad Laboratories (Hercules, CA).
1.2. Methods Electrophoretic procedures were carried out on PhastSystemTM (Pharmacia Biotechnology, Inc.; Piscataway, NJ) “mini-gels.” Native PAGE was run on 10–15 gradient gels (10–15% T, 2% C) using gel buffer strips containing 0.88 mol L-alanine, 0.25 mol Tris, pH 8.8. SDS-PAGE was run on both 4–15 gradient gels (4–15% T, 1–2% C) and high density homogenous gels with a separation zone of 20% T, 2% C. These gels also contain 30% (v/v) ethylene glycol to enhance resolution of low molecular weight proteins. The buffer strips used for SDSPAGE contain 0.2 mol tricine, 0.2 mol Tris, and 0.55% (w/v) SDS at pH 8.1. Native PAGE gels were run on b2GPI which was either untreated (other than dilution in the indicated buffer system) or treated with urea in the range 6–8 M with or without boiling. All native gels were run in the nonreduced state for 268 Vhr using bromophenol blue (Pharmacia) as a tracking dye. High molecular weight markers from Pharmacia were run in conjunction with these gels. SDSPAGE was run after treatment of b2GPI with 2%
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
133
SDS and 5% b-me (both w/v) in PBS containing 1 mM EDTA. These solutions were placed in a boiling water bath for 5 minutes prior to running. SDS-PAGE run conditions were 63 Vhr for 4–15 gels and 158 Vhr for the high density gels; tracking dye was also added to the specimens run in SDS under reducing conditions. Protein (1.7 mg) was applied from 4 ml capillary combs. All gels were stained in the PhastSystemTM staining chamber using silver stain reagents supplied by Pharmacia. Densitometric software (UN-SCAN-ITTM) for molecular weight determination and protein quantitation was obtained from Silk Scientific, Inc. (Orem, UT) and was applied to gel scans from a ScanJet 3c flat-bed scanner (Hewlett Packard Corp., Cupertino, CA). Western blots of purified b2GPI and b2GPI from whole serum (diluted 1:8 with PBS) were generated by transferring native PAGE gels to nitrocellulose via the PhastTransfer e electro-transfer system, using the protocol supplied by Pharmacia. Membranes were blocked with 3% (w/v) BSA in PBS (2 hours at 378C). The transferred b2GPI was visualized by incubation with rabbit anti-human b2GPI (0.01 mg/ ml) in BSA/PBS for 15 minutes at room temperature followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (0.03 mg/ ml) (15 minutes at room temperature). The membranes were stained with a-chloronaphthol/H2O2 using the protocol supplied by Bio-Rad. Atomic force microscopy (AFM) was performed using an ambient air scanning probe microscope (model DP) from Park Scientific Instruments (Sunnyvale, CA). Images were recorded with typical contact force loads of 0.5 nN using pyramidal Si3N4 probe tips mounted on triangular 0.03 (60.01) nm21 (nominal) Au-coated cantilevers. Samples of b2GPI with buffer and control samples of buffer solution were examined on freshly cleaved natural single crystal MoS2.
2. Results Typical SDS-PAGE electropherograms of b2GPI specimens are shown in Figure 1. Those run on a standard 4–15 gradient gel demonstrate two bands (Figure 1A), a major band at 54 kDa and a minor band which migrates near the electrophoretic front and which demonstrates a molecular weight of ,18.5 kDa. Based on densitometric data, this band
Fig. 1. (A) SDS-PAGE of b2GPI. Specimen from Crystal Chemical Corp. (right lane) run with markers (left lane) on 4–15 gradient gel. Note bands at 54 kDa and ,18.5 kDa. (B) SDS-PAGE of b2GPI from two sources: molecular weight determination of “,18.5 kDa” band. High-density gel with very low molecular weight markers (M) demonstrating an apparent molecular weight of ~8 kDa for the “,18.5 kDa” band. Lanes 1 and 2, b2GPI from Crystal Chemical Corp.; lanes 3 and 4, from Cappel Div., Organon Teknika Corp.
134
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
Fig. 2. Native PAGE of b2GPI under various conditions. Lane 1, MW markers as indicated; lane 2, b2GPI in PBS; lane 3, b2GPI in HEPES-buffered saline; lane 4, b2GPI in 6 M urea; lane 5, b2GPI in 7 M urea; lanes 6, 7, and 8 are b2GPI boiled in 6 M, 7 M, and 8 M urea, respectively. Composite of two gels with lanes 2 through 5 on a single gel and 1, 6, 7, and 8 from a second gel.
represents roughly 10% of the protein present in the preparation. To determine the molecular mass of the protein represented by this band, we used a high density homogenous gel for SDS-PAGE and a series of low mass standards. These experiments (Figure 1B) indicate a mass of 8 kDa for this protein from all three sources of b2GPI (two sources are shown in Figure 1B). Native PAGE was used to determine if the protein, in the absence of detergent or disulfide reducing agents, demonstrates a molecular mass consistent with a quarternary structure, (i.e., a subunit assembly). These data, presented in Figure 2, suggest that b2GPI migrates at a rate equivalent with a 320-kDa mass protein, based on the use of a
series of molecular mass standards. Urea, at 6–8 M and room temperature, is not sufficient to alter the apparent mass of b2GPI. However, boiling the protein in the presence of urea at these concentrations causes the apparent mass to decrease to roughly half that observed when the protein is untreated. Moreover, the very low molecular weight band also appears when the quarternary structure is disrupted. The western blots of serum b2GPI from five healthy control subjects (Figure 3) indicate that the high molecular mass observed in the commercially purified b2GPI is not an artifact of purification and suggest that the circulating form of human b2GPI in blood is also a multimeric complex.
Fig. 3. Western blots of b2GPI from serum. (A) Lanes marked CR, GB, MJ, RJ, and JM demonstrate b2GPI from sera from healthy subjects and are compared with transferred b2GPI from a commercial source separated on the native gel system. (B) Duplicate gel lane from which the standard b2GPI was transferred. Arrows on edges indicate the equivalence of position of the b2GPI in sera and in purified b2GPI.
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
Fig. 4. High-resolution AFM image of native b2GPI. Contact image on MoS2 single crystal. An aggregate of discrete particles is seen, each particle having a diameter of 30–35 nm, suggesting a molecular mass substantially greater than that expected for a 50-kDa protein.
The evidence for a large b2GPI complex presented by the electrophoretic data is strengthened by the images obtained through AFM. High-resolution images of b2GPI (Figure 4) demonstrate the presence of aggregates, each composed of at least 20 smaller particles. These smaller particles are typically 30–35 nm in diameter and are ovoid in shape.
3. Discussion Ever since the initial reports of the involvement of b2GPI in the autoantigen system for APS antibodies [1,2], a mechanism for the thrombotic manifestations of APS, based on the role of b2GPI, has been the goal of investigators in this field. The sine qua non of these studies is an understanding of the structure of b2GPI and the mechanism for its interaction both with phospholipid surfaces and with autoantibodies. These interactions are best understood in the context of the structure of the protein and one aspect of that structural information, namely the assembly of b2GPI into multisubunit complexes (the quarternary structure of b2GPI), has been addressed in this report.
135
Several previous studies have provided some evidence for the formation of b2GPI dimers. Sorice et al. [23] found that thiocarbamylation of lysine residues with phenyl isothiocyanate resulted in electrophoretic bands, immunoreactive with APS serum, at 50 and 100 kDa. Sheng et al. [21] observed a weak band at near 97 kDa in the western blot of an SDS-PAGE of b2GPI. It should be emphasized that both of these apparently dimeric bands were observed in the presence of SDS, suggesting a strong association between the monomer units. In the case of the thiocarbamylated b2GPI [23], we believe that a resonance thioether to thiol equilibrium could have resulted in disulfide bond formation, especially since their gels were run under nonreducing conditions. The other report [21] would indicate that b2GPI dimer is sufficiently stable to allow a fraction of it to withstand treatment with SDS. In our study we sought to investigate the migration of b2GPI in a native PAGE system, in which the protein would not be subjected to SDS or to reducing agents. Our data clearly indicate that b2GPI exists as a multimer. Although it migrates between the 232kDa and 440-kDa markers (as calculated: 320 kDa), we believe it is most likely a dimer. We base that opinion on: (1) the appearance of only one other faint band in lanes 2–5 of Figure 2 at roughly half the mass of the main band; (2) the recognition that actual molecular mass measurements in native gel systems are precarious in light of the contributions of both charge and mass to the migration characteristics of the protein; and (3) the high degree of glycosylation of b2GPI which probably retards migration. Moreover, when boiled in urea (lanes 6–8, Figure 2), the protein migrates with the faint band in lanes 2–5. AFM provides a physical method which appears to confirm our observations. Aggregates of b2GPI adsorbed on a freshly cleaved MoS2 crystal surface clearly show individual particles with a size of 30–35 nm. Even considering “tip broadening” [24] which might enlarge the measured particle size to some degree, this particle size is inconsistent with a protein of 50–55 kDa and suggestive of a much larger mass complex. The multimeric structure observed in commercially prepared b2GPI does not appear to be an artifact of purification. The protein is present in normal human serum and, when separated from
136
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
the sera of five healthy subjects, co-migrates with purified b2GPI. The appearance of the 8-kDa band was an unexpected finding. Although we have consistently observed a low mass band in the commerically prepared b2GPI which we routinely use in IgG antib2GPI assays [25], we previously thought that this was merely an impurity which co-purifies with b2GPI. However, it is clearly absent from native b2GPI or b2GPI treated with urea at room temperature. It appears both in SDS-treated protein and when the protein is boiled in urea, suggesting that it is either an integral subunit, a b2GPI-binding protein, or a degradation product of b2GPI. We consider the last of these options unlikely in that its mass of 8 kDa would suggest that its appearance would be accompanied by that of another band of ~40 kDa and this is not observed. In conclusion, we have provided electrophoretic evidence that b2GPI exists as a multi- subunit complex (most likely a dimer) which might also include an 8-kDa subunit or binding protein.
6.
7.
8.
9.
10.
References 1. Galli M, Comfurius P, Maasen C, Hemker HC, DeBaets MH, Van Breda-Vriesman PJC, Barbui T, Zwaal RFA, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990;335:1544–7. 2. McNeil HP, Simpson RJ, Chesterman CN, Krillis SA. Anti-phospholipid antibodies directed against a complex antigen that includes a lipid-binding inhibitor of coagulation b2 glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990;87:4120–4. 3. Matsuura E, Igarashi Y, Yasuda T, Triplett DA, Koike T. Anticardiolipin antibodies recognize b2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med 1994;179:457–62. 4. Cabral AR, Cabiedes J, Alarco´n-Segovia D. Antibodies to phospholipid-free b2-glycoprotein I in patients with primary antiphospholipid syndrome. J Rheumatol 1995;22:1894–8. 5. Cabiedes J, Cabral AR, Alarco´n-Segovia D. Clinical manifestations of the antiphospholipid syndrome in patients with systemic lupus ery-
11.
12.
13.
14.
15.
16.
thematosus associated more strongly with antib2-glycoprotein I than with antiphospholipid antibodies. J Rheumatol 1995;22:1899–1906. El-Kadi HS, Keil LB, DeBari VA. Analytical and clinical relationships between human IgG autoantibodies to b2 glycoprotein I and anticardiolipin antibodies. J Rheumatol 1995;22: 2233–7. Wagenkecht DR, McIntyre JA. Changes in b2-glycoprotein I antigenicity induced by phospholipid binding. Thromb Haemost 1993;69: 361–5. Keil LB, Galazka M, El-Kadi HS, Erickson EN Jr, DeBari VA. Binding of b2glycoprotein I to activated polystyrene and its recognition by human IgG autoantibodies. Biotechnol Appl Biochem 1995;22:305–13. Roubey RAS, Eisenberg RA, Harper MF, Winfield JB. “Anticardiolipin” autoantibodies recognize b2-glycoprotein I in the absence of phospholipid. Importance of ag density and bivalent binding. J Immunol 1995;154:954–60. Roubey RAS. Autoantibodies to phospholipid-binding plasma proteins: A new view of lupus anticoagulants and other “antiphospholipid” autoantibodies. Blood 1994;84:2854–67. Schousboe I. Characterization of the interaction between b2-glycoprotein I and mitochondria, platelets, liposomes and bile acids. Int J Biochem 1983;15:1393–1401. Schousboe I. b2-glycoprotein I: A plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood 1985;66: 1086–91. Nimpf J, Bevers EM, Bomans PHH, Till U, Wurm H, Kostner GM, Zwaal RFA. Prothrombinase activity of human platelets is inhibited by b2glycoprotein I. Biochim Biophys Acta 1986;884:142–9. Permpikul P, Rao LVM, Rapaport SI. Functional and binding studies of the roles of prothrombin and b2-glycoprotein I in the expression of lupus anticoagulant activity. Blood 1994;83:2878–92. Matsuda J, Gohchi K, Kawasugi K, Gotoh M, Saitoh N, Tsukamoto M. Inhibitory activity of anti-b2-glycoprotein I antibody on factor Va degradation by activated-protein C and its cofactor protein S. Am J Hematol 1995;49:89–91. Mori T, Takeya H, Nishioka J, Gabazza EC,
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
17.
18.
19.
20.
21.
Suzuki K. b2-glycoprotein I modulates the anticoagulant activity of activated protein C on the phospholipid surface. Thromb Haemost 1996; 75:49–55. Rand JH, Wu X-X, Andree HAM, Lockwood CJ, Guller S, Scher J, Harpel PC. Pregnancy loss in the antiphospholipid-antibody syndrome—a possible thrombogenic mechanism. New Engl J Med 1997;337:154–60. Lozier JA, Takahashi N, Putnam FW. Complete amino acid sequence of human plasma b2-glycoprotein I. Proc Natl Acad Sci USA 1984;81:3640–4. Kato H, Enjoji K. Amino acid sequence and location of the disulfide bonds in bovine b2-glycoprotein I: The presence of five sushi domains. Biochemistry 1991;30:11687–94. Steinkasserer A, Estaller C, Weiss EH, Sim RB, Day AJ. Complete nucleotide and deduced amino acid sequence of human b2-glycoprotein I. Biochem J 1991;277:387–91. Sheng Y, Sali A, Herzog H, Lahnstein J, Krilis SA. Site-directed mutagenesis of recombinant human b2-glycoprotein I identifies a cluster of
22.
23.
24.
25.
137
lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity. J Immunol 1996;157:3744–51. Walsh MT, Watzlawick H, Putnam FW, Schmid K, Brossmer R. Effect of the carbohydrate moiety on the secondary structure of b2-glycoprotein I. Implications for the biosynthesis and folding of glycoproteins. Biochemistry 1990;29:6250–7. Sorice M, Circella A, Griggi T, Garofalo T, Nicodemo G, Pittoni V, Pontieri GM, Lenti L, Valesini G. Anticardiolipin and anti-b2-GPI are two distinct populations of autoantibodies. Thromb Haemost 1996;75:303–8. Fritz M, Radmacher M, Cleveland JP, Allersma MW, Stewart RJ, Gieselmann R, Janmey P, Schmidt CF, Hansma PK. Imaging globular and filamentous proteins in physiological buffer solutions with tapping mode atomic force microscopy. Langmuir 1995;11:3529–35. Najmey SS, Erickson EN, Keil LB, DeBari VA. Antibodies to b2glycoprotein I: Standardized immunoassay and a reference interval for healthy controls. Proc Assoc Amer Phys 1996; 108:467–72.
REGULAR ARTICLE
A Stable, Multi-Subunit Complex of b2Glycoprotein I
Margaret Galazka1,3, Lynn B. Keil2,3, Joseph D. Kohles1, Jianmin Li1, Stephen P. Kelty1, Matthew Petersheim1 and Vincent A. DeBari2,3 1 Department of Chemistry, College of Arts and Sciences, and 2Department of Medicine, School of Graduate Medical Education, Seton Hall University, South Orange, New Jersey and 3 The Rheumatology Laboratory, St. Joseph’s Hospital and Medical Center, Paterson, New Jersey. (Received 24 September 1997 by Editor D.A. Triplett; revised/accepted 3 March 1998)
Abstract b2glycoprotein I (b2GPI) is a 54-kDa plasma protein which is recognized as an autoantigen for antibodies from patients with antiphospholipid syndrome (APS). SDS-PAGE (under reducing conditions) of b2GPI from three sources indicates that the 54-kDa b2GPI band is accompanied by a band corresponding to an 8-kDa protein. In the absence of detergent and reducing agents (native PAGE), b2GPI demonstrated a large complex (molecular mass ≈320 kDa) which is dissociable by boiling in 6–8 M urea, yielding several lower molecular mass bands, one of which corresponds to the 8-kDa protein observed in SDS-PAGE. Sera from five healthy adults demonstrated native b2GPI migration equivalent to the commercially purified protein. Atomic force microscopy (AFM) images of native b2GPI show aggregrates of particles each having a diameter of 30–35 nm. This is consistent with a globular unit the size of which would be substantially larger than that expected for a 54Abbreviations: AFM, atomic force microscopy; APS, antiphospholipid syndrome; b2GPI, b2glycoprotein I; b-me, b-mercaptoethanol; BSA, bovine serum albumin; HEPES, hydroxyethylpiperazine ethanesulfonate; PBS, phosphate-buffered saline; tricine, N-tris hydroxymethyl methylglycine; tris, tris hydroxymethylaminomethane. Corresponding author: Vincent A. DeBari, St. Joseph’s Hospital and Medical Center, 703 Main Street, Paterson, New Jersey 07503. Tel: (973) 754-3560; Fax: (973) 754-3555; E-mail: ,debariv@sjh mc.org..
kDa protein. These experiments suggest that the 54-kDa b2GPI monomer subunits exist as a multimeric complex with the 8-kDa protein. 1998 Elsevier Science Ltd. Key Words: b2glycoprotein I; Antiphospholipid syndrome; Phospholipids; Proteins; Electrophoresis; Atomic force microscopy
ince 1990, when it was discovered that b2 glycoprotein I (b2GPI) was a cofactor for anticardiolipin antibodies from patients with antiphospholipid syndrome (APS) [1,2], this protein has been the subject of intense investigation by clinical and molecular immunologists. In the intervening years, evidence has mounted that b2GPI is, itself, the target antigen for APS [3–6], presenting an epitope which is encrypted except when the protein is bound to anionic phospholipids or an analogous surface [3,7,8]. Alternatively, it has been proposed that the surface binding results in aggregate formation which may be required to achieve a suitable antigen density for high-affinity antibody binding [9,10]. The precise physiologic function of b2GPI has not yet been elucidated. b2GPI binds anionic phospholipids [11] and inhibits clotting via the intrinsic pathway [12]. It also binds platelets, presumably by a hydrophobic interaction with the steroid ring system [11], which results in the inhibition of prothrombinase activity [13]. Investigators have also proposed other mechanisms for the hypercoagula-
S
0049-3848/98 $19.00 1 .00 1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII S0049-3848(98)00047-4
132
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
ble state observed in APS patients, focusing on prothrombin and its activation system [14–16]. Recently Rand et al. [17] have provided evidence that the b2GPI–anti-b2GPI system may reduce annexin V levels on the surface of vascular endothelium, resulting in thrombosis. The primary structure of b2GPI was determined by Lozier et al. [18] and was found to be a single peptide chain of 326 amino acids, arranged as five short consensus repeats or “sushi” domains [19], relating it to the complement family of proteins. The fifth domain is critical for phospholipid binding and epitope presentation [20] and site-directed mutagenesis of b2GPI has confirmed that the sequence, Lys 282-Asn-Lys-Glu-Lys-Lys 287, is responsible for phospholipid binding [21]. The molecular mass of b2GPI based on its amino acid sequence is 40 kDa but SDS-PAGE of b2GPI preparations yields an estimated mass of slightly greater than 50 kDa due to extensive glycosylation at five sites on the second, third, and fourth domains [22]. We examined three commercial preparations of b2GPI by SDS-PAGE and observed a molecular mass of z54 kDa. This monomer may represent neither the circulating form of the protein nor the immunoreactive form which the protein assumes on a surface and which leads to its likely involvement in the pathogenesis of APS. Several recent reports [21,23] indicate that, under certain conditions, b2GPI may exist as a dimer. We examined the migration of b2GPI in a native PAGE system and report our observations, suggesting a quarternary structure for b2GPI.
1. Materials and Methods 1.1. Materials b2GPI was obtained from Crystal Chemical Corp. (Chicago, IL). This material and its preparation have been described previously [7] as have its immunological properties as they relate to human autoantibodies from APS patients [8]. Additionally, we obtained samples of b2GPI from Alexis Corp. (San Diego, CA) and from the Cappel Research Products Division of Organon Teknika Corp. (Durham, NC). Two of the three suppliers state a purity of >95% and an apparent molecular weight (by SDS-PAGE) of 48–55 kDa. The third
states that “one major band is present” with a molecular weight of 54 kDa. To compare the migration of purified b2GPI with the native serum protein, we studied sera from healthy adult subjects. These specimens had been stored in our serum bank at 2708C (618C). HEPES was obtained from Aldrich Chemical Corp. (Milwaukee, WI) and PBS (0.15 M NaCI, 0.010 M phosphate, pH 7.4) was from Sigma Chemical Corp. (St. Louis, MO). HEPES was used at 10 mM in 0.15 M NaCl (normal saline, from Baxter Healthcare Corp., Deerfield, IL). SDS, b-me, EDTA (disodium salt) and urea were obtained from Sigma Chemical Corp. Nitrocellulose membranes (0.45 mm pore size) were obtained from Schleicher & Schuell (Keene, NH). BSA (Cohn fraction V) was obtained from Boehringer Mannheim Corp. (Indiannapolis, IN). Rabbit antihuman b2GPI was from Cedarlane Laboratories Ltd. (Hornby, Ont., Canada). Horseradish peroxidase conjugated goat anti-rabbit IgG was obtained from Organon Teknika Corp. (West Chester, PA). Horseradish peroxidase conjugate substrate kit (a-chloronaphthol and H2O2) was obtained from Bio-Rad Laboratories (Hercules, CA).
1.2. Methods Electrophoretic procedures were carried out on PhastSystemTM (Pharmacia Biotechnology, Inc.; Piscataway, NJ) “mini-gels.” Native PAGE was run on 10–15 gradient gels (10–15% T, 2% C) using gel buffer strips containing 0.88 mol L-alanine, 0.25 mol Tris, pH 8.8. SDS-PAGE was run on both 4–15 gradient gels (4–15% T, 1–2% C) and high density homogenous gels with a separation zone of 20% T, 2% C. These gels also contain 30% (v/v) ethylene glycol to enhance resolution of low molecular weight proteins. The buffer strips used for SDSPAGE contain 0.2 mol tricine, 0.2 mol Tris, and 0.55% (w/v) SDS at pH 8.1. Native PAGE gels were run on b2GPI which was either untreated (other than dilution in the indicated buffer system) or treated with urea in the range 6–8 M with or without boiling. All native gels were run in the nonreduced state for 268 Vhr using bromophenol blue (Pharmacia) as a tracking dye. High molecular weight markers from Pharmacia were run in conjunction with these gels. SDSPAGE was run after treatment of b2GPI with 2%
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
133
SDS and 5% b-me (both w/v) in PBS containing 1 mM EDTA. These solutions were placed in a boiling water bath for 5 minutes prior to running. SDS-PAGE run conditions were 63 Vhr for 4–15 gels and 158 Vhr for the high density gels; tracking dye was also added to the specimens run in SDS under reducing conditions. Protein (1.7 mg) was applied from 4 ml capillary combs. All gels were stained in the PhastSystemTM staining chamber using silver stain reagents supplied by Pharmacia. Densitometric software (UN-SCAN-ITTM) for molecular weight determination and protein quantitation was obtained from Silk Scientific, Inc. (Orem, UT) and was applied to gel scans from a ScanJet 3c flat-bed scanner (Hewlett Packard Corp., Cupertino, CA). Western blots of purified b2GPI and b2GPI from whole serum (diluted 1:8 with PBS) were generated by transferring native PAGE gels to nitrocellulose via the PhastTransfer e electro-transfer system, using the protocol supplied by Pharmacia. Membranes were blocked with 3% (w/v) BSA in PBS (2 hours at 378C). The transferred b2GPI was visualized by incubation with rabbit anti-human b2GPI (0.01 mg/ ml) in BSA/PBS for 15 minutes at room temperature followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (0.03 mg/ ml) (15 minutes at room temperature). The membranes were stained with a-chloronaphthol/H2O2 using the protocol supplied by Bio-Rad. Atomic force microscopy (AFM) was performed using an ambient air scanning probe microscope (model DP) from Park Scientific Instruments (Sunnyvale, CA). Images were recorded with typical contact force loads of 0.5 nN using pyramidal Si3N4 probe tips mounted on triangular 0.03 (60.01) nm21 (nominal) Au-coated cantilevers. Samples of b2GPI with buffer and control samples of buffer solution were examined on freshly cleaved natural single crystal MoS2.
2. Results Typical SDS-PAGE electropherograms of b2GPI specimens are shown in Figure 1. Those run on a standard 4–15 gradient gel demonstrate two bands (Figure 1A), a major band at 54 kDa and a minor band which migrates near the electrophoretic front and which demonstrates a molecular weight of ,18.5 kDa. Based on densitometric data, this band
Fig. 1. (A) SDS-PAGE of b2GPI. Specimen from Crystal Chemical Corp. (right lane) run with markers (left lane) on 4–15 gradient gel. Note bands at 54 kDa and ,18.5 kDa. (B) SDS-PAGE of b2GPI from two sources: molecular weight determination of “,18.5 kDa” band. High-density gel with very low molecular weight markers (M) demonstrating an apparent molecular weight of ~8 kDa for the “,18.5 kDa” band. Lanes 1 and 2, b2GPI from Crystal Chemical Corp.; lanes 3 and 4, from Cappel Div., Organon Teknika Corp.
134
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
Fig. 2. Native PAGE of b2GPI under various conditions. Lane 1, MW markers as indicated; lane 2, b2GPI in PBS; lane 3, b2GPI in HEPES-buffered saline; lane 4, b2GPI in 6 M urea; lane 5, b2GPI in 7 M urea; lanes 6, 7, and 8 are b2GPI boiled in 6 M, 7 M, and 8 M urea, respectively. Composite of two gels with lanes 2 through 5 on a single gel and 1, 6, 7, and 8 from a second gel.
represents roughly 10% of the protein present in the preparation. To determine the molecular mass of the protein represented by this band, we used a high density homogenous gel for SDS-PAGE and a series of low mass standards. These experiments (Figure 1B) indicate a mass of 8 kDa for this protein from all three sources of b2GPI (two sources are shown in Figure 1B). Native PAGE was used to determine if the protein, in the absence of detergent or disulfide reducing agents, demonstrates a molecular mass consistent with a quarternary structure, (i.e., a subunit assembly). These data, presented in Figure 2, suggest that b2GPI migrates at a rate equivalent with a 320-kDa mass protein, based on the use of a
series of molecular mass standards. Urea, at 6–8 M and room temperature, is not sufficient to alter the apparent mass of b2GPI. However, boiling the protein in the presence of urea at these concentrations causes the apparent mass to decrease to roughly half that observed when the protein is untreated. Moreover, the very low molecular weight band also appears when the quarternary structure is disrupted. The western blots of serum b2GPI from five healthy control subjects (Figure 3) indicate that the high molecular mass observed in the commercially purified b2GPI is not an artifact of purification and suggest that the circulating form of human b2GPI in blood is also a multimeric complex.
Fig. 3. Western blots of b2GPI from serum. (A) Lanes marked CR, GB, MJ, RJ, and JM demonstrate b2GPI from sera from healthy subjects and are compared with transferred b2GPI from a commercial source separated on the native gel system. (B) Duplicate gel lane from which the standard b2GPI was transferred. Arrows on edges indicate the equivalence of position of the b2GPI in sera and in purified b2GPI.
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
Fig. 4. High-resolution AFM image of native b2GPI. Contact image on MoS2 single crystal. An aggregate of discrete particles is seen, each particle having a diameter of 30–35 nm, suggesting a molecular mass substantially greater than that expected for a 50-kDa protein.
The evidence for a large b2GPI complex presented by the electrophoretic data is strengthened by the images obtained through AFM. High-resolution images of b2GPI (Figure 4) demonstrate the presence of aggregates, each composed of at least 20 smaller particles. These smaller particles are typically 30–35 nm in diameter and are ovoid in shape.
3. Discussion Ever since the initial reports of the involvement of b2GPI in the autoantigen system for APS antibodies [1,2], a mechanism for the thrombotic manifestations of APS, based on the role of b2GPI, has been the goal of investigators in this field. The sine qua non of these studies is an understanding of the structure of b2GPI and the mechanism for its interaction both with phospholipid surfaces and with autoantibodies. These interactions are best understood in the context of the structure of the protein and one aspect of that structural information, namely the assembly of b2GPI into multisubunit complexes (the quarternary structure of b2GPI), has been addressed in this report.
135
Several previous studies have provided some evidence for the formation of b2GPI dimers. Sorice et al. [23] found that thiocarbamylation of lysine residues with phenyl isothiocyanate resulted in electrophoretic bands, immunoreactive with APS serum, at 50 and 100 kDa. Sheng et al. [21] observed a weak band at near 97 kDa in the western blot of an SDS-PAGE of b2GPI. It should be emphasized that both of these apparently dimeric bands were observed in the presence of SDS, suggesting a strong association between the monomer units. In the case of the thiocarbamylated b2GPI [23], we believe that a resonance thioether to thiol equilibrium could have resulted in disulfide bond formation, especially since their gels were run under nonreducing conditions. The other report [21] would indicate that b2GPI dimer is sufficiently stable to allow a fraction of it to withstand treatment with SDS. In our study we sought to investigate the migration of b2GPI in a native PAGE system, in which the protein would not be subjected to SDS or to reducing agents. Our data clearly indicate that b2GPI exists as a multimer. Although it migrates between the 232kDa and 440-kDa markers (as calculated: 320 kDa), we believe it is most likely a dimer. We base that opinion on: (1) the appearance of only one other faint band in lanes 2–5 of Figure 2 at roughly half the mass of the main band; (2) the recognition that actual molecular mass measurements in native gel systems are precarious in light of the contributions of both charge and mass to the migration characteristics of the protein; and (3) the high degree of glycosylation of b2GPI which probably retards migration. Moreover, when boiled in urea (lanes 6–8, Figure 2), the protein migrates with the faint band in lanes 2–5. AFM provides a physical method which appears to confirm our observations. Aggregates of b2GPI adsorbed on a freshly cleaved MoS2 crystal surface clearly show individual particles with a size of 30–35 nm. Even considering “tip broadening” [24] which might enlarge the measured particle size to some degree, this particle size is inconsistent with a protein of 50–55 kDa and suggestive of a much larger mass complex. The multimeric structure observed in commercially prepared b2GPI does not appear to be an artifact of purification. The protein is present in normal human serum and, when separated from
136
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
the sera of five healthy subjects, co-migrates with purified b2GPI. The appearance of the 8-kDa band was an unexpected finding. Although we have consistently observed a low mass band in the commerically prepared b2GPI which we routinely use in IgG antib2GPI assays [25], we previously thought that this was merely an impurity which co-purifies with b2GPI. However, it is clearly absent from native b2GPI or b2GPI treated with urea at room temperature. It appears both in SDS-treated protein and when the protein is boiled in urea, suggesting that it is either an integral subunit, a b2GPI-binding protein, or a degradation product of b2GPI. We consider the last of these options unlikely in that its mass of 8 kDa would suggest that its appearance would be accompanied by that of another band of ~40 kDa and this is not observed. In conclusion, we have provided electrophoretic evidence that b2GPI exists as a multi- subunit complex (most likely a dimer) which might also include an 8-kDa subunit or binding protein.
6.
7.
8.
9.
10.
References 1. Galli M, Comfurius P, Maasen C, Hemker HC, DeBaets MH, Van Breda-Vriesman PJC, Barbui T, Zwaal RFA, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990;335:1544–7. 2. McNeil HP, Simpson RJ, Chesterman CN, Krillis SA. Anti-phospholipid antibodies directed against a complex antigen that includes a lipid-binding inhibitor of coagulation b2 glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990;87:4120–4. 3. Matsuura E, Igarashi Y, Yasuda T, Triplett DA, Koike T. Anticardiolipin antibodies recognize b2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med 1994;179:457–62. 4. Cabral AR, Cabiedes J, Alarco´n-Segovia D. Antibodies to phospholipid-free b2-glycoprotein I in patients with primary antiphospholipid syndrome. J Rheumatol 1995;22:1894–8. 5. Cabiedes J, Cabral AR, Alarco´n-Segovia D. Clinical manifestations of the antiphospholipid syndrome in patients with systemic lupus ery-
11.
12.
13.
14.
15.
16.
thematosus associated more strongly with antib2-glycoprotein I than with antiphospholipid antibodies. J Rheumatol 1995;22:1899–1906. El-Kadi HS, Keil LB, DeBari VA. Analytical and clinical relationships between human IgG autoantibodies to b2 glycoprotein I and anticardiolipin antibodies. J Rheumatol 1995;22: 2233–7. Wagenkecht DR, McIntyre JA. Changes in b2-glycoprotein I antigenicity induced by phospholipid binding. Thromb Haemost 1993;69: 361–5. Keil LB, Galazka M, El-Kadi HS, Erickson EN Jr, DeBari VA. Binding of b2glycoprotein I to activated polystyrene and its recognition by human IgG autoantibodies. Biotechnol Appl Biochem 1995;22:305–13. Roubey RAS, Eisenberg RA, Harper MF, Winfield JB. “Anticardiolipin” autoantibodies recognize b2-glycoprotein I in the absence of phospholipid. Importance of ag density and bivalent binding. J Immunol 1995;154:954–60. Roubey RAS. Autoantibodies to phospholipid-binding plasma proteins: A new view of lupus anticoagulants and other “antiphospholipid” autoantibodies. Blood 1994;84:2854–67. Schousboe I. Characterization of the interaction between b2-glycoprotein I and mitochondria, platelets, liposomes and bile acids. Int J Biochem 1983;15:1393–1401. Schousboe I. b2-glycoprotein I: A plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood 1985;66: 1086–91. Nimpf J, Bevers EM, Bomans PHH, Till U, Wurm H, Kostner GM, Zwaal RFA. Prothrombinase activity of human platelets is inhibited by b2glycoprotein I. Biochim Biophys Acta 1986;884:142–9. Permpikul P, Rao LVM, Rapaport SI. Functional and binding studies of the roles of prothrombin and b2-glycoprotein I in the expression of lupus anticoagulant activity. Blood 1994;83:2878–92. Matsuda J, Gohchi K, Kawasugi K, Gotoh M, Saitoh N, Tsukamoto M. Inhibitory activity of anti-b2-glycoprotein I antibody on factor Va degradation by activated-protein C and its cofactor protein S. Am J Hematol 1995;49:89–91. Mori T, Takeya H, Nishioka J, Gabazza EC,
M. Galazka et al./Thrombosis Research 90 (1998) 131–137
17.
18.
19.
20.
21.
Suzuki K. b2-glycoprotein I modulates the anticoagulant activity of activated protein C on the phospholipid surface. Thromb Haemost 1996; 75:49–55. Rand JH, Wu X-X, Andree HAM, Lockwood CJ, Guller S, Scher J, Harpel PC. Pregnancy loss in the antiphospholipid-antibody syndrome—a possible thrombogenic mechanism. New Engl J Med 1997;337:154–60. Lozier JA, Takahashi N, Putnam FW. Complete amino acid sequence of human plasma b2-glycoprotein I. Proc Natl Acad Sci USA 1984;81:3640–4. Kato H, Enjoji K. Amino acid sequence and location of the disulfide bonds in bovine b2-glycoprotein I: The presence of five sushi domains. Biochemistry 1991;30:11687–94. Steinkasserer A, Estaller C, Weiss EH, Sim RB, Day AJ. Complete nucleotide and deduced amino acid sequence of human b2-glycoprotein I. Biochem J 1991;277:387–91. Sheng Y, Sali A, Herzog H, Lahnstein J, Krilis SA. Site-directed mutagenesis of recombinant human b2-glycoprotein I identifies a cluster of
22.
23.
24.
25.
137
lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity. J Immunol 1996;157:3744–51. Walsh MT, Watzlawick H, Putnam FW, Schmid K, Brossmer R. Effect of the carbohydrate moiety on the secondary structure of b2-glycoprotein I. Implications for the biosynthesis and folding of glycoproteins. Biochemistry 1990;29:6250–7. Sorice M, Circella A, Griggi T, Garofalo T, Nicodemo G, Pittoni V, Pontieri GM, Lenti L, Valesini G. Anticardiolipin and anti-b2-GPI are two distinct populations of autoantibodies. Thromb Haemost 1996;75:303–8. Fritz M, Radmacher M, Cleveland JP, Allersma MW, Stewart RJ, Gieselmann R, Janmey P, Schmidt CF, Hansma PK. Imaging globular and filamentous proteins in physiological buffer solutions with tapping mode atomic force microscopy. Langmuir 1995;11:3529–35. Najmey SS, Erickson EN, Keil LB, DeBari VA. Antibodies to b2glycoprotein I: Standardized immunoassay and a reference interval for healthy controls. Proc Assoc Amer Phys 1996; 108:467–72.