An A1?A1 mutant with improved binding and inhibition of b2GPI/antibody complexes in antiphospholipid syndrome
Alexey Kolyada, Ioannis Karageorgos, Pardeep Mahlawat and Natalia Beglova
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA
Keywords antiphospholipid syndrome; ApoER2; ApoH; lipoprotein receptor; b2GPI
N. Beglova, 330 Brookline Ave, CLS 941,
Boston, MA 02215, USA
Fax: +1 617 735 4000
Tel: +1 617 735 4009
E-mail: email@example.com (Received 16 October 2014, revised 1
December 2014, accepted 24 December 2014) doi:10.1111/febs.13185 b2 glycoprotein I (b2GPI) is the most common antigen for autoimmune antibodies in antiphospholipid syndrome (APS). Thrombosis is a clinical feature of APS. We created a molecule (A1?A1) that consists of two identical b2GPI-binding modules from ApoE receptor 2 (ApoER2). A1?A1 binds to b2GPI/antibody complexes, preventing their association with ApoER2 and anionic phospholipids, and reducing thrombus size in the mouse model of
APS. Here, we describe a mutant of A1?A1 (mA1?A1ND) with improved affinity for b2GPI. mA1?A1ND inhibits the binding of b2GPI to cardiolipin in the presence of anti-b2GPI antibodies, and inhibits the binding to phospholipids in plasma samples of APS patients, affecting the clotting time.
Reduction of the clotting time demonstrates the presence of soluble b2GPI/ antibody complexes in patients? plasma. These complexes either already exist in patients? plasma or form rapidly in the proximity to phospholipids. All members of the low-density lipoprotein receptor family bind b2GPI. Modeling studies of A1 in a complex with domain V of b2GPI (b2GPI-DV) revealed two possible modes of interaction of a ligand-binding module from lipoprotein receptors with b2GPI-DV. In both orientations, the ligand-binding module interferes with binding of b2GPI to anionic phospholipids; however, it interacts with two different but overlapping sets of lysine residues in b2GPI-DV, depending on the orientation.
Introduction b2 glycoprotein I (b2GPI) has an important pathological role in antiphospholipid syndrome (APS) .
Patients with thrombosis and/or pregnancy loss are diagnosed with APS if they have autoimmune antibodies to serum proteins that bind to anionic phospholipids in laboratory tests for APS . b2GPI is the major antigen for antiphospholipid antibodies [3?5]. Normally, b2GPI circulates in the blood at a high concentration and does not have a well-defined physiological function. b2GPI acquires prothrombotic properties in the presence of anti-b2GPI antibodies. Anti-b2GPI antibodies correlate with thrombosis in patients with
APS, and potentiate thrombosis in animal models of
In in vivo studies of mice, significant b2GPI deposition was detected only on the endothelium of the uterus, and the deposition pattern remained the same in mice with circulating anti-b2GPI antibodies .
In the presence of anti-b2GPI antibodies, b2GPI/ antibody complexes enhance thrombus size in rodent models of thrombosis, and activate endothelial cells, monocytes and platelets in vitro and in vivo [11?14].
Several receptors and cell-surface molecules, including toll-like receptor 4 (TLR4), Annexin II, ApoE receptor 2 (ApoER2) and other receptors of the lowdensity lipoprotein receptor (LDLR) family, GPIba and anionic phospholipids have been shown to bind b2GPI and contribute to cellular activation by
ApoER2, ApoE receptor 2; APS, antiphospholipid syndrome; DV, domain V; INR, international normalized ratio; ITC, isothermal titration calorimetry; LDLR, low-density lipoprotein receptor; SGU, standard IgG units; TMB, 3,30,5,50-tetramethylbenzidine; b2GPI, beta2-glycoprotein I. 864 FEBS Journal 282 (2015) 864?873 ? 2015 FEBS b2GPI/antibody complexes [15?22]. ApoER2 and
GPIba act together in activation of platelets by b2GPI/antibody complexes . Antibodies to b2GPI enhance the binding of b2GPI to cellular surfaces, and possibly crosslink several receptors. In vivo studies in mice confirmed the contribution of ApoER2,
TLR4 and Annexin II to the prothrombotic properties of b2GPI/antibody complexes [14,23?25]. In addition to cell-surface receptors, b2GPI interacts with proteins involved in blood coagulation and fibrinolysis [26?29]. Whether these interactions are modified by anti-b2GPI antibodies and their role in
APS has not been well established.
ApoER2 is a receptor for b2GPI/antibody complexes on endothelial cells and platelets [14,30]. It belongs to the LDLR family of receptors. Like other receptors of the same family, ApoER2 uses multiple small structurally homologous ligand-binding modules to interact with diverse ligands. The first ligand-binding module, A1, is critical for the binding of b2GPI to ApoER2 . We created a polypeptide consisting of two A1 modules connected by a flexible linker, and demonstrated that this molecule preferentially targets b2GPI/antibody complexes compared to b2GPI that is not bound to antibody . A1?A1 not only interferes with the binding of b2GPI/antibody complexes to ApoER2, but also prevents their association with anionic phospholipids, therefore inhibiting two potentially harmful activities of b2GPI/antibody complexes .
Most importantly, we demonstrated that infusion of
A1?A1 in mice abrogated the increase in thrombus size caused by the presence of b2GPI/antibody complexes .
In this study, we created a mutant of A1?A1 (mA1?A1ND) with improved affinity for b2GPI.
The rationale for the point mutation of Asn36 to
Asp in the b2GPI-binding region of mouse A1 was based on modeling of the complex between A1 and b2GPI. mA1?A1ND inhibits the binding of b2GPI/ antibody complexes to cardiolipin-coated surfaces, and decreases anti-b2GPI-dependent prolongation of clotting time in plasma samples of APS patients. All receptors of the LDLR family can bind b2GPI.
Modeling studies of A1 in a complex with domain V of b2GPI (b2GPI-DV) revealed two possible orientations of a ligand-binding module from lipoprotein receptors in the complex with b2GPIDV. In both orientations, the ligand-binding module interferes with the binding of b2GPI to anionic phospholipids; however, it interacts with two different but overlapping sets of lysine residues in b2GPIDV, depending on the orientation.