An allosteric redox switch in domain V of ß2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition.

ß2-glycoprotein I (ß2GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that ß2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to ß2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate ß2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of ß2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating ß2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.

Specific domain V reduction of beta-2-glycoprotein I induces protein flexibility and alters pathogenic antibody binding.

Beta-2-glycoprotein I (ß2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). ß2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of ß2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of ß2GPI. Atomic force microscopy images suggested that reduced ß2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced ß2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of ß2GPI reduction on the protein structure and its implications for antibody binding in APS patients.

Cardiolipin interacts with beta-2-glycoprotein I and forms an open conformation-Mechanisms analyzed using hydrogen/deuterium exchange.

Beta-2-glycoprotein I (ß2 GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of ß2 GPI can be recognized by the anti-ß2 GPI antibody. Here, we prepared the anionic di-oleoyl-phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the ß2 GPI. The conformational changes of ß2 GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry. The exchange level of sequences 21-27 significantly increased after ß2 GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequences 21-27 from the ring conformation. After ß2 GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequences 1-20, 21-27, 196-205, 273-279 and 297-306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequences 70-86, 153-162, 191-198, 196-205 and 273-279 indicated ß2 GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of ß2 GPI at sequences 21-27 and forms an intermediate conformation after 10 min of interaction. After a complete protein-lipid interaction, high negatively charged CL membrane induced a major conformation transition of ß2 GPI.

OxLDL/ß2GPI/anti­ß2GPI Ab complex induces inflammatory activation via the TLR4/NF­κB pathway in HUVECs.

Patients with antiphospholipid syndrome have been identified to have higher incidence rates of atherosclerosis (AS) due to the elevated levels of anti­ß2­glycoprotein I (ß2GPI) antibody (Ab). Our previous studies revealed that the anti­ß2GPI Ab formed a stable oxidized low­density lipoprotein (oxLDL)/ß2GPI/anti­ß2GPI Ab complex, which accelerated AS development by promoting the accumulation of lipids in macrophages and vascular smooth muscle cell. However, the effects of the complex on endothelial cells, which drive the initiation and development of AS, remain unknown. Thus, the present study aimed to determine the proinflammatory roles of the oxLDL/ß2GPI/anti­ß2GPI Ab complex in human umbilical vein endothelial cells (HUVECs) in an attempt to determine the underlying mechanism. Reverse transcription­quantitative PCR, enzymy­linked immunosorbent assay, western blotting and immunofluorescence staining were performed to detect the expressions of inflammation related factors and adhesion molecules. Monocyte­binding assay was used to investigate the effects of oxLDL/ß2GPI/anti­ß2GPI Ab complex on monocyte adhesion to endothelial cells. The results demonstrated that the oxLDL/ß2GPI/anti­ß2GPI Ab complex upregulated the expression of Toll­like receptor (TLR)4 and the levels of NF­κB phosphorylation in HUVECs, and subsequently enhanced the expression levels of inflammatory cytokines, including TNF­α, IL­1ß and IL­6, as well as those of adhesion molecules, such as intercellular adhesion molecule 1 and vascular adhesion molecule 1. In addition, the complex facilitated the recruitment of monocytes by promoting the secretion of monocyte chemotactic protein 1 in HUVECs. Notably, the described effects of the oxLDL/ß2GPI/anti­ß2GPI Ab complex in HUVECs were abolished by either TLR4 or NF­κB blockade. In conclusion, these findings suggested that the oxLDL/ß2GPI/anti­ß2GPI Ab complex may induce a hyper­inflammatory state in endothelial cells by promoting the secretion of proinflammatory cytokines and monocyte recruitment, which was discovered to be largely dependent on the TLR4/NK­κB signaling pathway.

Distinct and overlapping effects of ß2-glycoprotein I conformational variants in ligand interactions and functional assays.

One of the most abundant coagulation proteins is ß2-glycoprotein I (ß2GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) ß2GPI and converted it into an open conformation. The effectiveness of these procedures was assessed by Western blot and negative-staining electron microscopy. We found that in coagulation assays the open form of ß2GPI had a significant prolonging effect on fibrin formation in a dilute prothrombin time test (p < 0.001). In the dilute activated partial thromboplastin time test, both conformations had a significant prolonging effect (p < 0.001) but the open conformation was more effective. In a fluorescent thrombin generation assay both conformations slightly delayed thrombin generation with no significant effect on the quantity of formed thrombin. By using surface plasmon resonance assays, the equilibrium dissociation constants of both the open and closed conformations of ß2GPI showed a similar and strong affinity to isolated anti-ß2GPI autoantibodies (Kd closed ß2GPI = 5.17 × 10-8 M, Kd open ß2GPI = 5.56 × 10-8 M) and the open form had one order of magnitude stronger affinity to heparin (Kd = 0.30 × 10-6 M) compared to the closed conformation (Kd = 3.50 × 10-6 M). The two different forms of ß2GPI have distinct effects in functional tests and in ligand binding, which may considerably affect the intravascular events related to this abundant plasma protein in health and disease.

Beta2 glycoprotein I-derived therapeutic peptides induce sFlt-1 secretion to reduce melanoma vascularity and growth.

Melanoma, a form of skin cancer, is one of the most common cancers in young men and women. Tumors require angiogenesis to provide oxygen and nutrients for growth. Pro-angiogenic molecules such as VEGF and anti-angiogenic molecules such as sFlt-1 control angiogenesis. In addition, the serum protein, Beta2 Glycoprotein I (ß2-GPI) induces or inhibits angiogenesis depending on conformation and concentration. ß2-GPI binds to proteins and negatively charged phospholipids on hypoxic endothelial cells present in the tumor microenvironment. We hypothesized that peptides derived from the binding domain of ß2-GPI would regulate angiogenesis and melanoma growth. In vitro analyses determined the peptides reduced endothelial cell migration and sFlt-1 secretion. In a syngeneic, immunocompetent mouse melanoma model, ß2-GPI-derived peptides also reduced melanoma growth in a dose-dependent response with increased sFlt-1 and attenuated vascular markers compared to negative controls. Importantly, administration of peptide with sFlt-1 antibody resulted in tumor growth. These data demonstrate the therapeutic potential of novel ß2-GPI-derived peptides to attenuate tumor growth and endothelial migration is sFlt-1 dependent.

The J-elongated conformation of ß2-glycoprotein I predominates in solution: implications for our understanding of antiphospholipid syndrome.

ß2-Glycoprotein I (ß2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound ß2GPI to that in solution. ß2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, ß2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant ß2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound ß2GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of ß2GPI provides a strategy to block pathogenic aPLs in APS.

The role of beta-2-glycoprotein I in health and disease associating structure with function: More than just APS.

Beta-2-Glycoprotein I (ß2GPI) plays a number of essential roles throughout the body. ß2GPI, C-reactive protein and thrombomodulin are the only three proteins that possess the dual capability to up and down regulate the complement and coagulation systems depending upon external stimulus. Clinically, ß2GPI is the primary antigen in the autoimmune condition antiphospholipid syndrome (APS), which is typically characterised by pregnancy morbidity and vascular thrombosis. This protein is also capable of adopting at least two distinct structural forms, but it has been argued that several other intermediate forms may exist. Thus, ß2GPI is a unique protein with a key role in haemostasis, homeostasis and immunity. In this review, we examine the genetics, structure and function of ß2GPI in the body and how these factors may influence its contribution to disease pathogenesis. We also consider the clinical implications of ß2GPI in the diagnosis of APS and as a potentially novel therapeutic target.

Detection of anti-domain I antibodies by chemiluminescence enables the identification of high-risk antiphospholipid syndrome patients: A multicenter multiplatform study.

BACKGROUND: Classification of the antiphospholipid syndrome (APS) relies predominantly on detecting antiphospholipid antibodies (aPLs). Antibodies against a domain I (DI) epitope of anti-ß2glycoprotein I (ß2GPI) proved to be pathogenic, but are not included in the current classification criteria. OBJECTIVES: Investigate the clinical value of detecting anti-DI IgG in APS. PATIENTS/METHODS: From eight European centers 1005 patients were enrolled. Anti-cardiolipin (CL) and anti-ß2GPI were detected by four commercially available solid phase assays; anti-DI IgG by the QUANTA Flash® ß2GPI domain I assay. RESULTS: Odds ratios (ORs) of anti-DI IgG for thrombosis and pregnancy morbidity proved to be higher than those of the conventional assays. Upon restriction to patients positive for anti-ß2GPI IgG, anti-DI IgG positivity still resulted in significant ORs. When anti-DI IgG was added to the criteria aPLs or used as a substitute for anti-ß2GPI IgG/anti-CL IgG, ORs for clinical symptoms hardly improved. Upon removing anti-DI positive patients, lupus anticoagulant remained significantly correlated with clinical complications. Anti-DI IgG are mainly present in high-risk triple positive patients, showing higher levels. Combined anti-DI and triple positivity confers a higher risk for clinical symptoms compared to only triple positivity. CONCLUSIONS: Detection of anti-DI IgG resulted in higher ORs for clinical manifestations than the current APS classification criteria. Regardless of the platform used to detect anti-ß2GPI/anti-CL, addition of anti-DI IgG measured by QUANTA Flash® did not improve the clinical associations, possibly due to reduced exposure of the pathogenic epitope of DI. Our results demonstrate that anti-DI IgG potentially helps in identifying high-risk patients.

The IgA Isotype of Anti-ß2 Glycoprotein I Antibodies Recognizes Epitopes in Domains 3, 4, and 5 That Are Located in a Lateral Zone of the Molecule (L-Shaped).

Background: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with presence of anti-phospholipid antibodies (aPL). The APS classification criteria only consider the aPL of IgG/IgM isotype, however testing of aPL of IgA isotype is recommended when APS is suspected and consensus aPL are negative. IgA anti-ßeta-2 glycoprotein-I (B2GP1) has been clearly related with occurrence of thrombotic events. Antibodies anti-B2GP1 of IgG/M isotypes recognize an epitope in Domain 1 (R39-G43), the epitopes that recognize IgA anti-B2GP1 antibodies are not well-identified. Aim: To determine the zones of B2GP1 recognized by antibodies of IgA isotype from patients with APS symptomatology and positive for IgA anti-B2GP1. Methods: IgA antibodies to Domain-1(D1) and Domain-4/5(D4/5) of B2GP1 (ELISA) and epitope mapping on oligopeptide arrays of B2GP1 were evaluated in sera from a group of 93 patients with at least one thrombotic and with isolated positivity for IgA anti-B2GP1 antibodies (negative for other aPL). Results: A total of 47 patients (50.5%) were positive for anti-D4/5 and 23(25%) were positive for anti-D1. When peptide arrays were analyzed, three zones of B2GP1 reactivity were identified for more than 50% of patients. The center of these zones corresponds to amino acids 140(D3), 204(D4), and 264(D5). The peptides recognized on D3 and D4 contain amino acid sequences sharing high homology with proteins of microorganism that were previously related with a possible APS infectious etiology. In the three-dimensional structure of B2GP1, the three peptides, as the R39-G43 epitope, are located on the right side of the molecule (L-shape). The left side (J-shape) does not bind the antibodies. Conclusions: Patients with thrombotic APS clinical-criteria, and isolated IgA anti-B2GP1 positivity appear to preferentially bind, not to the D1 or D4/5 domains of B2GP1, but rather to three sites in D3, D4, and D5. The sites on D3 and D4 were previously described as the target identified by human monoclonal antibodies derived from patients that were capable of inducing APS in animal models. The localization of these epitopes opens a new route to explore to increase understanding of the patholophysiology of the APS and to propose new alternatives and therapeutic targets.

Structural and functional characterization of ß2 -glycoprotein I domain 1 in anti-melanoma cell migration.

We previously found that circulating ß2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of ß2 -glycoprotein I using structure-function analysis and mapped the critical region within the ß2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different ß2 -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified ß2 -glycoprotein I, as well as recombinant ß2 -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with ß2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for ß2 -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of ß2 -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of ß2 -glycoprotein I. This is the first study to show the therapeutic potential of ß2 -glycoprotein I D1 in the treatment of melanoma progression.

Simultaneous characterization of SNPs and N-glycans from multiple glycosylation sites of intact ß-2-glycoprotein-1 (B2GP1) by ESI-qTOF-MS.

The highly glycosylated ß-2-glycoprotein-1 (B2GP1), also called apolipoprotein H, is a 50 kDa human plasma protein with four or five N-glycosylation sites. Glycosylation of B2GP1 can impact auto antibody recognition leading to the development of antiphospholipid syndrome (APS), which can result in miscarriages or thrombosis. Next to its glycosylation different genetic variants are known to increase the risk of suffering from APS. Here we show that ESI-q/TOF-MS of intact B2GP1 can be used to analyze genetic variants and glycosylation simultaneously. After enrichment of B2GP1 from 16 different plasma samples and subsequent ESI-MS measurement of the intact protein, we detected five different SNPs in our samples either homozygous or heterozygous. The dominant glycan composition shows four biantennary, fully sialylated glycan structures, with a relative proportion of about 30%. We also detected compositions with one or two triantennary glycan structures in lower amounts and fucosylated species with one or two fucosyl residues. Two of our samples showed an unreported partially occupied fifth glycosylation site presumably arising from the presence of SNP variant S88N. Our method allows a fast determination of genetic variants and glycan compositions of human B2GP1 to be potentially used as diagnostic marker.

Oral administration of Domain-I of beta-2glycoprotein-I induces immunological tolerance in experimental murine antiphospholipid syndrome.

It is well established that the humoral immunity in antiphospholipid syndrome (APS) is presented by circulating pathogenic anti-ß2GPI autoantibodies targeting mainly domain I of the ß2GPI protein, playing a major role in the disease pathogenesis. Previously, we have demonstrated that treatment of experimental APS mice with tolerogenic dendritic cells loaded with domain-I was more efficient in tolerance induction than with the whole molecule or domain-V. In the current study we had orally administered a domain-I derivative of the ß2GPI molecule, as a new therapeutic approach to induce oral tolerance in this mouse model of APS. BALB/c mice immunized with ß2GPI, were fed with either domain-I, domain-V derivative or the complete ß2GPI protein. ß2GPI immunized mice developed experimental APS which were fed with domain-I significantly had decreased fetal loss (p < 0.004), a lower size of thrombi (p < 0.001) and lower circulating anti-ß2GPI Abs in comparison to mice fed with domain-V or PBS (p < 0.002). Likewise, Domain-I fed mice had a lowered inflammatory response, exhibited by decreased expression of inflammatory cytokines (IFNγ, IL-6, IL-17) and elevated production of IL-10 anti-inflammatory cytokine by splenocytes. Moreover, the anti-inflammatory response in the domain-I fed APS mice was associated with increased circulating miRNA variations (155, 146, 182, 98) by RT-PCR, which are associated with immunomodulation of the immune network. We propose that oral tolerance with domain-I can be a novel therapy for patients with APS.

Elevation of serum oxLDL/ß2-GPI complexes was correlated with diabetic microvascular complications in Type 2 diabetes mellitus patients.

BACKGROUND: High levels of oxLDL/ß2-GPI complexes might be a consequence of LDL atherogenic modification mediated by oxidative stress. We aimed to determine whether the levels of serum oxLDL/ß2-GPI complexes were correlated with diabetic microvascular complications in type 2 diabetes mellitus (T2DM) patients. METHODS: Levels of oxLDL/ß2-GPI complexes, oxLDL, routine lipid/lipoprotein parameters were measured in 100 healthy controls, 128 T2DM patients without any microvascular complications, and 172 T2DM patients with microvascular complications. Spearman's correlation, multivariable linear regression logistic regression analysis, and receiver operating characteristic (ROC) curve were performed. RESULTS: Levels of serum oxLDL/ß2-GPI complexes and oxLDL were significantly higher in T2DM patients with microvascular complications (oxLDL/ß2-GPI complexes: 1.10 ± 0.18 U/mL; oxLDL: 48.12 ± 7.24 mmol/L) than those in T2DM patients without microvascular complications (oxLDL/ß2-GPI complexes: 0.98 ± 0.16 U/mL; oxLDL: 41.45 ± 6.81 mmol/L) and controls (oxLDL/ß2-GPI complexes: 0.79 ± 0.15 U/mL; oxLDL: 27.85 ± 5.32 mmol/L). Variables that remained significantly associated with oxLDL/ß2-GPI complexes were oxLDL (ß = 0.568, P < 0.001), TC (ß = 0.312, P = 0.013) and microvascular complications (ß = 0.205, P = 0.027), which accounted for 58.3% of the variation of the level of oxLDL/ß2-GPI complexes in T2DM patients (R2 = 0.583). Logistic regression analysis demonstrated that elevation of oxLDL/ß2-GPI complexes (OR = 3.14, 95% CI: 1.04-9.46, P = 0.042) and oxLDL levels (OR = 3.02, 95% CI: 1.16-7.83, P = 0.023) were independently associated with occurrence of microvascular complications. Cutoff value of oxLDL/ß2-GPI for the presence of microvascular complications was 1.05 U/mL, and AUC area of ROC curve was 0.783 (95%CI: 0.713-0.853), yielding a sensitivity of 86.8% and specificity of 64.9%. CONCLUSIONS: Elevation of serum oxLDL/ß2-GPI complexes was associated with microvascular complications in T2DM patients.

PEGylated Domain I of Beta-2-Glycoprotein I Inhibits the Binding, Coagulopathic, and Thrombogenic Properties of IgG From Patients With the Antiphospholipid Syndrome.

APS is an autoimmune disease in which antiphospholipid antibodies (aPL) cause vascular thrombosis and pregnancy morbidity. In patients with APS, aPL exert pathogenic actions by binding serum beta-2-glycoprotein I (ß2GPI) via its N-terminal domain I (DI). We previously showed that bacterially-expressed recombinant DI inhibits biological actions of IgG derived from serum of patients with APS (APS-IgG). DI is too small (7 kDa) to be a viable therapeutic agent. Addition of polyethylene glycol (PEGylation) to small molecules enhances the serum half-life, reduces proteolytic targeting and can decrease immunogenicity. It is a common method of tailoring pharmacokinetic parameters and has been used in the production of many therapies in the clinic. However, PEGylation of molecules may reduce their biological activity, and the size of the PEG group can alter the balance between activity and half-life extension. Here we achieve production of site-specific PEGylation of recombinant DI (PEG-DI) and describe the activities in vitro and in vivo of three variants with different size PEG groups. All variants were able to inhibit APS-IgG from: binding to whole ß2GPI in ELISA, altering the clotting properties of human plasma and promoting thrombosis and tissue factor expression in mice. These findings provide an important step on the path to developing DI into a first-in-class therapeutic in APS.

The interaction of ß2-glycoprotein I with lysophosphatidic acid in platelet aggregation and blood clotting.

ß2-Glycoprotein I (ß2-GPI) is a plasma protein that binds to oxidized low-density lipoprotein (LDL) and negatively charged substances, and inhibits platelet activation and blood coagulation. In this study, we investigated the interaction of ß2-GPI with a negatively charged lysophosphatidic acid (LPA) in platelet aggregation and blood clotting. Two negatively charged lysophospholipids, LPA and lysophosphatidylserine, specifically inhibited the binding of ß2-GPI to oxidized LDL in a concentration-dependent manner. Intrinsic tryptophan fluorescence studies demonstrated that emission intensity of ß2-GPI decreases in an LPA-concentration-dependent manner without a shift in wavelength maxima. LPA specifically induced the aggregation of ß2-GPI in phosphate-buffered saline, and in incubated plasma and serum, both of which are known to accumulate LPA by the action of lecithin-cholesterol acyltransferase and lysophospholipase D/autotaxin. ß2-GPI aggregated by LPA did not inhibit activated von Willebrand factor-induced aggregation, and did not prolong the activated partial thromboplastin time in blood plasma, in contrast to non-aggregated ß2-GPI. These results suggest that ß2-GPI aggregated by the binding to LPA fails to inhibit platelet aggregation and blood clotting in contrast to non-aggregated ß2-GPI.

Lysine residues control the conformational dynamics of beta 2-glycoprotein I.

One of the major problems in the study of the dynamics of proteins is the visualization of changing conformations that are important for processes ranging from enzyme catalysis to signaling. A protein exhibiting conformational dynamics is the soluble blood protein beta 2-glycoprotein I (beta2GPI), which exists in two conformations: the closed (circular) form and the open (linear) form. It is hypothesized that an increased proportion of the open conformation leads to the autoimmune disease antiphospholipid syndrome (APS). A characteristic feature of beta2GPI is the high content of lysine residues. However, the potential role of lysine in the conformational dynamics of beta2GPI has been poorly investigated. Here, we report on a strategy to permanently open up the closed protein conformation by chemical acetylation of lysine residues using acetic acid N-hydroxysuccinimide ester (NHS-Ac). Specific and complete acetylation was demonstrated by the quantification of primary amino groups with fluoraldehyde o-phthalaldehyde (OPA) reagent, as well as western blot analysis with an anti-acetylated lysine antibody. Our results demonstrate that acetylated beta2GPI preserves its secondary and tertiary structures, as shown by circular dichroism spectroscopy. We found that after lysine acetylation, the majority of proteins are in the open conformation as revealed by atomic force microscopy high-resolution images. Using this strategy, we proved that the electrostatic interaction of lysine residues plays a major role in stabilizing the beta2GPI closed conformation, as confirmed by lysine charge distribution calculations. We foresee that our approach will be applied to other lysine-rich proteins (e.g. histones) undergoing conformational transitions. For instance, conformational dynamics can be triggered by environmental conditions (e.g. pH, ion concentration, post-translational modifications, and binding of ligands). Therefore, our study may be relevant for investigating the equilibrium of protein conformations causing diseases.

Thrombophilia Caused by Beta2-Glycoprotein I Deficiency: In Vitro Study of a Rare Mutation in APOH Gene.

This study aimed to explore the mechanism of a novel mutation (p.Lys38Glu) in apolipoprotein H (APOH) gene causing hereditary beta2-glycoprotein I (ß2GPI) deficiency and thrombosis in a proband with thrombophilia. The plasma level of ß2GPI was measured by ELISA and Western blotting, and anti-ß2GPI antibody by ELISA. Lupus anticoagulant (LA) was assayed using the dilute Russell viper venom time. Deficiency of the major natural anticoagulants including protein C (PC), protein S (PS), antithrombin (AT) and thrombomodulin (TM) was excluded from the proband. A mutation analysis was performed by amplification and sequencing of the APOH gene. Wild type and mutant (c.112A>G) APOH expression plasmids were constructed and transfected into HEK293T cells. The results showed that the thrombin generation capacity of the proband was higher than that of the other family members. Missense mutation p.Lys38Glu in APOH gene and LA coexisted in the proband. The mutation led to ß2GPI deficiency and thrombosis by impairing the protein production and inhibiting the platelet aggregation. It was concluded that the recurrent thrombosis of the proband is associated with the coexistence of p.Lys38Glu mutation in APOH gene and LA in plasma.