Chemical synthesis and characterization of wild‐type and biotinylated N‐terminal domain 1–64 of β2‐glycoprotein I

Pozzi, Nicola; Banzato, Alessandra; Bettin, Samuele; Bison, Elisa; Pengo, Vittorio; Filippis, Vincenzo De

doi:10.1002/pro.387

Cited by 23 publications

(20 citation statements)

References 52 publications

(86 reference statements)

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“…It is unlikely that the domains of protein H have adopted the short consensus repeat‐like conformation of domain I of β 2 ‐GPI, because protein H lacks disulfide bridges. Moreover, oxidized recombinant domain I of β 2 ‐GPI, but not reduced recombinant domain I, was recognized by APS patient antibodies, indicating the importance of intact disulfide bridges in the recognition of domain I of β 2 ‐GPI by patient antibodies [42]. Altogether, it seems highly unlikely that the antibody development after injection with protein H was attributable to molecular mimicry.…”

Section: Discussionmentioning

confidence: 99%

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Meijers

Ağar

et al. 2011

Journal of Thrombosis and Haemostasis

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Summary. Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-b 2 -glycoprotein I (b 2 -GPI) autoantibodies. b 2 -GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. b 2 -GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes-derived proteins to induce anti-b 2 -GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen-like protein A [SclA], and streptococcal collagen-like protein B [SclB]) were found to interact with b 2 -GPI. Only binding to protein H induces a conformational change in b 2 -GPI, thereby exposing a cryptic epitope for APS-related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody-related epitope in domain I of b 2 -GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti-protein H antibodies also generated anti-b 2 -GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in b 2 -GPI, resulting in the formation of antib 2 -GPI autoantibodies. This constitutes a novel mechanism for the formation of anti-b 2 -GPI autoantibodies.

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Section: Discussionmentioning

confidence: 99%

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Meijers

Ağar

et al. 2011

Journal of Thrombosis and Haemostasis

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“…Human α‐thrombin, PC, ProT, fibrinogen, activated factor X (FXa) and TM were from Haematologic Technologies (Essex Junction, VT, USA); HD1 and HD22 aptamers were from Primm (Milan, Italy); p ‐aminobenzamidine (PABA), N α ‐(2‐naphthyl‐sulfonyl‐glycyl)‐ d , l ‐ p ‐amidinophenylalanyl‐piperidine (NAPAP), lypopolysaccharide (LPS) and ecarin were from Sigma (St Louis, MO, USA); hirugen(54–65) peptide, fibrinogen γ′‐chain peptide(408–427), hirudin N‐terminal domain Hir(1–47) and PAR1(38–60) were chemically synthesized ; β 2 GPI was purified from human plasma and characterized (Fig. S1).…”

Section: Methodsmentioning

confidence: 99%

β2-Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions

Pozzi

Acquasaliente

Frasson

et al. 2013

Journal of Thrombosis and Haemostasis

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Glycoprotein I binds thrombin and selectively inhibits the enzyme procoagulant functions. J Thromb Haemost 2013; 11: 1093-102. Summary. Background: This work was aimed at characterizing the interaction of b 2-glycoprotein I (b 2 GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagu-lation cascade. Methods: The b 2 GPI-thrombin interaction was studied by surface plasmon resonance (SPR), fluores-cence, and molecular modeling. The effect of b 2 GPI on the procoagulant (fibrin generation and platelet aggrega-tion) and anticoagulant (protein C activation) functions of thrombin were investigated with turbidimetric, immu-nocytofluorimetric and enzymatic assays. Results: SPR and fluorescence data indicated that b 2 GPI tightly bound thrombin (K d = 34 nM) by interacting with both protease exosites, while leaving the active site accessible. This picture is fully consistent with the theoretical model of the b 2 GPI-thrombin complex. In particular, blockage of thrombin exosites with binders specific for exosite-1 (hiru-gen and HD1 aptamer) or exosite-2 (fibrinogen c′-peptide and HD22 aptamer) impaired the b 2 GPI-thrombin interaction. Identical results were obtained with thrombin mutants having one of the two exosites selectively compromised by mutation (Arg73Ala and Arg101Ala). Fluo-rescence measurements indicated that b 2 GPI did not affect the affinity of the enzyme for active site inhibitors, such as p-aminobenzamidine and the hirudin(1-47) domain, in agreement with the structural model. b 2 GPI dose-dependently prolonged the thrombin clotting time and ecarin clotting time in b 2 GPI-deficient plasma. b 2 GPI inhibited thrombin-induced platelet aggregation (IC 50 = 0.36 lM) by impairing thrombin cleavage of pro-tease-activated receptor 1 (PAR1) (IC 50 = 0.32 lM), both on gel-filtered platelets and in whole blood. Strikingly, b 2 GPI did not affect thrombin-mediated generation of the anticoagulant protein C. Conclusions: b 2 GPI functions as a physiologic anticoagulant by inhibiting the key proco-agulant activities of thrombin without affecting its unique anticoagulant function.

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“…The purity and chemical identity of each fragment were verified by SDS-PAGE and N-terminal sequencing. Domain I (1–60) was chemically synthesized and refolded as done before(57). Plasma-derived β 2 GPI (pβ 2 GPI) was purified using the perchloric acid method, as described previously(57).…”

Section: Methodsmentioning

confidence: 99%

“…Domain I (1–60) was chemically synthesized and refolded as done before(57). Plasma-derived β 2 GPI (pβ 2 GPI) was purified using the perchloric acid method, as described previously(57). MBB2 was produced as described before(13).…”

Section: Methodsmentioning

confidence: 99%

X-ray and solution structures of human beta-2 glycoprotein I reveal a new mechanism of autoantibody recognition

Ruben

Planer

Chinnaraj

et al. 2020

Preprint

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31Venous and arterial thromboses in patients suffering from the autoimmune disorder Antiphospholipid 32 Syndrome (APS) are caused by the presence of antiphospholipid antibodies (aPL). Emerging evidence 33 indicates that autoantibodies targeting the epitope R39-R43 in the N-terminal domain, Domain I (DI), of 34 b2-glycoprotein I (b2GPI) are among the most pathogenic aPL in patients with APS. How such 35 autoantibodies engage b2GPI at the molecular level remains incompletely understood. Here, we have 36 used X-ray crystallography, single-molecule FRET, and small-angle X-ray scattering to demonstrate that, 37 in the free form, under physiological pH and salt concentrations, human recombinant b2GPI adopts an 38 elongated, flexible conformation in which DI is exposed to the solvent, thus available for autoantibody 39 recognition. Consistent with this structural model, binding and mutagenesis studies revealed that the 40 elongated form interacts with a pathogenic anti-DI antibody in solution, without the need of phospholipids. 41Furthermore, complex formation was affected neither by the neighboring domains, nor by the presence 42 of the linkers, nor by the glycosylations. Since the pathogenic autoantibody requires residues R39 and 43 R43 for optimal binding, these findings challenge longstanding postulates in the field envisioning b2GPI 44 adopting immunologic inert conformations featuring inaccessibility of the epitope R39-R43 in DI and 45 support an alternative model whereby the preferential binding of anti-DI antibodies towards phospholipid-46 bound b2GPI arises from the ability of the pre-existing elongated form to bind to the membranes and then 47 oligomerize, processes that are likely to be supported by protein conformational changes. Interfering with 48 these steps may limit the pathogenic effects of anti-DI antibodies in APS patients. 503 Significance 51In the autoimmune disorder called Antiphospholipid Syndrome (APS), the presence of autoantibodies 52 targeting the plasma glycoprotein beta-2 glycoprotein I (b2GPI) is associated with arterial and venous 53 thrombosis as well as pregnancy complications. Understanding how b2GPI becomes immunogenic and 54 how autoantibodies in complex with b2GPI cause the blood to clot remains a top priority in the field. By 55 elucidating the structural architecture of b2GPI free in solution, our studies challenge longstanding 56 postulates in the field and shed new light on the pathogenic mechanisms of APS that may help the 57 development of new diagnostics and therapeutic approaches.58 59 4 Introduction 60β2GPI is a 50-kDa multi-domain glycoprotein that circulates in the plasma at a concentration of 0.2 61 mg/ml(1, 2)( Fig 1A). It acquired centerstage in hematology in 1990 when it was recognized by two 62 independent studies as the dominant antigen of antiphospholipid antibodies (aPL) in the Antiphospholipid 63 Syndrome (APS)(3-5), a life-threatening blood clotting disorder characterized by vascular thrombosis and 64 pregnancy morbidity(6). Autoantibodies against β2GPI (anti-β2...

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Chemical synthesis and characterization of wild‐type and biotinylated N‐terminal domain 1–64 of β2‐glycoprotein I

Cited by 23 publications

References 52 publications

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

β2-Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions

X-ray and solution structures of human beta-2 glycoprotein I reveal a new mechanism of autoantibody recognition

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