The J-elongated conformation of β2-glycoprotein I predominates in solution: implications for our understanding of antiphospholipid syndrome
2020; Elsevier BV; Volume: 295; Issue: 31 Linguagem: Inglês
10.1074/jbc.ra120.013939
ISSN1083-351X
AutoresEliza A. Ruben, William Planer, Mathivanan Chinnaraj, Zhiwei Chen, Xiaobing Zuo, Vittorio Pengo, Vincenzo De Filippis, Ravi Kumar Alluri, Keith R. McCrae, Paolo Macor, Francesco Tedesco, Nicola Pozzi,
Tópico(s)Cell Adhesion Molecules Research
Resumoβ2-Glycoprotein I (β2GPI) 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 β2GPI to that in solution. β2GPI 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, β2GPI 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 β2GPI 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 β2GPI 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 β2GPI provides a strategy to block pathogenic aPLs in APS. β2-Glycoprotein I (β2GPI) 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 β2GPI to that in solution. β2GPI 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, β2GPI 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 β2GPI 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 β2GPI 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 β2GPI provides a strategy to block pathogenic aPLs in APS. β2-Glycoprotein I (β2GPI) is a 50-kDa multidomain glycoprotein that circulates in the plasma at a concentration of ∼0.2 mg/ml (1Schultze H.E. Heide K. Haupt H. 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Phys. 2018; 20 (30176030): 26819-2682910.1039/c8cp03234cCrossref PubMed Google Scholar) using β2GPI purified from plasma using mild conditions, is the most abundant (>90%) protein conformation of β2GPI under physiological conditions, which is immunologically inert and incapable of reacting with aPLs (Fig. 1B, left). In contrast, the J-elongated form, originally described in 1999 by X-ray crystallography using β2GPI purified from plasma using the harsh oxidizing agent perchloric acid (33Schwarzenbacher R. Zeth K. Diederichs K. Gries A. Kostner G.M. Laggner P. Prassl R. Crystal structure of human beta2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome.EMBO J. 1999; 18 (10562535): 6228-623910.1093/emboj/18.22.6228Crossref PubMed Scopus (255) Google Scholar, 34Bouma B. de Groot P.G. van den Elsen J.M. Ravelli R.B. Schouten A. Simmelink M.J. Derksen R.H. Kroon J. Gros P. Adhesion mechanism of human beta(2)-glycoprotein I to phospholipids based on its crystal structure.EMBO J. 1999; 18 (10508150): 5166-517410.1093/emboj/18.19.5166Crossref PubMed Scopus (294) Google Scholar) and, more recently, observed by EM (29Agar C. van Os G.M. Morgelin M. Sprenger R.R. Marquart J.A. Urbanus R.T. Derksen R.H. Meijers J.C. de Groot P.G. Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome.Blood. 2010; 116 (20462962): 1336-134310.1182/blood-2009-12-260976Crossref PubMed Scopus (176) Google Scholar) and AFM (31Buchholz I. Nestler P. Koppen S. Delcea M. Lysine residues control the conformational dynamics of beta 2-glycoprotein I.Phys. Chem. Chem. Phys. 2018; 20 (30176030): 26819-2682910.1039/c8cp03234cCrossref PubMed Google Scholar) using "native" β2GPI subjected to high salt (i.e. 1.15 M NaCl) and high pH (11.5) or in complex with the mAb 3B7 (29Agar C. van Os G.M. Morgelin M. Sprenger R.R. Marquart J.A. Urbanus R.T. Derksen R.H. Meijers J.C. de Groot P.G. Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome.Blood. 2010; 116 (20462962): 1336-134310.1182/blood-2009-12-260976Crossref PubMed Scopus (176) Google Scholar), bacterial lipopolysaccharide (LPS) labeled with gold nanoparticles (30Agar C. de Groot P.G. Morgelin M. Monk S.D. van Os G. Levels J.H. de Laat B. Urbanus R.T. Herwald H. van der Poll T. Meijers J.C. Beta(2) glycoprotein I: a novel component of innate immunity.Blood. 2011; 117 (21454452): 6939-694710.1182/blood-2010-12-325951Crossref PubMed Scopus (74) Google Scholar), and protein H of Streptococcus pyogenes (35van Os G.M.A. Meijers J.C.M. Agar Ç. Seron M.V. Marquart J.A. Åkesson P. Urbanus R.T. Derksen R.H.W.M. Herwald H. Mörgelin M. D E Groot P.G. Induction of anti-beta2-glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes.J. Thromb. Haemost. 2011; 9 (21985124): 2447-245610.1111/j.1538-7836.2011.04532.xCrossref PubMed Scopus (28) Google Scholar), is believed to be the immunogenic conformation of the protein that interacts with aPLs, which appears when β2GPI binds to the membranes (Fig. 1B, right). Since the S-twisted conformation of the protein, inferred by small-angle X-ray scattering (SAXS) (32Hammel M. Kriechbaum M. Gries A. Kostner G.M. Laggner P. Prassl R. Solution structure of human and bovine beta(2)-glycoprotein I revealed by small-angle X-ray scattering.J. Mol. Biol. 2002; 321 (12139935): 85-9710.1016/S0022-2836(02)00621-6Crossref PubMed Scopus (76) Google Scholar), was not detected by EM, AFM, or X-ray crystallography, this model also predicts that the S-twisted form represents a transient, unreactive intermediate state that the protein populates while transitioning between the J and O forms (Fig. 1B, central). Although very popular in the field and highly endorsed by the scientific community, there are two important caveats for this commonly accepted model. First, the three structures have been obtained using different protein preparations and experimental techniques. Second, the O-circular form has never been documented in solution. Thus, while consensus exists regarding the fact the J-form is the structure of β2GPI bound to aPLs (29Agar C. van Os G.M. Morgelin M. Sprenger R.R. Marquart J.A. Urbanus R.T. Derksen R.H. Meijers J.C. de Groot P.G. Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome.Blood. 2010; 116 (20462962): 1336-134310.1182/blood-2009-12-260976Crossref PubMed Scopus (176) Google Scholar) and perhaps to the membranes (30Agar C. de Groot P.G. Morgelin M. Monk S.D. van Os G. Levels J.H. de Laat B. Urbanus R.T. Herwald H. van der Poll T. Meijers J.C. Beta(2) glycoprotein I: a novel component of innate immunity.Blood. 2011; 117 (21454452): 6939-694710.1182/blood-2010-12-325951Crossref PubMed Scopus (74) Google Scholar), unresolved issues remain: 1) which conformation exists and predominates in solution, and 2) what is the conformational pathway leading to the bound state. Encouraged by our recent results with prothrombin (36Chinnaraj M. Planer W. Pengo V. Pozzi N. Discovery and characterization of 2 novel subpopulations of aPS/PT antibodies in patients at high risk of thrombosis.Blood Adv. 2019; 3 (31175129): 1738-174910.1182/bloodadvances.2019030932Crossref PubMed Scopus (10) Google Scholar, 37Chinnaraj M. Chen Z. Pelc L.A. Grese Z. Bystranowska D. Di Cera E. Pozzi N. Structure of prothrombin in the closed form reveals new details on the mechanism of activation.Sci. Rep. 2018; 8 (29440720): 294510.1038/s41598-018-21304-1Crossref PubMed Scopus (12) Google Scholar, 38Chinnaraj M. Planer W. Pozzi N. 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Chem. 2003; 278 (12807892): 33831-3383810.1074/jbc.M212655200Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), an alternative mechanism to explain how negatively charged phospholipids enhance the affinity toward aPLs without requiring opening of the protein structure or relocation of the glycosylations away from DI is proposed, and its implications for our understanding of APS is discussed here. To get a better grasp of the structural architecture of β2GPI under conditions relevant to physiology, we set out to perform structural and biophysical studies of fully glycosylated human recombinant β2GPI (hrβ2GPI). Two versions of the proteins were successfully expressed and purified under native conditions at high yield and purity. The first version, called LT-β2GPI, contained a long multifunctional cleavable tag at the N terminus, located right before the natural N-terminal sequence 1GRTC4 (Fig. 2A and Fig. S1). The tag was then cleaved with enterokinase to generate the intact, mature protein (hrβ2GPI). Removal of the tag was confirmed by N-terminal sequencing (Fig. 2B). The second version, called ST-β2GPI, contains a shorter, noncleavable purification tag at the N terminus that, based on our previous work (37Chinnaraj M. Chen Z. Pelc L.A. Grese Z. Bystranowska D. Di Cera E. Pozzi N. Structure of prothrombin in the closed form reveals new details on the mechanism of activation.Sci. Rep. 2018; 8 (29440720): 294510.1038/s41598-018-21304-1Crossref PubMed Scopus (12) Google Scholar), is expected not to affect the conformational properties of the protein (Fig. 2B and Fig. S1). ST-β2GPI was made to eliminate the enterokinase cleavage step, which was very laborious and not as efficient as expected. The presence of the short tag was confirmed by N-terminal sequencing and accounted for the different electrophoretic mobilities observed between recombinant and plasma purified protein before and after enzymatic removal of the N-glycosylations (Fig. 2B). To evaluate the functional integrity of the recombinant proteins, LT-β2GPI, hrβ2GPI, and ST-β2GPI were tested in several biochemical assays. β2GPI purified from human plasma using the perchloric acid method (pβ2GPI) was used as a control. Using surface plasmon resonance (SPR), a technique that allows us to measure association (on) and dissociation (off) rate constants in real time, we found that all variants interacted avidly with liposomes containing negatively charged phospholipids, such as phosphatidylserine, yet they failed to interact with phospholipids made entirely of phosphatidylcholine (Fig. 2, C and D). Importantly, the values of the affinity constants were similar for all the constructs and consistent with published data (28Bevers E.M. Zwaal R.F. Willems G.M. The effect of phosph
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