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Complement Activation and Thrombin Generation by MBL Bound to Β2 Complement Activation and Thrombin Generation by MBL Bound to β 2-Glycoprotein I This information is current as Paolo Durigutto, Paolo Macor, Nicola Pozzi, Chiara of September 27, 2021. Agostinis, Fleur Bossi, Pier Luigi Meroni, Claudia Grossi, Maria O. Borghi, William Planer, Peter Garred and Francesco Tedesco J Immunol published online 5 August 2020 http://www.jimmunol.org/content/early/2020/07/31/jimmun Downloaded from ol.2000570 Supplementary http://www.jimmunol.org/content/suppl/2020/07/31/jimmunol.200057 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 5, 2020, doi:10.4049/jimmunol.2000570 The Journal of Immunology Complement Activation and Thrombin Generation by MBL Bound to b2-Glycoprotein I Paolo Durigutto,*,1 Paolo Macor,†,1 Nicola Pozzi,‡ Chiara Agostinis,x Fleur Bossi,x Pier Luigi Meroni,* Claudia Grossi,* Maria O. Borghi,*,{ William Planer,‡ Peter Garred,‖ and Francesco Tedesco* b2-Glycoprotein I (b2-GPI) is an abundant plasma glycoprotein with unknown physiological function and is currently recognized as the main target of antiphospholipid Abs responsible for complement activation and vascular thrombosis in patients with antiphospholipid syndrome (APS). In this study, we provide evidence that mannose-binding lectin (MBL) binds to b2-GPI in Ca++ and a dose-dependent manner and that this interaction activates complement and promotes complement-dependent throm- bin generation. Surprisingly, a significant binding was observed between MBL and isolated domains II and IV of b2-GPI, whereas the carbohydrate chains, domain I and domain V, were not involved in the interaction, documenting a noncanonical binding mode Downloaded from between MBL and b2-GPI. Importantly, this interaction may occur on endothelial cells because binding of MBL to b2-GPI was detected on the surface of HUVECs, and colocalization of MBL with b2-GPI was observed on the endothelium of a biopsy specimen of a femoral artery from an APS patient. Because b2-GPI–mediated MBL-dependent thrombin generation was in- creased after priming the endothelium with TNF-a, our data suggests that this mechanism could play an important yet unrec- ognized role under physiological conditions and may be upregulated in pathological situations. Moreover, the complement activation and the procoagulant effects of the b2-GPI/MBL complex may contribute to amplify similar activities of anti–b2- http://www.jimmunol.org/ GPI Abs in APS and possibly act independently of Abs, raising the issue of developing appropriate therapies to avoid recurrences and disability in patients at risk for these clinical conditions. The Journal of Immunology, 2020, 205: 000–000. he 50-kDa protein b2-glycoprotein I (b2-GPI) is syn- has greatly increased over the last two decades after the discovery thesized in the liver and circulates in the blood at ∼200 that it serves as a preferential target for Abs present in patients T mg/ml (1). Although the function of this protein is largely with antiphospholipid syndrome (APS) (5). Animal models of unknown, the finding that individuals and mice with b2-GPI de- APS have clearly shown that these Abs mediate the onset of ficiency lead an apparently healthy life suggests that its role is not thrombosis and adverse pregnancy outcomes that represent the by guest on September 27, 2021 critical under physiologic conditions (2–4). The interest in b2-GPI two main clinical manifestations of the syndrome (6–8). In vitro studies have revealed that b2-GPI binds to several cell types, in- cluding platelets, monocytes, endothelial cells, and trophoblasts, *Laboratorio di Immuno-Reumatologia, Istituto Auxologico Italiano, Istituto di interacting with various receptors differently expressed on the cell Ricerca e Cura a Carattere Scientifico, Cusano Milanino, 20095 Milan, Italy; †Department of Life Sciences, University of Trieste, 34127 Trieste, Italy; ‡Edward A. targets (9–12). However, analysis of the in vivo distribution of the Doisy Department of Biochemistry and Molecular Biology, Saint Louis University b x purified protein has shown that 2-GPI localizes on decidual en- School of Medicine, St. Louis, MO 63104; Istituto Materno-Infantile, Istituto dothelial cells and extravillous trophoblast, whereas its deposition di Ricerca e Cura a Carattere Scientifico, Burlo Garofolo, 34137 Trieste, Italy; {Department of Clinical Sciences and Community Health, University of Milan, on the endothelium of the other vascular districts required priming ‖ 20122 Milan, Italy; and Laboratory of Molecular Medicine, Department of Clinical with LPS (13). b2-GPI is structurally organized into five domains, Immunology, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark four of which are composed of 60 amino acids also found in 1P.D. and P.M. contributed equally to this work complement control proteins and the fifth domain characterized by an extra stretch of positively charged amino acids that allows ORCIDs: 0000-0003-3862-4970 (P.D.); 0000-0003-3079-4019 (P.M.); 0000-0003- 2309-7100 (N.P.); 0000-0002-8315-2258 (C.A.); 0000-0002-3394-1451 (P.L.M.); binding of the protein to anionic phospholipids (14). Clinical and 0000-0003-0811-2472 (C.G.); 0000-0001-8967-9678 (M.O.B.); 0000-0003-2860- animal studies have provided convincing evidence that the path- 4343 (W.P.); 0000-0002-2876-8586 (P.G.); 0000-0003-2462-0493 (F.T.). ogenic Abs are directed against the DI domain that is exposed Received for publication May 15, 2020. Accepted for publication July 4, 2020. after the deposition of b2-GPI on the cell surface (15, 16). This work was supported by Grant 20C003_2010 Ricerca Corrente from the Italian The complement system plays a critical role in mediating anti–b Ministry of Health (P.L.M.), National Institutes of Health Research Grant HL150146 (N.P.), and Grant DFF-6110-00489 from the Danish Research Council for Indepen- 2-GPI–induced clot formation and pregnancy loss as revealed by dent Research (P.G.). the failure of these Abs to cause vascular thrombi and pregnancy Address correspondence and reprint requests to Prof. Francesco Tedesco, Laboratorio loss in complement-deficient animals (8). This conclusion is also di Immuno-Reumatologia, Istituto Auxologico Italiano, Istituto di Ricerca e Cura a supported by the finding that complement-activation products are Carattere Scientifico, Via Giuseppe Zucchi 18, Cusano Milanino, 20095 Milan, Italy. E-mail address: [email protected] deposited on the vascular endothelium at the site of thrombus The online version of this article contains supplemental material. formation (17) and in the placentae collected from APS patients Abbreviations used in this article: AP, alkaline phosphatase; APS, antiphospholipid and animal models of APS (8). Ab-mediated complement acti- syndrome; BMP-1, bone morphogenetic protein-1; BTP, tolloid-like proteinase; vation through the classical pathway in this pathological condition CRD, carbohydrate-recognition domain; b2-GPI, b2-glycoprotein I; MBL, mannose- leads to the release of C5a and the assembly of the terminal binding lectin; NHS, normal human serum; VBS, veronal-buffered saline. complement complex involved in the onset of pregnancy loss and Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 thrombus formation (6, 7). www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000570 2 b2-GPI/MBL COMPLEX ACTIVATES COMPLEMENT AND THROMBIN In this work, we have investigated the ability of b2-GPI to of enzyme mix and 50 ml of buffer 1 for 30 min at room temperature trigger complement activation independently of Ab and to pro- and then transferred to 37˚C for 48 h or until completion. The reac- mote biological effects associated with complement-activation tion was monitored by taking samples at 0, 1, 2, 3, 8, 24, and 48 h and analysis by SDS-PAGE. Lack of sugars was verified by Pierce products. Our working hypothesis was that the protein may Glycoprotein Staining Kit. The mixture was then purified by size bind mannose-binding lectin (MBL) because of the high carbo- exclusion chromatography to recover the deglycosylated protein that hydrate content that accounts for ∼20% w/w of the molecular typically elutes ∼1 ml after the glycosylated protein because of the mass (18, 19) and activates the lectin pathway. MBL has long smaller hydrodynamic radius. Recombinant deglycosylated human b2-GPI was obtained by been known to contribute to host defense, particularly in early blocking O-andN-glycosylations. Residues T130, N143, N164, N174, childhood, targeting mannose and N-acetylglucosamine oligo- and N234 were mutated to serine (S) or glutamine (Q) by site-directed saccharides expressed on infectious agents (20). In addition, mutagenesis to generate the b2-GPI mutant T130S/N143Q/N164Q/ MBL-mediated activation of the lectin pathway has been im- N174Q/N234Q. After sequence verification, deglycosylated b2-GPI plicated in the development of various pathological condi- was expressed in HEK293 cells and purified from the media following the protocol developed for the wild-type protein. The purity of each tions, including ischemia/reperfusion (21), autoimmune diseases preparation was .98%, as judged by SDS-PAGE and reverse phase (22, 23), and IgA nephropathies (24). More recently, activation HPLC.
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