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The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech

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The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech This information is current as Siew Siew Pang, Lakshmi C. Wijeyewickrema, Lilian Hor, of September 29, 2021. Sheareen Tan, Emilie Lameignere, Edward M. Conway, Anna M. Blom, Frida C. Mohlin, Xuyu Liu, Richard J. Payne, James C. Whisstock and Robert N. Pike J Immunol 2017; 199:3883-3891; Prepublished online 23 October 2017; Downloaded from doi: 10.4049/jimmunol.1700158 http://www.jimmunol.org/content/199/11/3883 References This article cites 35 articles, 9 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/199/11/3883.full#ref-list-1 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 29, 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech Siew Siew Pang,*,1 Lakshmi C. Wijeyewickrema,†,1 Lilian Hor,† Sheareen Tan,* Emilie Lameignere,‡ Edward M. Conway,‡ Anna M. Blom,x Frida C. Mohlin,x Xuyu Liu,{ Richard J. Payne,{ James C. Whisstock,*,1 and Robert N. Pike†,1 Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is ac- tivated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and Downloaded from MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway. The Journal of Immunology, 2017, 199: 3883–3891. http://www.jimmunol.org/ omplement is vital to host immunity (1) and an effector is constitutively active through a so-called “tickover” mechanism system that facilitates the elimination of invading path- and is amplified when Factors D and B are activated following the C ogens, but it must be tightly controlled to avoid inflam- binding of C3b to foreign surfaces (5). Once activated, all three mation and tissue damage (2). The complement system is pathways converge at C3 and progress to the formation of the activated via three pathways (3). The classical pathway is initiated membrane attack complexes (or C5b–C9) on a target membrane (6). by binding of the C1 complex and its associated serine proteases, From a therapeutic perspective, selective inhibition of the different C1r and C1s, to ligands, such as Ag-bound Igs. The lectin pathway complement-activation pathways to attenuate disease would ideally is initiated by the MASP-1 and MASP-2 proteases associated with be required to avoid compromising the immune status of patients. In by guest on September 29, 2021 lectins, such as mannose-binding lectin (4). The alternative pathway this regard, the classical and lectin pathways present more suitable targets, given that the alternative pathway acts as an amplification loop for both of these pathways (7). Accordingly, the C1r/C1s and *Department of Biochemistry and Molecular Biology and Australian Research Coun- cil Centre of Excellence in Advanced Molecular Imaging, Biomedicine Discovery MASP-1/MASP-2 serine protease systems present as ideal targets to Institute, Monash University, Melbourne, Victoria 3800, Australia; †Department of inhibit the classical and lectin pathways, respectively. Biochemistry and Genetics and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, La Trobe Institute for Molecular Science, La Trobe Previously, a novel 17-kDa C1s inhibitor, designated BD001 University, Melbourne, Victoria 3086, Australia; ‡Centre for Blood Research, Department (which we have termed gigastasin in this article), derived from the of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, British x giant Amazonian leech (Haementaria sp.) (8) was identified and Columbia V6T 1Z3, Canada; Division of Medical Protein Chemistry, Department of { Translational Medicine, Lund University, Malmo¨ SE-221 00, Sweden; and School of found to inhibit the classical pathway. However, its role in the Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia lectin pathway was not assessed. Gigastasin includes a highly 1S.S.P., L.C.W., J.C.W., and R.N.P. contributed equally. acidic C terminus containing three sulfotyrosine (sY) residues, ORCIDs: 0000-0003-0557-5447 (S.S.P.); 0000-0001-6084-4887 (L.C.W.); 0000- which is similar to the stretch of amino acids found in complement 0002-2967-0134 (L.H.); 0000-0003-0081-0305 (E.M.C.); 0000-0002-1348-1734 (A.M.B.); 0000-0003-4096-3090 (X.L.); 0000-0002-2083-0269 (R.N.P.). component C4, the primary substrate for C1s and MASP-2. This part of C4 has more recently been shown to bind to C1s (9) and Received for publication February 6, 2017. Accepted for publication September 27, 2017. MASP-2 (10) via highly positively charged areas on the protease This work was supported by National Health and Medical Research Council of surface, termed exosites (anion-binding exosites [ABEs]). Australia Project Grant 1082090 (to R.N.P.). In this study, we have shown that gigastasin is a potent inhibitor of The coordinates and structure factors presented in this article have been sub- C1s and MASP-2, is less active against MASP-1, and is a poor mitted to the Protein Data Bank (https://www.rcsb.org/pdb/results/results.do? inhibitor of C1r. We have further determined the structure of gig- tabtoshow=Unreleased&qrid=4A0C606A) under accession number 5UBM. astasin in complex with its most preferred target, C1s, and dem- Address correspondence and reprint requests to Prof. Robert N. Pike or Prof. James C. Whisstock, La Trobe Institute of Molecular Science, Melbourne, VIC 3086, Aus- onstrated that the mechanism of inhibition involves contact with the tralia (R.N.P.) or Department of Biochemistry and Molecular Biology and ARC activation loop, the active site, and the ABE of the protease. Our data Centre of Excellence in Advanced Molecular Imaging, Biomedicine Discovery In- stitute, Monash University, Melbourne, VIC 3800, Australia (J.C.W.). E-mail ad- reveal that contact with the ABE regions of C1s and MASP-2 is vital dresses: [email protected] (R.N.P.) or [email protected] (J.C.W.) to its inhibitory activity. Finally, we used deposition assays to assess Abbreviations used in this article: ABE, anion-binding exosite; MBL, mannose- the effect of gigastasin. Interestingly, in contrast to our predictions binding lectin; NHS, normal human serum; PDB, Protein Data Bank; RT, room from the protease-inhibition kinetics, these data reveal that activation temperature; sY, sulfotyrosine. of the lectin pathway was more potently inhibited by gigastasin than Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 was classical pathway activation. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700158 3884 GIGASTASIN, A NOVEL COMPLEMENT PROTEASE INHIBITOR Materials and Methods data were indexed, integrated, and scaled using MOSFLM (14) and Materials SCALA (15) from the CCP4 package (16). The complex structure was solved by molecular replacement [using Z–K–SBzl, Boc–LGR–AMC, and Boc–VPR–AMC were purchased from PHASER from the CCP4 suite (17)] using the C1s SP domain (1ELV, residue Bachem. 4,49-Dithiodidyridine was purchased from Sigma-Aldrich. Normal 410–668) (18) as the probe in the space group P3121. Each asymmetric unit human serum (NHS) and C1q-depleted serum were from Complement contains one single C1s/gigastasin complex. After the initial refinement [using Technology (Tyler, TX). Mannose-binding lectin (MBL)-deficient serum was REFMAC5 (19)] with the C1s SP domain, the additional densities of the from the Statens Serum Institut (Copenhagen, Denmark). Murine mono- missingCCP1andCCP2domains,aswellas gigastasin, were clearly visible. clonal anti-C4d Ab was from Quidel (San Diego, CA). Goat anti-mouse The missing structures were built in manually using COOT (20), with re- secondary Ab (Alexa Fluor 488 conjugate) was from Life Technologies. finement cycles using Phenix (21) or the CCP4 suite (16). The progress of Human dermal microvascular endothelial cells (HMEC-1) were a kind gift refinement was monitored using the Rfree value, and the final refinement from the Centers for Disease Control and Prevention (Atlanta, GA). statistics are listed in Table II. Coordinates and structure factors were sub- mitted to the Protein Data Bank (PDB) under ID 5UBM (https://www.rcsb. Purification of active plasma-derived C1s and gigastasin org/pdb/results/results.do?tabtoshow=Unreleased&qrid=4A0C606A).
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