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Thromb Haemost 2014; 111(01): 29-40
DOI: 10.1160/TH13-04-0340
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

Tina M. Kousted
1   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
,
Karsten Skjoedt
2   Department of Cancer and Inflammation, University of Southern Denmark, Odense, Denmark
,
Steen V. Petersen
3   Department of Biomedicine, Aarhus University, Aarhus, Denmark
,
Claus Koch
2   Department of Cancer and Inflammation, University of Southern Denmark, Odense, Denmark
,
Lars Vitved
2   Department of Cancer and Inflammation, University of Southern Denmark, Odense, Denmark
,
Maja Sochalska
1   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
,
Céline Lacroix
1   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
,
Lisbeth M. Andersen
1   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
,
Troels Wind
1   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
,
Peter A. Andreasen
1   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
,
Jan K. Jensen
1   Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
› Author Affiliations Financial support: This study was supported by the Alfred Nielsen’s Foundation; The Cancer Research Foundation of 1989; the Danish Cancer Society; Danish National Research Foundation (26–331–6); The Faculty of Science and Technology, Aarhus University; The Research Council for Production and Technology (09–072885); The Lundbeck Foundation.
Further Information

Publication History

Received: 26 April 2013

Accepted after major revision: 24 August 2013

Publication Date:
21 November 2017 (online)

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Summary

Protease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1’s protease inhibitory activity.

Keywords

Cancer - glia-derived nexin - SERPIN E2 - serine protease - inhibitory antibody

 

  • References

  • 1 Irving JA, Pike RN, Lesk AM. et al. Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 2000; 10: 1845-1864.
    CrossrefPubMedGoogle Scholar
  • 2 Ye S, Goldsmith EJ. Serpins and other covalent protease inhibitors. Curr Opin Struct Biol 2001; 11: 740-745.
    CrossrefPubMedGoogle Scholar
  • 3 Wind T, Hansen M, Jensen JK. et al. The molecular basis for anti-proteolytic and non-proteolytic functions of plasminogen activator inhibitor type-1: roles of the reactive centre loop, the shutter region, the flexible joint region and the small serpin fragment. Biological Chem 2002; 383: 21-36.
    CrossrefPubMedGoogle Scholar
  • 4 Huntington JA. Shape-shifting serpins--advantages of a mobile mechanism. Trends Biochem Sci 2006; 31: 427-435.
    CrossrefPubMedGoogle Scholar
  • 5 Huntington JA. Serpin structure, function and dysfunction. J Thromb Haemost 2011; 9 (Suppl. 01) 26-34.
    CrossrefPubMedGoogle Scholar
  • 6 Wallace A, Rovelli G, Hofsteenge J. et al. Effect of heparin on the glia-derived-nexin-thrombin interaction. Biochem J 1989; 257: 191-196.
    CrossrefPubMedGoogle Scholar
  • 7 Rovelli G, Stone SR, Guidolin A. et al. Characterization of the heparin-binding site of glia-derived nexin/protease nexin-1. Biochemistry 1992; 31: 3542-3549.
    CrossrefPubMedGoogle Scholar
  • 8 Herndon ME, Stipp CS, Lander AD. Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding. Glycobiology 1999; 9: 143-155.
    CrossrefPubMedGoogle Scholar
  • 9 Richard B, Bouton MC, Loyau S. et al. Modulation of protease nexin-1 activity by polysaccharides. Thromb Haemost 2006; 95: 229-235.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 10 Scott RW, Bergman BL, Bajpai A. et al. Protease nexin. Properties and a modified purification procedure. J Biol Chem 1985; 260: 7029-7034.
    CrossrefPubMedGoogle Scholar
  • 11 Evans DL, McGrogan M, Scott RW. et al. Protease specificity and heparin binding and activation of recombinant protease nexin I. J Biol Chem 1991; 266: 22307-22312.
    CrossrefPubMedGoogle Scholar
  • 12 Stone SR, Brown-Luedi ML, Rovelli G. et al. Localization of the heparin-binding site of glia-derived nexin/protease nexin-1 by site-directed mutagenesis. Biochemistry 1994; 33: 7731-7735.
    CrossrefPubMedGoogle Scholar
  • 13 Li W, Huntington JA. Crystal structures of protease nexin-1 in complex with heparin and thrombin suggest a 2-step recognition mechanism. Blood 2012; 120: 459-467.
    CrossrefPubMedGoogle Scholar
  • 14 Jensen JK, Dolmer K, Gettins PG. Specificity of binding of the low density lipo-protein receptor-related protein to different conformational states of the clade E serpins plasminogen activator inhibitor-1 and proteinase nexin-1. J Biol Chem 2009; 284: 17989-17997.
    CrossrefPubMedGoogle Scholar
  • 15 Kousted TM, Jensen JK, Gao S. et al. Protease Nexin 1– a serpin with a possible pro-invasive role in cancer. In: Behrendt N. editor. Matrix Proteases in Health and Disease. Wiley-VCH Verlag GmbH & Co; 2012. pp. 251-281.
    CrossrefGoogle Scholar
  • 16 Dupont DM, Madsen JB, Kristensen T. et al. Biochemical properties of plasminogen activator inhibitor-1. Frontiers Biosci 2009; 14: 1337-1361.
    CrossrefPubMedGoogle Scholar
  • 17 Bouton MC, Boulaftali Y, Richard B. et al. Emerging role of serpinE2/protease nexin-1 in hemostasis and vascular biology. Blood 2012; 119: 2452-2457.
    CrossrefPubMedGoogle Scholar
  • 18 Boulaftali Y, Francois D, Venisse L. et al. Endothelial Protease Nexin-1 Is a Novel Regulator of A Disintegrin and Metalloproteinase 17 Maturation and Endothelial Protein C Receptor Shedding via Furin Inhibition. Arterioscler Thromb Vasc Biol 2013; 33: 1647-1654.
    CrossrefPubMedGoogle Scholar
  • 19 Selbonne S, Azibani F, Iatmanen S. et al. In vitro and in vivo antiangiogenic properties of the serpin protease nexin-1. Mol Cell Biol 2012; 32: 1496-1505.
    CrossrefPubMedGoogle Scholar
  • 20 McKee CM, Xu D, Cao Y. et al. Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma. J Clin Invest 2012; 122: 4025-4036.
    CrossrefPubMedGoogle Scholar
  • 21 Bijnens AP, Gils A, Stassen JM. et al. The distal hinge of the reactive site loop and its proximity: a target to modulate plasminogen activator inhibitor-1 activity. J Biol Chem 2001; 276: 44912-44918.
    CrossrefPubMedGoogle Scholar
  • 22 Bijnens AP, Ngo TH, Gils A. et al. Elucidation of the binding regions of PAI-1 neutralizing antibodies using chimeric variants of human and rat PAI-1. Thromb Haemost 2001; 85: 866-874.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 23 Verhamme I, Kvassman JO, Day D. et al. Accelerated conversion of human plasminogen activator inhibitor-1 to its latent form by antibody binding. J Biol Chem 1999; 274: 17511-17517.
    CrossrefPubMedGoogle Scholar
  • 24 Naessens D, Gils A, Compernolle G. et al. Elucidation of the epitope of a latency-inducing antibody: identification of a new molecular target for PAI-1 inhibition. Thromb Haemost 2003; 90: 52-58.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 25 Dupont DM, Blouse GE, Hansen M. et al. Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C. J Biol Chem 2006; 281: 36071-36081.
    CrossrefPubMedGoogle Scholar
  • 26 Gorlatova NV, Elokdah H, Fan K. et al. Mapping of a conformational epitope on plasminogen activator inhibitor-1 by random mutagenesis. Implications for serpin function. J Biol Chem 2003; 278: 16329-16335.
    PubMedGoogle Scholar
  • 27 Schousboe SL, Egelund R, Kirkegaard T. et al. Vitronectin and substitution of a beta-strand 5A lysine residue potentiate activity-neutralization of PA inhibitor-1 by monoclonal antibodies against alpha-helix F. Thromb Haemost 2000; 83: 742-751.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 28 Wind T, Jensen MA, Andreasen PA. Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type-1: implications for antibody-mediated PAI-1-neutralization and vitronectin-binding. Eur J Biochem 2001; 268: 1095-1106.
    CrossrefPubMedGoogle Scholar
  • 29 Bijnens AP, Gils A, Knockaert I. et al. Importance of the hinge region between alpha-helix F and the main part of serpins, based upon identification of the epitope of plasminogen activator inhibitor type 1 neutralizing antibodies. J Biol Chem 2000; 275: 6375-6380.
    CrossrefPubMedGoogle Scholar
  • 30 Naessens D, Gils A, Compernolle G. et al. Elucidation of a novel epitope of a substrate-inducing monoclonal antibody against the serpin PAI-1. J Thromb Haemost 2003; 1: 1028-1033.
    CrossrefPubMedGoogle Scholar
  • 31 Debrock S, Declerck PJ. Identification of a functional epitope in plasminogen activator inhibitor-1, not localized in the reactive center loop. Thromb Haemost 1998; 79: 597-601.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 32 Komissarov AA, Declerck PJ, Shore JD. Mechanisms of conversion of plasminogen activator inhibitor 1 from a suicide inhibitor to a substrate by monoclonal antibodies. J Biol Chem 2002; 277: 43858-43865.
    CrossrefPubMedGoogle Scholar
  • 33 Gao S, Krogdahl A, Sorensen JA. et al. Overexpression of protease nexin-1 mRNA and protein in oral squamous cell carcinomas. Oral Oncol 2008; 44: 309-313.
    CrossrefPubMedGoogle Scholar
  • 34 Durocher Y, Perret S, Kamen A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 2002; 30: E9.
    CrossrefPubMedGoogle Scholar
  • 35 Bager R, Kristensen TK, Jensen JK. et al. Urokinase-type plasminogen activator-like proteases in teleosts lack genuine receptor-binding epidermal growth factor-like domains. J Biol Chem 2012; 287: 27526-27536.
    CrossrefPubMedGoogle Scholar
  • 36 Tom R, Bisson L, Durocher Y. Transfection of HEK293-EBNA1 Cells in Suspension with Linear PEI for Production of Recombinant Proteins. CSH Protocols. 2008 2008: pdb prot4977.
    PubMedGoogle Scholar
  • 37 Jensen JK, Wind T, Andreasen PA. The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand. FEBS Lett 2002; 521: 91-94.
    CrossrefPubMedGoogle Scholar
  • 38 Jensen JK, Thompson LC, Bucci JC. et al. Crystal structure of plasminogen activator inhibitor-1 in an active conformation with normal thermodynamic stability. J Biol Chem 2011; 286: 29709-29717.
    CrossrefPubMedGoogle Scholar
  • 39 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497.
    CrossrefPubMedGoogle Scholar
  • 40 Reading CL. Theory and methods for immunization in culture and monoclonal antibody production. J Immunol Methods 1982; 53: 261-291.
    CrossrefPubMedGoogle Scholar
  • 41 Nielsen LS, Andreasen PA, Grondahl-Hansen J. et al. Monoclonal antibodies to human 54,000 molecular weight plasminogen activator inhibitor from fibrosarcoma cells--inhibitor neutralization and one-step affinity purification. Thromb Haemost 1986; 55: 206-212.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 42 Munch M, Heegaard C, Jensen PH. et al. Type-1 inhibitor of plasminogen activators. Distinction between latent, activated and reactive centre-cleaved forms with thermal stability and monoclonal antibodies. FEBS Lett 1991; 295: 102-106.
    PubMedGoogle Scholar
  • 43 Futamura A, Gettins PG. Serine 380 (P14) --> glutamate mutation activates antithrombin as an inhibitor of factor Xa. J Biol Chem 2000; 275: 4092-4098.
    CrossrefPubMedGoogle Scholar
  • 44 Lawrence DA, Olson ST, Palaniappan S. et al. Engineering plasminogen activator inhibitor 1 mutants with increased functional stability. Biochemistry 1994; 33: 3643-3648.
    CrossrefPubMedGoogle Scholar
  • 45 Berkenpas MB, Lawrence DA, Ginsburg D. Molecular evolution of plasminogen activator inhibitor-1 functional stability. EMBO J 1995; 14: 2969-2977.
    CrossrefPubMedGoogle Scholar
  • 46 De Taeye B, Gils A, Declerck PJ. The story of the serpin plasminogen activator inhibitor 1: is there any need for another mutant?. Thromb Haemost 2004; 92: 898-924.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 47 Carrell RW, Stein PE. The biostructural pathology of the serpins: critical function of sheet opening mechanism. Biol Chem Hoppe-Seyler 1996; 377: 1-17.
    PubMedGoogle Scholar
  • 48 Jensen JK, Gettins PG. High-resolution structure of the stable plasminogen activator inhibitor type-1 variant 14–1B in its proteinase-cleaved form: a new tool for detailed interaction studies and modeling. Protein Sci 2008; 17: 1844-1849.
    CrossrefPubMedGoogle Scholar
  • 49 Dementiev A, Dobo J, Gettins PG. Active site distortion is sufficient for proteinase inhibition by serpins: structure of the covalent complex of alpha1-proteinase inhibitor with porcine pancreatic elastase. J Biol Chem 2006; 281: 3452-3457.
    CrossrefPubMedGoogle Scholar
  • 50 Rubinstein ND, Mayrose I, Halperin D. et al. Computational characterization of B-cell epitopes. Mol Immunol 2008; 45: 3477-3489.
    CrossrefPubMedGoogle Scholar