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

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  • Tina Mostrup Kousted, Denmark
  • K Skjoedt, Department of Cancer and Inflammation, University of Southern Denmark
  • ,
  • S V Petersen
  • C Koch, Department of Cancer and Inflammation, University of Southern Denmark
  • ,
  • Lars Vitved, Cancer- og Inflammationsforskning, Denmark
  • M Sochalska, Department of Molecular Biology and Genetics, Aarhus University
  • ,
  • Céline Lacroix
  • ,
  • Lisbeth Moreau Andersen, Denmark
  • Troels Wind, Denmark
  • Peter A Andreasen, Denmark
  • Jan Kristian Jensen
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 stable 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 á-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.
Original languageEnglish
JournalThrombosis and Haemostasis
Volume111
Issue1
Pages (from-to)29-40
ISSN0340-6245
DOIs
Publication statusPublished - 2 Oct 2013

    Research areas

  • cancer, serine protease, glia-derived nexin, serpin E2, inhibitory antibody

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