期刊
THROMBOSIS AND HAEMOSTASIS
卷 111, 期 1, 页码 29-40出版社
SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN
DOI: 10.1160/TH13-04-0340
关键词
Cancer; glia-derived nexin; SERPIN E2; serine protease; inhibitory antibody
资金
- Alfred Nielsen's Foundation
- Cancer Research Foundation of 1989
- Danish Cancer Society
- Danish National Research Foundation [26-331-6]
- Faculty of Science and Technology, Aarhus University
- Research Council for Production and Technology [09-072885]
- Lundbeck Foundation
Protease nexin-1 (PN-1) belongs to the serpin. family and is an in, hibitor 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, PM-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 alpha-helix F with beta-strand 3A and the loop connecting alpha-helix A with beta-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, PM-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1s protease inhibitory activity.
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