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
