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

Abstract

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.

Selective neutralization of the serpin protease nexin‐1 by a specific monoclonal antibody

“…Furthermore, cleaved PN‐1 remained, represented by the slightly faster migrating band, indicating that PN‐1 could still be cleaved by thrombin in the presence of MA‐48H11. These observations suggest that binding of MA‐48H11 to PN‐1 resulted in substrate behaviour of PN‐1 toward thrombin, as previously shown for other neutralizing monoclonal anti‐PN‐1 antibodies (Kousted et al , ). In contrast, the 120‐kDa plasmin/PN‐1 complex was still formed in the presence of MA‐48H11, indicating that MA‐48H11 did not affect the plasmin/PN‐1 complex formation (Fig B).…”

“…Complex formation between PN-1 and plasmin results in the formation of a 120-kDa band ( Fig 2B). The formation of thrombin/PN-1 complexes was accompanied by the presence of a band migrating slightly faster than PN-1, corresponding to the P1-P'1 cleaved form of PN-1, as previously described (Kousted et al, 2014). Thrombin/PN-1 complex formation was inhibited in the presence of MA-48H11 (Fig 2A), as evidenced by the disappearance of the 77-kDa thrombin/PN-1 complex.…”

A slow-cycling subpopulation of melanoma cells with highly invasive properties

“… 25 For validation, we blocked the anti-proteolytic activity of SerpinE2 with a specific monoclonal antibody. 26 Neutralization of SerpinE2 reduced melanoma invasion in a dose-dependent manner in all four models tested ( Figure 4b ; Supplementary Figure 7B ). SerpinE2 knockdown with shRNA ( Supplementary Figure 8 ; Supplementary Table 8 ) also confirmed the inhibition of invasion ( Figure 4c ).…”

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour

“…This suggested that its binding site was situated in a region of the molecule only indirectly associated with the inhibitory mechanism [44]. In comparison, antibodies interacting with β-sheet A of protease nexin-1 were found to almost completely abolish inhibitory activity through direct interference with the RCL insertion mechanism [37]. Variable effects on inhibitory activity have been seen in studies with serpin mutants, dependent on the identity of the target protease [45,57–59].…”

“…Antibodies that decrease PAI-1 stability have also been found that interact with regions associated with conformational transitions: the C-sheet upon strand 1C displacement [33] and helix D [34]. Others that suppress inhibitory activity have been noted to interact directly with components of the inhibitory apparatus, localising to the vicinity of helix F of PAI-1 — which is known to move during RCL insertion [38] — and the lower portion of β-sheet A that accepts the incoming RCL during inhibition by protease nexin-1 [37]. Each of these epitopes is distinct from the one presented here, and collectively they span a significant proportion of the serpin fold.…”