The physiological roles of PAI-2 are not yet well understood. This study describes the generation and characterisation of "relaxed" PAI-2 by insertion of a synthetic reactive site loop (RSL) peptide, mimicking the conformation of PAI2 induced by inhibition of its target protease urokinase (uPA)1. The generation of a monoclonal antibody to specifically detect this conformation of PAI-2 provides an immunological probe for precise localisation of in vivo sites (i.e. skin, placenta, tumours) in which PAI-2 is actively inhibiting uPA or other target proteases and may lead to a better understanding of the physiological and/or pathological roles of this molecule2. The ability of a panel of different length RSL peptides to induce "relaxed" PAI2 were examined by urea denaturation, fluorimetry, CD spectroscopy and activity assays. PAI-2:RSL complexes were refractory to urea denaturation, indicating a significant increase in conformational stability of PAI-2 following peptide insertion. Additionally, a significant shift in the CD spectra of PAI-2 was observed following incubation with 14mer RSL peptide. RSL insertion converted PAI-2 from an inhibitor to a substrate of uPA. It is likely that the synthetic RSL peptide occupies a similar, if not identical, position in -sheet A to that which the RSL of PAI-2 would occupy following stable complex formation with uPA or other proteases. Binding of the synthetic RSL peptide to this site may block insertion of the RSL of PAI-2 following cleavage at the PI-PI' bond and prevent trapping of the protease in a stable complex, hence preventing inhibition. We did not observe the formation of relaxed PAI-2 by incubation with any RSL peptides lacking the P14 (Thr367) and P13 (Glug) residues, suggesting that either/both of these residues are critical for insertion of strand s4A into -sheet A of PAI-2 during protease inhibition. Understanding the mechanism of action of serpins is critical not only for understanding how this family of proteins functions but also to assist in the identification of cognate target proteases where the physiological activity of particular serpins is unknown.
|Number of pages||1|
|Journal||Fibrinolysis and Proteolysis|
|Issue number||SUPPL. 1|
|Publication status||Published - 1998|