The physiological role(s) of PAI-2 are unclear. Although this serpin may be either a u-PA or a t-PA inhibitor, other intracellular roles (e.g. protection against apoptosis) are entirely plausible. Because PAI-2 "knockout" studies show normal gross morphology, fecundity, placentation, inheritance, epidermal architecture, wound healing and sensitivity to bacterial products1 there has been considerable recent conjecture concerning its authentic physiological roles. This biochemical study determines those reactive centre loop residues critical for "driving" the process of serpin strand insertion and the subsequent "relaxation" of PAI-2-utilising urea stability studies, fluorimetry, CD spectroscopy and activity assays after incubation with RCL peptides. In addition, reactivity against a mAb (2H5) which specifically detect epitopes associated with the "relaxed" form but not the native "stressed" confor-mation of PAI-221 have been employed to confirm these biochemical studies. Our results show that only authentic 14mer PAI-2 RCL peptides (not any shorter RCL peptides nor scrambled 14mer peptides) induce resistance to urea denaturation and loss of serpin inhibitory activity, corresponding with changes in the CD spectra. No changes were observed following incubation with PAI-2 RCL peptides where either the P14 residue was mutated or removed or with scrambled peptides. Mutating only the P13 position in full length 14mer also was not found to affect effective insertion. Insertion of the 14mer converted the "relaxed" PAI-2 from a uPA inhibitor to a substrate of uPA as the endogenous PI-PI' peptide bond could still be cleaved. Our data suggest that the PAI-2 P14 residue (i.e. Thr367) is absolutely critical for driving strand s4A insertion into -sheet A during protease inhibition. Recent crystal structure data confirm the importance of interaction around P14 in this process.4.
|Number of pages||1|
|Journal||Fibrinolysis and Proteolysis|
|Issue number||SUPPL. 1|
|Publication status||Published - 2000|