PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-2 (PAI-2) IS A SPONTANEOUSLY POLYMERIZING SERPIN - BIOCHEMICAL-CHARACTERIZATION OF THE RECOMBINANT INTRACELLULAR AND EXTRACELLULAR FORMS

Plasminogen-activator inhibitor type 2 (PAI-2) is a specific inhibitor of plasminogen activators (PA) that exists in an intracellular, low-m olecular-mass form and a secreted, high-molecular-mass form that varie s with respect to glycosylation. Here we have developed expression sys tems for both forms of PAI-2 and biochemically characterised the purif ied proteins. In order to obtain efficient secretion, we constructed a n artificial signal sequence and fused it to the coding region of PAI- 2. With this construct, more than 90% of PAI-2 was secreted as a glyco sylated, 60-kDa molecular-mass form, but the level of expression was l ow and unstable. To obtain higher expression of secreted PAI-2, a nove l expression vector based on the Semliki-forest-virus replicon was use d. Secreted PAI-2 was purified to homogeneity and N-terminal sequence analysis showed that the artificial signal peptide was correctly remov ed. The intracellular, non-glycosylated form of PAI-2 was expressed in Escherichia coli and purified to homogeneity. Both the secreted and t he intracellular forms of PAI-2 were found to inhibit plasminogen acti vators by forming SDS-resistant complexes and the second-order rate co nstants were similar for both forms, ranging over 2.4-2.7 X 10(6) M-1 s-1 for urokinase-type PA, 2.5-2.7 x 10(5) M-1 s-1 for two-chain tissu e-type PA and 0.8-1.2 X 10(4) M-1 s-1 for single-chain tissue-type PA. None of the purified PAI-2 forms bound to vitronectin. Circular-dichr oism spectral analysis revealed that PAI-2 has a CD spectrum that rese mbles oval-bumin more than PA-inhibitor type 1, confirming the greater similarity between these two members of the serine-protease inhibitor family. Similar to what has been described for the Z-form of alpha1-a ntitrypsin, purified PAI-2 was found to spontaneuosly form polymers du ring incubation at room temperature. Attempts to convert PAI-2 to a st able locked conformation resembling the conformation of latent PAI-1 b y treatment with diluted guanidinium chloride were unsuccessful. Inste ad, this treatment enhanced the formation of PAI-2 polymers, possibly by the loop-sheet polymerisation mechanism described for alpha1-antitr ypsin.