INTRACELLULAR POLYMERIZATION OF THE SERPIN PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-2

Plasminogen activator inhibitor type 2 (PAI-2) is synthesized in two m olecular forms: an intracellular, nonglycosylated form and an extracel lular, glycosylated form. The bitopological distribution of PAI-2 is c aused by an inefficient internal secretion signal. In addition, the se cretion efficiency of PAI-2 seems to differ, depending on the cell typ e, differentiation state, and culture conditions. In recombinant cell clones designed for the synthesis of the secreted form of PAI-2, the f raction of secreted PAI-2 decreased with increasing expression levels. Subcellular fractionation of cell clones with higher expression level s revealed that PAI-2 accumulating in the cell was mainly associated w ith the organelles of the secretory pathway. Electrophoresis under non denaturating conditions revealed that the PAI-2 retained at higher exp ression levels was mainly polymerized. Polymers of PAI-2 were also det ected in cytosolic extracts prepared from human placenta and phorbol e ster-stimulated U 937 cells, indicating that intracellular polymerizat ion of PAI-2 may occur in the cytosols of cells that normally express PAI-2 under physiological conditions. When purified PAI-2 or cellular extracts were incubated at 37 degrees C for 24 h most of the PAI-2 pro tein was found to polymerize. Polymer formation was prevented by the a ddition of synthetic peptides with sequences corresponding to residues P2 to P14 in the reactive center loop of PAI-2 and antithrombin. Thes e synthetic peptides also caused dissociation of prepolymerized purifi ed PAI-2 and PAI-2 polymers in cellular extracts. Incubation with unre lated peptides of the same size had no effect on polymer formation or dissociation of preformed polymers, indicating that polymerization of PAI-2 occurs by the loop-sheet mechanism. Taken together, our data sug gest that the wild-type form of PAI-2, like some natural pathological genetic variants of alpha(1)-antitrypsin, antithrombin, and C1 inhibit or readily polymerizes intracellularly and that polymerization may lea d to a reduced secretion efficiency.