Abstract
Plasminogen activator inhibitor type 2 (PAI-2) is synthesized in two molecular forms: an intracellular, nonglycosylated form and an extracellular, glycosylated form. The bitopological distribution of PAI-2 is caused by an inefficient internal secretion signal. In addition, the secretion efficiency of PAI-2 seems to differ, depending on the cell type, differentiation state, and culture conditions. In recombinant cell clones designed for the synthesis of the secreted form of PAI-2, the fraction of secreted PAI-2 decreased with increasing expression levels. Subcellular fractionation of cell clones with higher expression levels revealed that PAI-2 accumulating in the cell was mainly associated with the organelles of the secretory pathway. Electrophoresis under nondenaturating conditions revealed that the PAI-2 retained at higher expression levels was mainly polymerized. Polymers of PAI-2 were also detected in cytosolic extracts prepared from human placenta and phorbol ester-stimulated U 937 cells, indicating that intracellular polymerization 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 protein was found to polymerize. Polymer formation was prevented by the addition of synthetic peptides with sequences corresponding to residues P2 to P14 in the reactive center loop of PAI-2 and antithrombin. These synthetic peptides also caused dissociation of prepolymerized purified PAI-2 and PAI-2 polymers in cellular extracts. Incubation with unrelated 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 suggest that the wild-type form of PAI-2, like some natural pathological genetic variants of alpha1-antitrypsin, antithrombin, and C1 inhibitor readily polymerizes intracellularly and that polymerization may lead to a reduced secretion efficiency.
Highlights
Polymers of Plasminogen activator inhibitor type 2 (PAI-2) were detected in cytosolic extracts prepared from human placenta and phorbol esterstimulated U 937 cells, indicating that intracellular polymerization of PAI-2 may occur in the cytosols of cells that normally express PAI-2 under physiological conditions
By measuring the PAI-2 content in cellular extracts and conditioned medium we found that the secretion efficiency of PAI-2 varied with the level of expression, such that the secretion efficiency was lower when the expression level was higher
About 50% of PAI-2 was secreted from Chinese hamster ovary (CHO) cells with the highest PAI-2 expression, while 98% of PAI-2 was secreted from cells with low levels of expression
Summary
Materials—Restriction enzymes, T4 DNA ligase, and polynucleotide kinase were from Boehringer Mannheim (Germany). To determine the expression level and the secretion efficiency of PAI-2, about 80% confluent cultures (8 ϫ 105 cells) of PAI-2 expressing stable cell clones or cells infected with SFV expression construct were grown for 24 h at 37 °C After this period conditioned medium was collected, and corresponding cellular extracts (1 ml) were prepared (Ny et al, 1989) and assayed for PAI-2 antigen by specific enzyme-linked immunosorbent assay (Biopool) and ECL Western blotting (Amersham) as described by the suppliers. Purified recombinant PAI-2 (Mikus et al, 1993) (0.1 mg/ml) was incubated in 10 mM Tris-HCl buffer, pH 7.5, with a 100-fold molar excess of the synthetic peptide at 37 °C for 24 h. Galactosyl transferase was only detected in a fraction containing organelles of the secretory pathway and not in the homogenate and nuclear pellet
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