A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization

Abstract

Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.

Fig. 1. Ribbon model of PAI-2. The model was built with co-ordinates from Brookhaven Protein Data Bank (accession No. 1BY7) using the Insight II program (Biosym. Technologies, San Diego, CA). The cysteines present in the X-ray structure are shown as red sticks, the CD-loop (not solved by the X-ray structure) is drawn schematically as a red line, and Cys79 is marked as a red sphere.

Fig. 2. Influence of alkylation of thiol groups in wt PAI-2 on its polymerization and activity. (A) Polymerization of wt PAI-2 tested by non-denaturing PAGE followed by western blot (each lane contains 2 µg of PAI-2). Lane 1, wt PAI-2 pre-polymerized in 50 mM Tris–HCl buffer pH 8.0, containing 0.14 M NaCl at 37°C for 16 h; lane 2, pre-polymerized PAI-2 modified by reduction with 100 mM DTT followed by alkylation with excess iodoacetamide (designed as ‘blocked’ PAI-2); lane 3, ‘blocked’ PAI-2 mixed with a 1.5 molar excess of uPA; lane 4, ‘blocked’ PAI-2 incubated at 37°C for 24 h; lane 5, ‘blocked’ PAI-2 as in lane 4 but incubated with uPA. The positions of monomers, dimers and trimers are marked with arrows; the bracket shows a gel region with PAI-2–uPA complex. (B) Inhibitory activities of native wt PAI-2 and the ‘blocked’ PAI-2 before (black bars) and after (dashed bars) incubation at 37°C for 24 h, tested by chromogenic activity assay.

Fig. 3. Polymerization and activity tests of PAI-2 mutants with a cysteine substituted by serine. (A) Purified PAI-2 mutants (‘minus’ lanes) and their complexes with uPA (‘plus’ lanes) analyzed by SDS–PAGE under reducing conditions and Coomassie Blue stained. The positions of intact and cleaved PAI-2, A-chain of uPA and the PAI-2–uPA complex are marked. (B) Non-denaturing PAGE analysis of wt PAI-2 and the mutants before (‘minus’ lanes) and after (‘plus’ lanes) incubation for 24 h at 37°C, followed by western blot and ECL detection. The positions of PAI-2 monomers (M), dimers (D), trimers (Tr) and tetramers (Te) are marked. (C) Relative inhibitory activities of wt PAI-2, and C79S and C161C mutants of PAI-2 assayed before (black bars) and after (dashed bars) incubation at 37°C for 24 h, tested by chromogenic activity assay. The activity prior to the incubation for each PAI-2 form was assumed to be 100%.

Fig. 4. Molecular sieving of wt PAI-2 and the C161S mutant. The PAI-2 proteins pre-incubated in 50 mM Tris–HCl pH 7.4 for 24 h at 37°C were analyzed on a Sephacryl S-200 (Pharmacia, Uppsala, Sweden) column (1 × 120 cm) equilibrated with the above buffer. The positions of ovalbumin monomers (M) and dimers (D) are marked with arrows. The continuous line represents the OD at 280 nm; the dotted line corresponds to inhibitory activity measured by chromogenic assay.

Fig. 5. Importance of the C79–C161 disulfide bond for PAI-2 polymerization. PAI-2 mutants were analyzed by non-denaturing PAGE followed by western blot. Lane 1, PAI-2 mutant with the K87E substitution in the CD-loop; lane 2, PAI-2 mutant with the F81K substitution in the CD-loop; lane 3, 79cys/161cys mutant; lane 4, 79cys/161cys mutant reduced with DTT and labeled with sulfhydryl-specific BODIPY.

Fig. 6. Annealing of synthetic peptides into wt PAI-2 and the mutants. (A) Wt PAI-2 and the mutants were incubated with different molar excesses of a synthetic peptide homologous to RCL of PAI-2 (in 50 mM Na-phosphate buffer pH 5.9, with 20% glycerol) for 6 h at 37°C. As a control, a synthetic peptide homologous to the CD-loop was used. After the incubation, the PAI-2 samples were mixed with a 1.5 molar excess of uPA and analyzed by SDS–PAGE under reducing conditions, followed by western blot and ECL detection of PAI-2 antigen. The annealed PAI-2 was quantified as relative amount of the cleaved PAI-2 in relation to PAI-2 antigen in each lane. Inset: a western blot where the RCL peptide was annealed into wt PAI-2. Lane 1, wt PAI-2 pre-incubated without peptide and then reacted with uPA; lanes 2–6, wt PAI-2 pre-incubated with a 6-, 12-, 25-, 50- and 100-fold molar excess of the RCL peptide (respectively) and then reacted with uPA (*, cleaved PAI-2; **, PAI-2–uPA complex); lane 7, intact PAI-2. (B) Kinetics of annealing of the peptide homologous to RCL of PAI-2. The wt PAI-2 and the mutants were incubated with a 100-fold molar excess of the RCL peptide and samples were analyzed at different time points as described above. For (A) and (B), annealing of the RCL peptide into: wt PAI-2 (open circles), C5S (open squares), C145S (open triangles), C79S (filled circles), C161S (filled squares), C5S7C79S (filled upright triangles) and C5S/C161S mutants (filled inverted triangles). Annealing of the control peptide into wt PAI-2 is marked by diamonds.

Fig. 7. Probing hydrophobic cavities in polymerizing and non-polymerizing forms of PAI-2 by bis-ANS binding. The bis-ANS was mixed with a monomeric wt PAI-2 and C79S mutant, and the fluorescence was measured by exciting at 370 nm and measuring emitted light at 520 nm. (A) Kinetics of bis-ANS binding. (B) Calculated rate of bis-ANS binding to PAI-2 (mean value of three experiments).

Fig. 8. Polymerization of PAI-2 in vivo. Wt PAI-2 and PAI-2 fused with signal peptide derived from PAI-1 were overexpressed in CHO cells using the SFV expression system. Cell extracts in 50 mM phosphate buffer pH 5.9 were fractionated by SDS–PAGE (A) and non- denaturing PAGE (B) followed by western blot and ECL. In both panels: lane 1, wt PAI-2; lane 2, PAI-2 fused with the external secretion signal.

Fig. 9. Three interconvertible forms of PAI-2. Under reducing conditions, when disulfide bonds cannot be formed, PAI-2 exists in stable monomeric form with a closed A-β-sheet. This form of PAI-2 resembles that of other native serpins. However, under oxidative conditions, when the disulfide bond between C79 (located in the middle of the CD-loop) and C161 (located at the bottom of helix F) can form, PAI-2 converts into a polymerogenic conformation with an open A-β-sheet. The gap in the A-β-sheet makes PAI-2 an acceptor for the RCL of another PAI-2 molecule, leading to spontaneous polymerization by the loop–sheet mechanism. Both the polymerogenic conformation of PAI-2 and the polymers can convert to the stable monomeric form following reduction.