title: VLHL plasminogen activator inhibitor spontaneously reactivates from the latent to active form.
abstract: Wild-type plasminogen activator inhibitor type 1 (PAI-1) is a fast-acting uPA and tPA inhibitor with half-life of 1-2 h. Recombinant PAI-1 with two mutations, Q197C and G355C, shows a very long half-life (VLHL). An introduced disulfide bridge holds together two central, parallel strands of beta-sheet A, preventing their separation to incorporate residues P4-P14 during the serpin's transition into latency. An active PAI-1 is usually described as a single structure with the reactive center loop (RCL) with P1-P1' (R369-M370) extended far from the bulk of the serpin's body. We have found that VLHL PAI-1 exists in several active forms that travel with different electrophoretic mobilities. Under aerobic conditions, two distinct active forms are observed. Upon reduction of cysteines, the VLHL mutant converts into the latent form, which spontaneously reactivates into a fully or partially active serpin, with yet another mobility. Utilizing electrophoresis, zymography (to check PAI-1 activity toward uPA) and theoretical calculations for molecular modeling, we have characterized active 1, 2, 3 and latent conformers of VLHL PAI-1 and their behaviors at normal and elevated temperatures, and in normal or reducing environments. VLHL PAI-1 activity is not affected, and the molecules do not polymerize unless reduced and/or heated. VLHL PAI-1 associates into dimers and bigger oligomers when -SH groups become available for oxidation and formation of intra- or intermolecular -S-S- bridges between conformers of different shapes and activities. We postulate that the active structures differ in RCL conformation and their position in relation to the gate region and the rest of the molecule.
authors.affiliation: Urology Research Center, University of Toledo, Health Science Campus, 3065 Arlington, Toledo, OH 43614-5807, USA. jerzy.jankun@utoledo.edu
authors.forename: Jerzy
authors.initials: J
authors.lastname: Jankun
authors.forename: Ansari M
authors.initials: AM
authors.lastname: Aleem
authors.forename: Steve H
authors.initials: SH
authors.lastname: Selman
authors.forename: Venkatesha
authors.initials: V
authors.lastname: Basrur
authors.forename: Ewa
authors.initials: E
authors.lastname: Skrzypczak-Jankun
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D006207:Half-Life; D006358:Hot Temperature; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D011108:Polymers; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors