Characterization of a reduced form of plasma plasminogen as the precursor for angiostatin formation

Abstract

Plasma plasminogen is the precursor of the tumor angiogenesis inhibitor, angiostatin. Generation of angiostatin in blood involves activation of plasminogen to the serine protease plasmin and facilitated cleavage of two disulfide bonds and up to three peptide bonds in the kringle 5 domain of the protein. The mechanism of reduction of the two allosteric disulfides has been explored in this study. Using thiol-alkylating agents, mass spectrometry, and an assay for angiostatin formation, we show that the Cys462-Cys 541 disulfide bond is already cleaved in a fraction of plasma plasminogen and that this reduced plasminogen is the precursor for angiostatin formation. From the crystal structure of plasminogen, we propose that plasmin ligands such as phosphoglycerate kinase induce a conformational change in reduced kringle 5 that leads to attack by the Cys541 thiolate anion on the Cys53 6 sulfur atom of the Cys512-Cys536 disulfide bond, resulting in reduction of the bond by thiol/disulfide exchange. Cleavage of the Cys512-Cys536 allosteric disulfide allows further conformational change and exposure of the peptide backbone to proteolysis and angiostatin release. The Cys462-Cys541 and Cys512-Cys536 disulfides have -/+RHHook and - LHHook configurations, respectively, which are two of the 20 different measures of the geometry of a disulfide bond. Analysis of the structures of the known allosteric disulfide bonds identified six other bonds that have these configurations, and they share some functional similarities with the plasminogen disulfides. This suggests that the -/+RHHook and-LHHook disulfides, along with the-RHStaple bond, are potential allosteric configurations. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.