title: 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 Cys(462)-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 Cys(541) thiolate anion on the Cys(536) sulfur atom of the Cys(512)-Cys(536) disulfide bond, resulting in reduction of the bond by thiol/disulfide exchange. Cleavage of the Cys(512)-Cys(536) allosteric disulfide allows further conformational change and exposure of the peptide backbone to proteolysis and angiostatin release. The Cys(462)-Cys(541) and Cys(512)-Cys(536) 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.
authors: From the Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia and.
authors: Diego
authors: D
authors: Butera
authors: Troels
authors: T
authors: Wind
authors: Angelina J
authors: AJ
authors: Lay
authors: Julia
authors: J
authors: Beck
authors: Francis J
authors: FJ
authors: Castellino
authors: Philip J
authors: PJ
authors: Hogg
mesh_terms: D000494:Allosteric Regulation; D043168:Angiostatins / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D005341:Fibrinolysin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D010958:Plasminogen / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D011498:Protein Precursors; D013438:Sulfhydryl Compounds; D043168:Angiostatins; D010958:Plasminogen; D005341:Fibrinolysin; D003545:Cysteine
keywords: Allosteric Regulation; Angiogenesis; Disulfide; Plasmin; Plasminogen