title: Elimination of the Cys558-Cys566 bond in Lys78-plasminogen--effect on activation and fibrin interaction.
abstract: Plasminogen contains a unique disulphide bond, Cys558-Cys566, responsible for the cyclic nature of the peptide sequence surrounding the activation site at Arg561-Val562. A recombinant [Ser558, Ser566]-Lys78-plasminogen variant was produced in which the two cysteine residues were replaced by serine residues. The variant was used to study the functional implications of removing the structural restrains imposed to the activation loop by this disulphide bond. Elimination of the Cys558-Cys566 bond attenuated activation by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), but resulted in an increased susceptibility to cleavage by trypsin and plasma kallikrein. Two opposite effects on the interaction of plasminogen with streptokinase were produced by modification of this bond; (a) attenuation of the rate at which the active complex with streptokinase was formed and (b) a 7.5-fold increase in plasminogen activation catalysed by this complex. Activation by tPA in the presence of fibrin, in contrast to activation in its absence, was not attenuated by elimination of this disulphide bond. However, the activation rate as a function of plasminogen concentration followed a different saturation curve, and the fibrin degradation pattern was changed. The results suggest that the Cys558-Cys566 disulphide bond is of importance for the specificity of plasminogen. This applies to its activation and also to its role in subsequent fibrin clot degradation.
authors.affiliation: Novo Nordisk A/S, Health Care Discovery, Protein Chemistry, Bagsvaerd, Denmark. vili@novo.dk
authors.forename: V
authors.initials: V
authors.lastname: Linde
authors.forename: L S
authors.initials: LS
authors.lastname: Nielsen
authors.forename: D C
authors.initials: DC
authors.lastname: Foster
authors.forename: L C
authors.initials: LC
authors.lastname: Petersen
mesh_terms: D000818:Animals; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D008239:Lysine; D010958:Plasminogen / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine; D012697:Serine Endopeptidases / Q000378:metabolism / Q000494:pharmacology; D013300:Streptokinase / Q000378:metabolism / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D012694:Serine; D005337:Fibrin; D010958:Plasminogen; D013300:Streptokinase; D012697:Serine Endopeptidases; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine; D003545:Cysteine