Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis |
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Authors: | Weening-Verhoeff EJD; Quax PHA; van Leeuwen RTJ; Rehberg EF; Marotti KR; Verheijien JH |
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Affiliation: | Gaubius Institute TNO PO Box 612, 2300 AP Leiden, The Netherlands
1The Upjohn Company, Molecular Biology Department Kalamazoo, MI, USA |
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Abstract: | Modification of glutamic and aspartic acid residues of tissue-typeplasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimideleads to a decrease in affinity for lysine and fibrin, to adecrease of plasminogen activation activity in the presenceof a fibrin mimic, but leaves amidolytic activity and plasminogenactivation without fibrin mimic unaffected. Experiments withkringle-2 ligands and a deletion mutant of t-PA (K2P) suggeststhat glutamic or aspartic acid residues in K2 of t-PA are involvedin stimulation of activity, lysine binding and fibrin binding.Mutant t-PA molecules were constructed by site-directed mutagenesisin which one or two of the five aspartic or glutamic acid residuesin K2 were changed to asparagine or glutamine respectively.Mutation of Asp236 and/or Asp238 leads to t-PA molecules with3- to 4-fold lower specific activity in the presence of fibrinmimic and having no detectable affinity for lysine analogs.However, fibrin binding was not influenced. Mutation of Glu254also leads to a 3- to 4-fold lower activity, but to a much smallerreduction of lysine or fibrin binding. Residues Asp236 and Asp238are both essential for binding to lysine derivatives, whileGlu254 might be involved but is not essential. Residues Asp236,Asp238 and Glu254 are all three involved in stimulation of activity.Remarkably, mutation of residues Asp236 and/or Asp238 appearsnot to influence fibrin binding of t-PA whereas that of Glu254does. |
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Keywords: | fibrin binding/ kringle/ lysine binding/ mutant/ tissue-type plasminogen activator |
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