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Construction of Urokinase Mutant Glu154-mtcu-PA and Characterization of Its Properties
Gui-Hong Peng1, Zhong Ma, Yu-Ming Xue
1Department of Biochemistry and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China.
Abstract:
The recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a mutant constructed by in vitro site-specific mutagenesis of Argl54 in rscu-PA to Glul54 (Glul54-mscu-PA) were both expressed in E. coli. The expressed products were both purified to homogeneity by in vitro denaturation and renaturation, followed by Zn(2+) selective precipitation and immuno-affinity chromatography. The plasmin sensitivity assay indicated that the activation of this single chain Glul54-mscu-PA by plasmin was essentially identical to that of rscu-PA. After activation by plasmin, the kinetic constants against synthetic substrate S2444 of the resulted two chain form of Glul54-mscu-PA (Glul54-mtcu-PA) and that of rscu-PA (rtcu-PA) were 87 &mgr;M and 80 &mgr;M, respectively, which indicated that the catalytic active site of the Glul54-mtcu-PA was not changed by the mutation. Whereas, both (125)I-fibrin plasma-clot lysis and fibrinogenolysis in plasma showed that the Glul54-mtcu-PA possessed a better affinity and selectivity for fibrin than rtcu-PA, even better than rscu-PA.
Insights
A novel mutant of recombinant single-chain urokinase-type plasminogen activator (rscu-PA) showed enhanced fibrin affinity and selectivity. This Glul54-mscu-PA mutant offers improved potential for thrombolytic therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Thrombolysis
Background:
- Recombinant single-chain urokinase-type plasminogen activator (rscu-PA) is a key thrombolytic agent.
- Site-specific mutagenesis can alter protein function and therapeutic potential.
Purpose of the Study:
- To investigate the functional impact of a specific mutation (Arg154 to Glu154) in rscu-PA.
- To evaluate the fibrin affinity and selectivity of the engineered Glul54-mscu-PA mutant.
Main Methods:
- Expression and purification of rscu-PA and Glul54-mscu-PA in E. coli.
- Plasmin activation assays and kinetic analysis using synthetic substrate S2444.
- In vitro plasma clot lysis and fibrinogenolysis assays using radiolabeled fibrin.
Main Results:
- The Glul54-mscu-PA mutant exhibited plasmin activation kinetics similar to wild-type rscu-PA.
- The catalytic activity of the mutated two-chain form (Glul54-mtcu-PA) remained comparable to wild-type (rtcu-PA).
- Glul54-mtcu-PA demonstrated significantly improved affinity and selectivity for fibrin compared to rtcu-PA and rscu-PA.
Conclusions:
- The Arg154 to Glu154 mutation in rscu-PA enhances fibrin binding without compromising catalytic activity.
- Glul54-mscu-PA represents a promising candidate for developing more effective thrombolytic agents.
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