Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low M_r Single-Chain Urokinase-Type Plasminogen Activator

 

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Summary

In an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M _r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).

The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi ¹²⁵I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.

These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

• References

• 1 Günzler WA, Steffens GJ, Ötting F, Kim SM A, Frankus E, Flohe L. The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain. Hoppe-Seyler’s Z Physiol Chem 1982; 363: 1155-65
  PubMedGoogle Scholar
• 2 Holmes WE, Pennica D, Blaber M, Rey MW, Günzler WA, Steffens GJ, Heyneker HL. Cloning and expression of the gene for prourokinase in Escherichia coli. Biotechnology 1985; 3: 923-9
  CrossrefPubMedGoogle Scholar
• 3 Günzler WA, Steffens GJ, Ötting F, Buse G, Flohé L. Structural relationship between human high and low molecular mass urokinase. Hoppe-Seyler’s Z Physiol Chem 1982; 363: 133-41
  PubMedGoogle Scholar
• 4 Pannell R, Gurewich V. Activation of plasminogen by single-chain urokinase or by two-chain urokinase – a deinuusLiatiun that single-chain urokinase has a low catalytic activity (pro-urokinase). Blood 1987; 69: 22-6
  PubMedGoogle Scholar
• 5 Urano T, de Serrano VS, Gaffney PJ, Castellino FJ. The activation of human Glu¹-plasminogen by human single-chain urokinase. Arch Biochem Biophys 1988; 264: 222-30
  CrossrefPubMedGoogle Scholar
• 6 Lijnen HR, Van Hocf B, Dc Cock F, Collen D. The meclumism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro. Blood 1989; 73: 1864-72
  PubMedGoogle Scholar
• 7 Lijnen HR, Van Hoef B, Nelles L, Collen D. Plasminogen activation with single-chain urokinase-type plasminogen activator (scu-PA). Studies with active site mutageiiized plasminogen (Ser⁷⁴⁰ → Ala) and plasmin resistant scu-PA (Lys¹⁵⁸ → Glu). J Biol Chem 1990; 265: 5232-6
  PubMedGoogle Scholar
• 8 Petersen LC, Lund LR, Nielsen LS, Dano K, Skriver L. One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity. J Biol Chem 1988; 263: 11189-95
  PubMedGoogle Scholar
• 9 Lijnen HR, Zamarron C, Blaber M, Winkler ME, Collen D. Activation of plasminogen by pro-urokinase. I. Mechanism. J Biol Chem 1986; 261: 1253-8
  PubMedGoogle Scholar
• 10 Gurewich V, Pannell R, Louie S, Kelley P, Suddith RL, Greenlee R. Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species. J Clin Invest 1984; 73: 1731-9
  CrossrefPubMedGoogle Scholar
• 11 Zamarron C, Lijnen HR, Van Hoef B, Collen D. Biological and thrombolytic properties of proenzyme and active forms of human urokinase. I. Fibrinolytic and fibrinogenolytic properties in human plasma in vitro of urokinases obtained from human urine or by recombinant DNA technology. Thromb Haemost 1984; 52: 19-23
  PubMedGoogle Scholar
• 12 PRIMI Trial Study Group. Randomized double-blind trial of recombinant pro-urokinase against streptokinase in acute myocardial infarction. Lancet 1989; 1: 863-8
  PubMedGoogle Scholar
• 13 Lijnen HR, Collen D. Comparative fibrinolytic properties of staphy-lokinase and streptokinase in animal models of venous thrombosis. Thromb Haemost 1991; 66: 88-110
  Article in Thieme ConnectPubMedGoogle Scholar
• 14 Stump DC, Lijnen HR, Collen D. Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator. J Biol Chem 1986; 261: 17120-6
  PubMedGoogle Scholar
• 15 Wijngaards G, Rijken DC, Van Wezel AL, Groeneveld E. Van der Velden CAM. Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture. Thromb Res 1986; 42: 749-60
  CrossrefPubMedGoogle Scholar
• 16 Lijnen HR, Nelles L, Holmes WE, Collen D. Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu¹⁴⁴) of recombinant single chain urokinase-type plasminogen activator. J Biol Chem 1988; 263: 5594-8
  PubMedGoogle Scholar
• 17 Stump DC, Kieckens L, De Cock F, Collen D. Pharmacokinetics of single-chain forms of urokinase-type plasminogen activator. J Pharmacol Exper Therap 1987; 242: 245-50
  PubMedGoogle Scholar
• 18 Bennett WF, Paoni NF, Keyt BA, Botstein D, Jones AJ S, Presta L, Wurm FM, Zoller MJ. High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem 1991; 266: 5191-201
  PubMedGoogle Scholar
• 19 Keyt B, Baldini L, Brady K, Meunier A, Spellman M, Bennett W. Slowly clearing mutants of tissue plasminogen activator with conserved fibrinolytic activity. Fibrinolysis 1992; 6 suppl (Suppl. 02) 26 (Abstract 64)
  PubMedGoogle Scholar
• 20 Degen SJ F, Davie EW. Nucleotide sequence of the gene for human prothrombin. Biochemistry 1987; 26: 6165-77
  CrossrefPubMedGoogle Scholar
• 21 Lijnen HR, Van Hoef B, Collen D. On the role of the carbohydrate side chains of human plasminogen in its interaction with α₂-antiplasmin and fibrin. Eur J Biochem 1981; 120: 149-54
  CrossrefPubMedGoogle Scholar
• 22 Nelles L, Lijnen HR, Collen D, Holmes WE. Characterization of recombinant human single-chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of Lys¹⁵⁸ . J Biol Chem 1987; 262: 5682-9
  PubMedGoogle Scholar
• 23 Declerck PJ, Van Keer L, Verstreken M, Collen D. An enzyme-linked immunosorbent assay for urokinase-type plasminogen activator (u-PA) and mutants and chimeras containing the serine protease domain of u-PA. Thromb Haemost 1992; 67: 95-100
  Article in Thieme ConnectPubMedGoogle Scholar
• 24 Stanssens P, Opsomer C, Mc Keown YM, Kramer W, Zabeau M, Fritz HJ. Efficient olignucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res 1989; 17: 4441-54
  CrossrefPubMedGoogle Scholar
• 25 Maniatis T, Fritsch EF, Sarnbrook J. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory. Cold Spring Harbor, NY: 1982: p. 200
  Google Scholar
• 26 Gorman C, Padmanabhan R, Horvard BH. (DNA rlrming A practical approach; vol 2). Glover DM. (ed) Oxford, UK: IRL Press; 1985: p. 143
• 27 Summers MD, Smith GE. A manual of methods for Baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station. Texas A & M University; 1987. Bulletin: (; 1555). 1-56
  Google Scholar
• 28 Lijnen HR, Van Hoef B, Collen D. Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator. Biochim Biophys Acta 1986; 884: 402-8
  CrossrefPubMedGoogle Scholar
• 29 Astrup T, Mullertz S. Fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-51
  CrossrefPubMedGoogle Scholar
• 30 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54
  CrossrefPubMedGoogle Scholar
• 31 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Hematol 1957; 17: 237-46
  CrossrefPubMedGoogle Scholar
• 32 Gibaldi M, Perrier DP. Pharmacokinetics. Marcel Dekker; New York: 1983: 45-111
  Google Scholar
• 33 Stassen JM, Vanlinthout I, Lijnen HR, Collen D. A hamster pulmonary embolism model for the evaluation of thrombolytic agents. Fibrinolysis 1990; 4: 15-21
  PubMedGoogle Scholar
• 34 Kuiper J, Rijken DC, De Munk GA W, van Berkel TJ C. In vivo and in vitro interaction of high and low molecular weight single-chain urokinase-type plasminogen activator with rat liver cells. J Biol Chem 1992; 267: 1589-95
  PubMedGoogle Scholar
• 35 Lijnen HR, Li X-K, Nelles L, Hu M-H, Collen D. Biochemical properties of recombinant single chain urokinase-type plasminogen activator mutants with deletion of Asn 2 through Phe 157 and/or substitution of Cys 279 with Ala. Eur J Biochem 1992; 205: 701-9
  CrossrefPubMedGoogle Scholar
• 36 Li X-K, Lijnen HR, Nelles L, Hu M-H, Collen D. Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator. Biochim Biophys Acta 1992; 1159: 37-43
  CrossrefPubMedGoogle Scholar
• 37 Holvoet P, Laroche Y, Stassen JM, Lijnen HR, Van Hoef B, De Cock F, Van Houtven A, Gansemans Y, Matthyssens G, Collen D. Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody fused to single-chain urokinase. Blood 1993; 81: 696-703
  PubMedGoogle Scholar