Human Pro-urokinase Research Articles

Pharmacokinetics and biochemical properties of human pro-urokinase variants carrying the deletion in epidermal growth factor-like domain

Abstract Two variants of pro-urokinase (pro-UK), Δ11–32pro-UK lacking the first and the second loops (Cys11Asn32) within the EGF-like domain and δ33–42pro-UK lacking the third loop (Cys33Cys42), were prod in Chinese hamster ovary cells. They were compared with natural pro-UK (n-pro-UK) and Δ10–42pro-UK lacking entire EGF-like domain (Hiramatsu et al1) in terms of clearance rate and biochemical properties. Fibrinolytic activity half-lives of these mutants in rat circulation after bolus administration were 7.1 min for Δ11–32pro-UK and 6.8 min for Δ33–42pro-UK, which were about 3.5 times longer than that of n-pro-UK (2.0 min) and slightly shorter than that of Δ10–42pro-UK (7.6 min). The amidolytic activity of these deletion mutants were almost the same as n-pro-UK in terms of both Km and kcat (3.4–4.O × 10−4 M, and 8.4–10.O × 103 min−1 respectively), suggesting that the protease domain was not affected by the deletion in EGF-like domain. It was also found that the binding ability to fibrin-celite was decreased by the absence of the third loop of the EGF-like domain. In addition, conversion from the single chain form to the two chain form by the action of plasmin was slowed in proportion to the size of the deletion in the EGF-like domain.

Optimization of pro‐urokinase secretion from recombinant Saccharomyces cerevisiae

Secretion of a nonglycosylated form of human pro-urokinase, also known as single-chain urinary plasminogen activator (scu-PA), from Saccharomyces cerevisiae is described. A "supersecreting" yeast strain harboring multiple copies of integrated plasmids was grown batchwise and at constant respiratory quotient (RQ) in 20-L fermenters. Because the promoters used to drive expression of the pro-urokinase genes are not tightly regulated, secretion into the culture supernatant was growth associated. Although the final cell density achieved in the perturbed-batch fermentation (45 g dry wt/L) was less than that observed in the RQ-controlled culture (77 g dry wt/L), the scu-PA titer in the perturbed-batch fermentation (1863 IU/mL) was nearly twice that attained at constant RQ (1108 IU/mL). The effects on cell growth and scu-PA titer of other process variables (pH, temperature, phosphate concentration, and medium composition) are also discussed.

Isolation and characterization of human pro-urokinase and its mutants accumulated within the yeast secretory pathway

Human pro-urokinase (pro-UK) and two pro-UK deletion mutants, one lacking the epidermal growth factor(EGF)-like domain, and the other lacking both the EGF-like domain and the kringle domain, were produced in Saccharomyces cerevisiae. This was done using the yeast GAL7 promoter and the prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). Although biologically active and heavily glycosylated pro-UKs were secreted into the culture medium, the amounts were extremely small. On the other hand, large amounts of pro-UKs of a single-chain form were accumulated inside the cells, exceeding 3-4% of total cellular proteins. The intracellular pro-UKs were N-glycosylated, probably with a single core carbohydrate unit, and amino acid sequencing of their N termini revealed that the secretion signal of MPR was correctly processed. Biologically active pro-UKs were recovered in high yields by means of solubilization with 4.5 M guanidine.HCl and subsequent dialysis for refolding. The refolded yeast pro-UK was indistinguishable from human kidney-derived pro-UK in terms of specific enzymatic activity and its secondary structure, as determined by circular dichroism spectroscopy.

A bifunctional vector system for controlled expression and subsequent release of the cloned gene product by phi X174 lysis protein-E.

A new bifunctional Escherichia coli cloning vector, pSB50, is presented. The plasmid allows controlled expression of a gene of interest under control of the lac promoter and the subsequent release of the cloned product by the use of bacteriophage phi X174 lysis protein-E, the gene of which is under control of the phage Lambda pL promoter. To ensure optimal repression of the Lambda pL promoter and the lac promoter in plasmid pSB50, E. coli strain UB89-1 was constructed which carries a chromosomal copy of the lambda cl857 repressor allele and the laclq1 allele, respectively. Here, we employ the E-based lysis system to release human prourokinase to the culture medium.

Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase

A new post-translational modification site in the growth factor domain of urinary type plasminogen activator has been identified. A glycopeptide containing the monosaccharide, fucose, covalently linked directly to the peptide backbone has been isolated from the tryptic digest of pro-urokinase expressed in a mouse hybridoma cell line Sp 2/0 Ag 14. The glycopeptide was isolated by semi-preparative reversed phase high performance liquid chromatography. The identity of a fucose containing peptide was confirmed by carbohydrate analysis, amino acid analysis and plasma desorption mass spectrometry (PDMS). A combination of these methodologies showed an equimolar ratio of peptide and fucose in the glycopeptide. This modification is not detected without mass spectrometry because the fucose residue is hydrolyzed under standard acidic conditions of amino acid composition and N-terminal sequence analysis. The site of attachment of fucose to the peptide has been localized towards the N-terminus (within first 23 amino acids) of the protein. Also, the carbohydrate composition of recombinant pro-urokinase is reported.

Activation of pro-urokinase by the human T cell-associated serine proteinase HuTSP-1

The human T cell-associated serine proteinase-1 (HuTSP-1) is expressed by activated T lymphocytes and is exocytosed upon their interaction with target cells. Here, we report that HuTSP-1 is able to convert single-chain human pro-urokinase into the active two-chain enzyme. Time-dependent activation by HuTSP-1 of recombinant human pro-urokinase as well as natural pro-urokinase derived from human melanoma cells was demonstrated in a chromogenic assay specific for active urokinase type plasminogen activator and in immunoblotting experiments revealing the conversion of single-chain into two-chain urokinase. Control experiments excluded plasmin as the activating agent. These data suggest a novel pathway for plasmin generation during T cell-mediated processes such as immune responses and extravasation of immune cells.

A new method for the synthesis of a structural gene

A novel method of synthesizing a structural gene or gene fragment, consisting of the first synthesis of a single-stranded DNA (ssDNA), has been developed. As a preliminary test of this method, four synthetic genes or gene fragments have been synthesized. The first one with 396 base pairs (b.p.) codes for the mature rbcS from wheat, the next two with 370 and 342 b.p. respectively, for two half molecules of a gene for trichosanthin and the last one with 315 b.p. for the N-terminal 1-102 residues of human prourokinase. In all these syntheses, a plus-stranded DNA of the target gene was generally assembled by a stepwise or one step T4 DNA ligase reaction of six oligonucleotides (A, *pB, *pC, *pD, *pE and *pF) of 30-71 nucleotides long in the presence of two terminal complementary oligonucleotides (Ab' and eF') and three short inter-fragment complementary oligonucleotides (bc, cd and de). After purification, the synthetic ssDNA was inserted into a cloning vector, pWR13. The resulting product was directly used to transform a host cell. The structure of the cloned synthetic gene was confirmed by DNA sequence analysis.

Combined action of purothionin and thioredoxin on the refolding of recombinant pro-urokinase.

We examined the synergistic effects of purothionin on the thioredoxin-catalyzed refolding of recombinant human pro-urokinase. We reported here, the combined action of these two proteins on the recombinant protein refolding

The Differential Glycosylation of Human Pro-Urokinase from Various Recombinant Mammalian Cell Lines Does Not Affect Activity and Binding to PAI-1

Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function. All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs. No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the KM and kcat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1. In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.

Effects of protein disulfide-isomerase on the refolding of human pro-urokinase cloned and expressed in escherichia coli.

Protein disulfide-isomerase (PDI) was studied for the first time as a stimulator of refolding of recombinant proteins produced in bacteria. Experiments with reduced or scrambled ribonuclease as a substrate showed that PDI was active in low concentrations of the protein denaturants which were generally used in the refolding systems of recombinant proteins. Crude human pro-urokinase cloned and expressed in Escherichia coli was tested as an example of a recombinant protein, and PDI accelerated its refolding. The time needed to reach 50% maximum pro-urokinase refolding was shortened by half by the addition of PDI. Reduced pro-urokinase was further stimulated to refold by this enzyme. Evidence that PDI catalyzes the refolding of pro-urokinase through thiol-disulfide exchange reactions was also obtained from the repression of such stimulation by a PDI inhibitor, bacitracin. The results suggested that this refolding system can be used for the refolding of recombinant proteins produced in bacteria.

Enhanced expression of human pro-urokinase cDNA in Escherichia coli.

Human pro-urokinase cDNA was isolated from the cDNA library constructed from human kidney mRNA using the dC/dG homopolymer tailing method and Okayama-Berg method with pBR322 as a vector. A mature polypeptide starting with Ser was produced in Escherichia coli under the control of the tac promoter and the Shine-Dalgarno sequence of the catechol 2, 3-oxygenase gene derived from Pseudomonas putida. By replacing the sequence coding for N-terminal eight amino acids of pro-urokinase with the synthetic DNA oligomer, the bacterial pro-urokinase had a molecular weight of 47, 000 daltons and accounted for 15% of the insoluble fraction of E. coli proteins in induced cells. Its biological activity was restored by renaturing the bacterial product. The activity of bacterial pro-urokinase was 450 IU/ml culture.

Activation of plasminogen by pro-urokinase. I. Mechanism.

The mechanism of the activation of plasminogen by recombinant pro-urokinase (Rec-pro-UK), obtained by expression of the human pro-urokinase gene in Escherichia coli, was investigated in purified systems. In mixtures of Rec-pro-UK and plasminogen, both active urokinase and plasmin are quickly generated. Addition of plasmin inhibitors (aprotinin or alpha 2-antiplasmin) abolishes the conversion of Rec-pro-UK to urokinase but not the activation of plasminogen to plasmin, suggesting that Rec-pro-UK activates plasminogen directly. Human plasma competitively inhibits the activation of plasminogen by pro-urokinase with a Ki of 0.2% (v/v). This explains the relative stability of Rec-pro-UK in plasma and the lack of activation of the plasma fibrinolytic system in the absence of fibrin. The competitive inhibition by plasma is abolished by the addition of CNBr-digested fibrinogen although Rec-pro-UK has no specific affinity for fibrin. These findings suggest that the fibrin specificity of the activation of plasminogen by pro-urokinase is due to neutralization by fibrin of the competitive inhibition exerted by plasma and not to fibrin-enhanced activation of plasminogen.

Coronary thrombolysis in dogs with intravenously administered human pro-urokinase.

Coronary thrombolysis was induced by infusion of highly purified human pro-urokinase isolated from a transformed kidney cell line (ACHN) or by infusion of urokinase of urinary origin in anesthetized dogs with 1-hr-old clots in the left anterior descending coronary artery. The clots were induced with a copper coil and thrombolysis was detected by repeat coronary angiography. Intravenous infusion of pro-urokinase at a rate of 10 micrograms/kg/min for 30 min in two dogs did not induce thrombolysis, which was only obtained after 8 and 15 min of its subsequent intracoronary administration. Intravenous infusion of pro-urokinase at a rate of 20 micrograms/kg/min for 30 min in four dogs induced coronary thrombolysis within 23 +/- 2 min (mean +/- SEM). This was not associated with systemic fibrinolytic activation because the alpha 2-antiplasmin and fibrinogen levels did not decrease. Intravenous infusion of urokinase at a rate of 10 micrograms/kg/min for 30 min elicited thrombolysis in four of seven dogs within an average of 19 +/- 2 min. In the other three dogs thrombolysis was only obtained within 11 +/- 3 min of its subsequent intracoronary infusion. Administration of urokinase was associated with systemic fibrinolytic activation as evidenced by a decrease of alpha 2-antiplasmin to about 10% and of fibrinogen to 43 +/- 13% of the preinfusion value. It is concluded that intravenous infusion of pro-urokinase at a sufficiently high rate produces coronary thrombolysis without systemic fibrinolysis in dogs.

Characterization of the high-affinity interaction between human plasminogen and pro-urokinase.

Activation of human Glu-plasminogen, Lys-plasminogen and low-Mr plasminogen (lacking lysine-binding sites) by pro-urokinase (pro-UK), obtained from a human lung adenocarcinoma cell line (Calu-3, ATCC), obeys Michaelis-Menten kinetics. Activation occurs with a comparable affinity (Km 0.40-0.77 microM), while the catalytic rate constant (kcat) is comparable for Glu-plasminogen (0.0022s-1) and low-Mr plasminogen (0.0034 s-1), but is somewhat higher for Lys-plasminogen (0.0106 s-1). The rate of activation of plasminogen by pro-UK is not significantly influenced by the presence of 6-aminohexanoic acid, purified fragments LBS I or LBS II or histidine-rich glycoprotein, indicating that the high affinity of pro-UK for plasminogen is not mediated via the high-affinity lysine-binding site of plasminogen located in kringles 1-3 (LBS I) nor via the low-affinity lysine-binding site comprised within kringle 4 (LBS II). The site(s) in plasminogen involved in the high-affinity interaction with pro-UK thus appear to be located within the low-Mr plasminogen moiety.