Plasminogen Activation by Pro-Urokinase in Complex with its Receptor - Dependence on a Tripeptide (Spectrozyme Plasmin) (original) (raw)
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Biochemistry, 1991
The question whether single-chain urokinase-type plasminogen activator (Sc-uPA) possesses an enzymatic activity has been a subject of intense investigation for a number of years but still remains unresolved. Recent studies from several laboratories suggest that Sc-uPA or its plasmin-resistant mutants obtained by site-directed mutagenesis possess significant, albeit low, amidolytic and plasminogen activator activities, ranging from 0.1% to 1% of that observed for two-chain urokinase (Tc-uPA). In an effort to characterize these putative intrinsic activities, Sc-uPA was repeatedly treated with dansyl-Glu-Gly-Arg chloromethyl ketone (dansyl-EGRck) or diisopropyl fluorophosphate (DFP) (0.1-0.25 mM added thrice over a period of 24 h at 0 "C). This treatment exhaustively inactivated the Tc-uPA contaminant but did not affect Sc-uPA, as evidenced by the lack of significant incorporation of radiolabeled inhibitor in Sc-uPA and full activation of the inhibitor-treated Sc-uPA by plasmin. Assayed in the presence of excess DFP or dansyl-EGRck to ensure trapping of any Tc-uPA generated in the assay mixture, Sc-uPA (84 pg/mL, 10500 latent units/mL) did not elicit any detectable cleavage of the chromogenic substrate S-2444 (detection limit 0.1 unit of Tc-uPA/mL). However, if the Tc-uPA inhibitors were removed prior to assay, a trace amount of amidolytic activity invariably reappeared in the Sc-uPA preparation. Incorporation experiments with [3H]DFP suggested that the appearance of this amidolytic activity was due to formation of Tc-uPA. Plasminogen activator assay of DFP-and dansyl-EGRck-treated Sc-uPA (0.45-2.25 pM), performed in the presence of these inhibitors and Trasylol (10 pM) to ensure entrapment of any Tc-uPA or plasmin generated in the reaction mixture, showed no significant cleavage of 1251-labeled plasminogen (detection limit 0.1 nM). However, if dansyl-EGRck and DFP were removed from the inhibitor-treated Sc-uPA and the assay was performed in the presence of Trasylol alone, there was significant cleavage of 1251-plasminogen due to contamination by Tc-uPA. Fibrin, a positive effector of plasminogen activation by Tc-uPA or Sc-uPA preparations in the absence of DFP and dansyl-EGRck, did not promote cleavage of plasminogen or S-2444 by Sc-uPA in the presence of the Tc-uPA inhibitors. The present findings indicate that, under conditions stringently excluding Tc-uPA contamination, neither recombinant human Sc-uPA expressed in Chinese hamster ovary cells nor Sc-uPA secreted by fetal kidney cells or a transformed line of kidney cells shows measurable amidolytic activity above the detection limit of 0.001 7% or plasminogen activator activity above the detection limit of 0.01% of Tc-uPA activity. These studies suggest that the intrinsic activities ascribed to Sc-uPA or its plasmin-resistant mutants arise from small amounts of Tc-uPA, possibly generated from Sc-uPA by the action of traces of contaminating proteases that are not susceptible to inactivation by usual inhibitors of trypsin-like serine proteases. U r o k i n a s e 4 ype plasminogen activator (uPA)' is synthesized and secreted by cells as a 50-kDa single-chain glycoprotein that is variously referred to as single-chain urokinase (Sc-uPA) or prourokinase [review by Lijnen et al. (1987a) and references cited therein]. Sc-uPA is converted to two-chain urokinase (Tc-uPA) following cleavage of the Lys'S8-Ile'S9 peptide bond by plasmin (Wun et al., 1982; Nielsen et al., 1982), plasma kallikrein, or some other serine proteases (Ichinose et al., 1986).
Fibrinolysis and Proteolysis, 1997
Urokinase-type plasminogen activator (uPA) is a serine protease which has been implicated in numerous physiological and pathological processes, e.g. tissue remodelling, embryogenesis, fibrinolysis, and tumour spread, uPA protease binds to a specific high-affinity receptor (uPAR; CD87) on normal and tumour cells. This binding is mediated by the growth factor domain of uPA and thus independent of its proteolytic activity. An ELISA-type, solid-phase microtitre plate assay is presented, designed for the quantitation of such uPA molecules capable of binding to the receptor uPAR. This solid-phase uPA-ligand binding assay makes use of the specific, high-affinity interaction of uPA with uPAR. This assay format is different from the common uPA-ELISA which measures uPA antigen but not uPAR-reactivity. Recombinant soluble uPAR (CHO-uPAR), attached to the well of a microtitre plate, serves as the capture molecule for uPA. uPA-containing samples are added to allow binding of uPA to immobilized uPAR. Receptor-bound uPA is then detected by reaction of uPA with biotinylated monoclonal antibody no. 377 (American Diagnostica, Greenwich, CT, USA) directed to the kringle domain of uPA followed by avidin-peroxidase. The solid-phase uPA-ligand binding assay detects various forms of uPA: pro-uPA, HMW-uPA, ATF, GFD but does not react with the low molecular weight form of uPA (LMW-uPA) lacking the uPAR-reactive domain, uPA molecules in which the uPAR-binding domain has been impaired by proteolysis and uPA/uPAR complexes are also excluded from detection. The high sensitivity of the solid-phase uPAligand binding assay (lower limit 2 pM = 0.1 ng uPA/ml) allowed to measure reactive uPA (and fractions thereof) in the supernatants of cultured ovarian cancer cells, in extracts of ovarian and breast cancer tissues, in placenta tissue extracts and in malignant ascites. The solid-phase uPA-ligand binding assay was also used to screen the receptor binding reactivity of recombinant human uPA-polypeptides synthesized by yeast cells and that of synthetic uPA-peptides. The uPAR-blocking capability of uPA-peptides uPA14-32, uPA14-32/H29A, and uPA14-32/N32A, determined by the solidphase uPA-ligand binding assay, was confirmed by flow cytofluorometric analysis employing fluorescent pro-uPA (FITCpro-uPA) and the uPAR-rich promyeloid cell line U937.
Biochemistry, 1999
The binding of the zymogenic form of urokinase-type plasminogen activator (pro-uPA) to its specific cellular receptor, uPAR, leads to a large potentiation of plasmin generation. This is dependent on the concurrent cellular binding of plasminogen, and is completely abrogated by the plasminogen lysinebinding site ligand, 6-aminohexanoic acid. Previous data have provided circumstantial evidence for the formation of specific complexes to mediate the kinetically favorable reciprocal interactions between the protease and zymogen components [Ellis, V., and Danø, K. (1993) J. Biol. Chem. 268, 4806-4813]. To further investigate the formation of these putative complexes, we have studied the effect of various lysinebinding site ligands on the binding and activation of plasminogen on U937 cells. Lysine-binding site ligands resembling internal lysine residues, such as N R -acetyl-L-lysine methyl ester, were found to specifically inhibit uPAR-mediated cell-surface plasminogen activation at concentrations up to 40-fold lower than those inhibiting the cellular binding of 125 I-labeled plasminogen (IC 50 s 300 µM vs 8.5 mM). By contrast, 6-aminohexanoic acid, resembling a C-terminal lysine residue, did not display this disparity (IC 50 s 25 vs 30 µM). These lysine analogues were also found to compete a non-active-site interaction between uPA and plasminogen, detected by surface plasmon resonance (K d 50 nM), at concentrations correlating with their effect on cell-surface plasminogen activation, suggesting that this interaction is part of the kinetic mechanism. Consistent with this, synthetic peptides corresponding to the sequence uPA 149-158 (GQKTLRPRFK) and uPA 149-157 (GQKTLRPRF) specifically abolished the amplification of cell-surface plasminogen activation. These data demonstrate that a novel non-active-site interaction between uPA and plasminogen is necessary for the assembly and efficiency of cell-surface plasminogen activation complexes.
Journal of Histochemistry & Cytochemistry, 1997
The urokinase-type plasminogen activator (uPA) is a serine protease that plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA receptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA using a conjugation procedure that did not significantly alter its binding characteristics to the uPAR. Thirty nM FITC-uPA efficiently stains 2 ϫ 10 5 uPAR-transfected mouse cells in suspension, as determined by flow cytometric analysis. One g of FITC-uPA efficiently stains 2 ϫ 10 5 uPAR transfectants grown on slides and analyzed by fluorescence optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde only slightly reduced the efficiency of staining of both transfectants and cell lines. These characteristics allow the use of FITC-uPA in both static and dynamic morphological studies of uPAR-expressing cells. (J Histochem Cytochem 45:
ELECTROPHORESIS, 1995
The receptor for urokinase‐type plasminogen activator (uPAR) may contribute to the invasive and metastatic capacity of tumor cells by focusing the serine protease urokinase‐type plasminogen activator (uPA) to the cell surface. uPA activates plasminogen to plasmin which in turn degrades extracellular matrix proteins or activates other proteases. Mature uPAR is a heavily glycosylated protein of about 284 amino acids attached to the plasma membrane via a glycosyl‐phosphatidylinositol (GPI) anchor. A set of different polyclonal uPAR antibodies has been generated in order to investigate the role of uPAR in tumor spreading in more detail. For this purpose, uPAR (lacking the GPI anchor) was expressed in E. coli and Chinese hamster ovary (CHO) cells. Recombinant uPAR from E. coli (corresponding to amino acids 1–284 of human uPAR) was expressed with an N‐terminal histidine‐tag insertion and purified by nickel chelate affinity chromatography. Soluble uPAR, synthesized by CHO cells (correspond...
European Journal of Biochemistry, 1986
Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded.
European Journal of Biochemistry, 1996
The amino-terminal fragment of human uPA (ATF; amino acids I-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae. Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His, tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR. The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR. All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate. Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine. Lys23, Tyr24, Phe25, Ile28, and Trp30 were important determinants for uPAR binding. A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATE Comparable results to those with the yeast ATF mutants were obtained. In a different set of experiments, those amino acids of the uPARbinding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thrl8 by alanine or Asn32 by serine had hardly any effect, replacement of Am22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR. Deletion of either Va120, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants. Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity. The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated UPNUPAR interaction.
A direct, plasmin-independent assay for plasminogen activator
Thrombosis Research, 1984
A direct assay for plasminogen activator (PA) was developed. It employed polyacrylamide gel electrophoresis in the oresence of sodium dodecyl sulfate and fi-mercaptoethanol to monitor PA-mediated conversion of single chain, lz51-plasminogen to two chain plasmin.