Human Plasminogen Activator Research Articles

Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.

Smad proteins play a key role in the intracellular signalling of transforming growth factor beta (TGF beta), which elicits a large variety of cellular responses. Upon TGF beta receptor activation, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transcriptional regulation is largely unknown. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGF beta. Here we report the identification of Smad3/Smad4 binding sequences, termed CAGA boxes, within the promoter of the human PAI-1 gene. The CAGA boxes confer TGF beta and activin, but not bone morphogenetic protein (BMP) stimulation to a heterologous promoter reporter construct. Importantly, mutation of the three CAGA boxes present in the PAI-1 promoter was found to abolish TGF beta responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGF beta. In addition, TGFbeta induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Furthermore, bacterially expressed Smad3 and Smad4 proteins, but not Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGF beta-responsive regions of several other genes suggests that this may be a widely used motif in TGF beta-regulated transcription.

Ecotin: a serine protease inhibitor with two distinct and interacting binding sites

The interaction between ecotin and target proteases with trypsin-like specificity has been systematically dissected to understand the structural basis of ecotin’s broad inhibitory specificity and the role of the secondary binding site. Site-directed and region-specific mutagenesis were preformed at ecotin’s primary site P1 residue (84), the C-terminal dimer interface (133 to 142), and two surface loops of the secondary binding site (67 to 70, 108 to 113). Substitutions at the P1 position resulted in less than fivefold difference in the potency of ecotin binding to rat trypsin, suggesting that the extended binding site is important in binding. A ten amino acid C-terminal truncation variant showed threefold weaker self-association but remained a dimer. The interactions of the secondary binding site of ecotin with bovine trypsin, rat trypsin and human urokinase-type plasminogen activator (uPA) were investigated with alanine substitutions in ecotin at Trp67, Gly68, Tyr69, Asp70, Arg108, Asn110, Lys112 and Leu113, which formed contacts between the inhibitor and protease. By combining these mutations at the secondary binding site with mutations in the primary binding site the molecular recognition between ecotin and its target serine proteases was probed. The contrast in the K ivalues of the various ecotin variants towards bovine trypsin, rat trypsin and human uPA established the role of ecotin’s secondary binding site in recognizing these homologous serine proteases. Ecotin binds to proteases with a chymotrypsin fold through a combination of primary and secondary site surface loops and is amenable to redesign of its potency and specificity for this class of enzymes.

Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator

Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design. An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA). The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 Å from the enzyme active site. Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop. The ecotin 60X 4library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds. Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence. Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70. The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT). A second generation library, ecotin M84R+60X 4including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin. Panning against rat trypsin resulted in enrichment but no consensus sequence. Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70. Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT. Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin. The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold.

3 X-ray crystal structure of single chain human tPA and vampire bat plasminogen activator help to explain the activation mechanism of tPA

3 X-ray crystal structure of single chain human tPA and vampire bat plasminogen activator help to explain the activation mechanism of tPA

Theory of kinetic partitioning in protein folding with possible applications to prions

This study focuses of the phenomenon of kinetic partitioning when a polypeptide chain has two ground-state conformations, one of which is kinetically more reachable than the other. We designed sequences for lattice model proteins with two different conformations of equal energy corresponding to the global energy minimum. Folding simulations revealed that one of these conformations was indeed much more kinetically accessible than the other. We found that the number and strength of local contacts in the ground-state conformation are the major factors that determine which conformation is reached faster; the greater the number of local contacts, the more kinetically reachable a conformation is. We present simple statistical-mechanical arguments to explain these findings. Our results may be relevant in explaining the phenomenology of such proteins as human plasminogen activator inhibitor-1 (PAI-1), photosystem II, and prions.

Theory of kinetic partitioning in protein folding with possible applications to prions

This study focuses on the phenomenon of kinetic partitioning when a polypeptide chain has two ground-state conformations, one of which is kinetically more reachable than the other. We designed sequences for lattice model proteins with two different conformations of equal energy corresponding to the global energy minimum. Folding simulations revealed that one of these conformations was indeed much more kinetically accessible than the other. We found that the number and strength of local contacts in the ground-state conformation are the major factors that determine which conformation is reached faster; the greater the number of local contacts, the more kinetically reachable a conformation is. We present simple statistical–mechanical arguments to explain these findings. Our results may be relevant in explaining the phenomenology of such proteins as human plasminogen activator inhibitor-1 (PAI-1), photosystem II, and prions. Proteins 31:335–344, 1998. © 1998 Wiley-Liss, Inc.

Cloning and high-level expression of plasminogen activator inhibitor-1 cDNA derived from human glomerular mesangial cells

Translation reading frame of human plasminogen activator inhibitor-1 (PAI-1) cDNA was amplified from total RNA extracted from glomerular mesangial cells by using reverse-transcription polymerase chain reaction (RT-PCR) and inserted into plasmid pUC19 in a sense orientation. Sequencing results revealed that PAI-1 cDNA had new initial codon ATG, but no signal peptide sequences, and translation reading frame was in agreement with PAI-1 cDNA derived from human endothelial cells. PAI-1 cDNA determined by sequencing was inserted into prokaryotic expression plasmid pBV220, and recombinant plasmid pBV220-PAI-l which had high-level expression inEscherichia coli was obtained. Recombinant PAI-1 protein attained to approximately 45 % of total bacterial proteins. Western blotting showed that there was a specific band in the region of 43.0 ku. Latent recombinant PAI-1 with 97 % purity was obtained from inclusion bodies after denaturation, renaturation and purification by FPLC. Recombinant PAI-1 activated by treatment with 4.0 mol/L guanidinium-hydrochloride had significant inhibition activity to u-PA and shared bioactivity in common with natural PAI-1.

Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.

Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.

Pharmacokinetics and biodistribution of recombinant human plasminogen activator inhibitor type 2 (PAI-2) in control and tumour xenograft-bearing mice

Summary Objective: Previous studies have shown that the endogenous overexpression of human plasminogen activator inhibitor type-2 (PAI-2) in tumour cells decrease their ability to grow and metastasise. The therapeutic utility of PAI-2, however, remains poorly evaluated. In this study the kinetics and biodistribution of recombinant human PAI-2 were determined in a xenograft Nu/Nu mouse model of human colon cancer and compared to control mice (i.e. without a human xenograft tumour). Design: 125I-labelled recombinant PAI-2 was injected intravenously and the pharmacokinetics and biodistribution determined in control Nu/Nu mice or mice carrying a subcutaneous HCT116 human colon cancer xenograft. Results: The clearance of 125I-PAI-2 from mouse plasma was found to be a biphasic process and radioactivity was excreted via the urine in a degraded form. While radioactivity corresponding to intact PAI-2 localized to tumour xenograft tissues quickly (after 1 min, peaking after 30–60 min at 1.3% of injected dose), the majority of radioactivity localized to major organs (e.g., liver, kidneys) 5 min after intravenous injection of 125I-PAI-2 in both tumour bearing and control mice. However, radioactivity was cleared more rapidly from these organs when compared to tumour tissues. Moreover, multiple injections of 125I-PAI-2 resulted in an increased uptake of radioactivity in tumour xenografts without accompanying increases in the liver. Furthermore, the clearance rate was markedly increased by the presence of a tumour xenograft indicating the requirement to undertake pharmacokinetic studies in diseased as well as control animals. Conclusion: Our results provide important information concerning the pharmacokinetics and biodistribution of PAI-2 and indicates that the presence of a tumour may affect these parameters. Moreover, the localization and accumulation of PAI-2 in tumour tissues argues for a potential therapeutic use in human cancer.

Synthesis of a neoglycoprotein containing the Lewis X analogous trisaccharide β- d-Gal pNAc-( [formula omitted]4)[ α- l-Fuc p-( [formula omitted]3)]- β- d-Glc pNAc

The trisaccharide allyl glycoside 36 and related disaccharide part structures have been prepared using the 2-trichloroacetamido-2-deoxy- α- d-galactopyranosyl trichloroacetimidate derivative 9 as glycosyl donor under promotion with TMSOTf or Sn(OTf) 2, respectively, to produce the β-( 1 → 4) linkage to suitably protected glucosamine derivatives in fair yields. Fucosylation was effected employing the ethyl 1-thio glycosyl donor 20 in the presence of IDCP. Deprotection of the intermediates afforded the disaccharide allyl glycosides β- d-Gal pNAc-( 1 → 4)- β- d-Glc pNAc 13, β- d-Gal pNClAc-( 1 → 4)- β- d-Glc pNAc 14, α- l-Fuc p-( 1 → 3)- β- d-Glc pNAc 24, α- l-Fuc p-( 1 → 4)- β- d-Glc pNAc 31 and the branched trisaccharide allyl glycoside β- d-Gal pNAc-( 1 → 4)[ α- l-Fuc p-( 1 → 3)]- β- d-Glc pNAc 36. The trisaccharide which corresponds to a structural motif occurring in N-glycoprotein glycans from human urokinase, human recombinant protein C, phospholipase A 2 as well as O-glycans, was converted into a neoglycoprotein following introduction of a cysteamine-derived spacer group and subsequent activation with thiophosgene.

The 3'-untranslated region of the urokinase gene enhances the expression of chimeric genes in cultured cells and correlates with specific brain expression in transgenic mice.

Transgenic mice were previously described, carrying the cDNA of the human or murine protease urokinase-type plasminogen activator (uPA) while linked to the cell-specific promoter of the alphaA-crystallin gene. Surprisingly, these mice produced transgenic uPA in an ectopic manner specifically in the brain. Here we tested the possibility that this ectopic expression could have been contributed primarily by the uPA transgenic moiety. Several experimental approaches have been used. (a) Constructs consisting of uPA cDNA linked to the cell-specific promoters of the alphaA-crystallin or insulin genes yielded active uPA after transfection into cells where these promoters are thought to be inactive. (b) When reporter genes were inserted into these constructs between the promoter and the cDNA, the cDNA enhanced the chimeric reporter expression 5-50-fold. This effect was obtained upon stable or transient construct transfection into four different cell types. (c) Reporter enhancement also took place in the presence of the homologous uPA gene promoter. (d) Mapping of the cDNA through deletion-substitution analysis has detected fragments mediating positive or negative effects on reporter expression, all fragments residing in the 3'-untranslated region (3'UTR) of the uPA gene that was included in the cDNA. Some fragments exhibited cell-specific effects. One fragment (2002/2187) behaved like a classical transcriptional enhancer, enhancing reporter expression from different positions and orientations. (e) Transgenic mice have now been generated that carry a transgene consisting of the alphaA-crystallin promoter, the luciferase reporter gene and mouse uPA cDNA. Among four transgenic lines producing luciferase activity in the eye lens, three lines exhibited ectopic luciferase activity exclusively in the brain, where luciferase mRNA was localized through in situ hybridization. From these results we conclude that the 3'UTR of the uPA gene contains sequences capable of exerting variable effects on gene expression, including transcriptional enhancement. In addition, uPA cDNA correlates with transgenic brain expression. Therefore, we suggest that the 3'UTR of the uPA gene is involved in brain expression of the transgenes containing uPA cDNA as well as of the normal uPA gene.

Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis.

A novel low-molecular-weight inhibitor, AR-H029953XX, was developed from a known fibrinolytic compound, flufenamic acid, which prevented complex formation of human plasminogen activator inhibitor type 1 (PAI-1) with tissue plasminogen activator (tPA) by inhibition of PAI-1. To explore the binding site for AR-H029953XX, mutants of human PAI-1 were constructed by site-directed mutagenesis and were then expressed in CHO cells, purified, activated, and characterized. (1) PAI-1 with mutations in the reactive center loop: L1-PAI-1 (P10, Ser337Glu) had stability and activity similar to those of wild-type PAI-1 (wt-PAI-1), and L2-PAI-1 (P12, Ala335Glu) was highly stable but was a substrate for tPA. (2) PAI-1 with mutations near the binding epitope for the strongly inhibiting monoclonal antibody CLB-2C8: C1-PAI-1 (Phe114Glu), C2-PAI-1 (Val121Phe), C3-PAI-1 (Arg76Glu/Arg115Glu/Arg118Glu), and C4-PAI-1 (Arg115Glu) were all comparable in activity and stability to wt-PAI-1. AR-H029953XX (Ki = 25 microM) prevented complex formation between tPA and active wt-PAI-1 as well as that with mutants L1-, L2-, C1-, C2-, and C4-PAI-1. AR-H029953XX also inhibited binding of these PAI-1 variants to the antibody CLB-2C8, as measured by surface plasmon resonance. In contrast, AR-H029953XX had almost no inhibitory effect on the complex formation of tPA with C3-PAI-1. Moreover, AR-H029953XX had no effect on the binding rate of CLB-2C8 to C3-PAI-1, or on the binding to latent PAI-1 or to cleaved L2-PAI-1. The binding site of AR-H029953XX thus appears to be located in the neighborhood of the postulated epitope for CLB-2C8, near residues Arg76 and/or Arg118. This specific domain of the PAI-1 molecule might thus also be important for the mechanism of inhibitory activity toward tPA. Moreover, the structure of this region in active PAI-1 has to be different from the corresponding regions in latent and cleaved PAI-1.

The human plasminogen activator inhibitor type I gene promoter targets to kidney.

The plasminogen activator inhibitor type 1 (PAI-1) gene encodes the physiological inhibitor of tissue-type and urokinase-type plasminogen activators and is induced by cytokines such as transforming growth factor-beta (TGF-beta). Studies have identified DNA sequence elements within the first 1.3 kb of the 5'-upstream DNA that mediate cytokine responsiveness in transfected cells in vitro. However, the DNA sequences that mediate PAI-1 expression in vivo have not yet been delineated. To define these regulatory sequences, we generated transgenic mice that expressed a hybrid gene comprising sequences between -1,272 and +75 of the human PAI-1 gene ligated to a LacZ reporter gene. Transgene expression detected in two independent lines was observed only in kidney from embryonic day 13 to adult and was seen primarily in proximal tubule cells of the outer medulla. Transgene expression and activity were unchanged in response to TGF-beta and remained restricted to kidney. Thus we have identified a promoter region within the PAI-1 gene that targets transgene expression to kidney but, unlike the native promoter, is unresponsive to TGF-beta in the experimental protocol used.

Plasminogen Activation by Streptokinase via a Unique Mechanism

The mechanism of human plasminogen (HPlg) activation by streptokinase (SK)-type activator was investigated with recombinant truncated SK peptides. An enzyme-substrate intermediate of HPlg.SK. HPlg ternary complex was demonstrated by a sandwich-binding experiment. Formation of the ternary complex was saturable, HPlg-specific, and inhibited by 6-aminocaproic acid. Three interaction sites between SK and HPlg were demonstrated. SK-(220-414) bound to HPlg with two binding sites: one to the micro-HPlg region, the catalytic domain of HPlg, and one to the kringle 1-5 region, with Kd values of 1.50 x 10(-7) and 2.44 x 10(-6) M, respectively. SK-(16-251) bound to a single site on the kringle 1-5 region of HPlg with a Kd of 4.09 x 10(-7) M. SK-(220-414) and SK-(16-251) competed for binding on the same or nearby location on the human kringle 1-5 domain. Combination of SK-(220-414) and SK-(16-251), but not either peptide alone, could effectively activate HPlg. In addition, SK-(16-251) dose-dependently enhanced the activation of HPlg by SK-(16-414), while the HPlg activation by SK-(16-414) was inhibited by SK-(220-414). We conclude that the HPlg that binds to the COOH-terminal domains of SK functions as an enzyme to catalyze the conversion of substrate HPlg that binds to the NH2-terminal domain of SK to human plasmin.

Binding of Human Single Chain Urokinase to Chinese Hamster Ovary Cells and Cloning of Hamster u-PAR

The plasminogen activator, urokinase (u-PA), interacts with the u-PA receptor (u-PAR) which results in enhanced plasminogen activation on cell surfaces. The u-PAR is comprised of three homologous domains of approximately 90 amino acids, defined by the pattern of disulfide bonds. Domain 1 (amino acids 1-87) binds the ligand. Within this domain, Y57, and a site between residues 47 and 53, have been suggested as ligand contact points. Intradomain interactions also contribute to the interaction of u-PA and u-PAR. The interaction of u-PA with its receptor exhibits some species specificity. Previous studies have shown that human u-PA does not bind to the murine u-PAR and murine u-PA does not recognize human u-PAR. However, human u-PA does interact with bovine cells with high affinity. To further examine the interaction of the human ligand with the u-PAR of a different species, we characterized the binding of human 125I single chain u-PA (scu-PA) to hamster cells. Chinese Hamster Ovary (CHO) cells bound human scu-PA with high affinity and capacity (Kd = 1.13 +/- 0.8 nM; Bmax = 5.45 +/- 0.98 x 10(4) sites/cell). In ligand blotting with human 125I-scu-PA, major bands migrating with apparent Mr's of 74, 49 and 38 kDa were observed. The cDNA of hamster u-PAR was cloned and a single 1.4 kb mRNA species identified in Northern blots of CHO cell RNA. For comparison, we also cloned u-PAR cDNA from human THP-1 cells. Our human sequence was identical to those published for U937 and endothelial cells. These sequences were aligned with the published sequences for the murine, bovine and rat u-PAR's to obtain a consensus sequence for five species. The cysteine residues could be aligned for all species. Y57, which has been suggested as a ligand contact point was also conserved across species. In addition, 5 of the 7 amino acids between amino acids 47 and 53 were conserved in all species. Gly283, the most likely glycosyl-phosphatidyl inositol attachment site, was also conserved in all species. The conservation of these amino acid residues across all five species, attests to their importance in u-PAR function. In addition, the results of our studies suggest that the hamster may be a useful small animal model for studies of human urokinase function.

A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity

The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR/CD87) in cell migration and invasion is well substantiated. Recently, uPA has been shown to be essential in cell migration, since uPA-/- mice are greatly impaired in inflammatory cell recruitment. We have shown previously that the uPA-induced chemotaxis requires interaction with and modification of uPAR/CD87, which is the true chemoattracting molecule acting through an unidentified cell surface component which mediates this cell surface chemokine activity. By expressing and testing several uPAR/CD87 variants, we have located and functionally characterized a potent uPAR/CD87 epitope that mimics the effects of the uPA-uPAR interaction. The chemotactic activity lies in the region linking domains 1 and 2, the only protease-sensitive region of uPAR/CD87, efficiently cleaved by uPA at physiological concentrations. Synthetic peptides carrying this epitope promote chemotaxis and activate p56/p59(hck) tyrosine kinase. Both chemotaxis and kinase activation are pertussis toxin sensitive, involving a Gi/o protein in the pathway.

Studies on the relationship between urokinase plasminogen activator (uPA) and human sperm motility.

To clarify the role of urokinase plasminogen activator (uPA) in the mechanisms of regulating sperm motility and the ability of fertilizing, we investigated the quantities and activities of uPA in human seminal plasma and on the membrane of spermatozoa. Semens were harvested from 22 infertile patients with asthenospermia and 20 healthy fertile men according to WHO standards. To quantify the membrane-bound uPA in the samples, polyclonal antibodies against human urokinase were employed by means of a sandwich ELISA. The uPA activities in seminal plasma and on the surface of spermatozoa were determined using A-garose-Fibrine-Plate method and the experiment of immunological identification with polyclonal antibodies against urokinase. In lysates of spermatozoa, significantly lower levels of uPA (23.1 +/- 7.35 mu/10(6) cells) and uPA activity (5.13 +/- 3.85 mu/10(6) cells) were found in patient group as compared to healthy fertile men exhibiting normospermia (29.89 +/- 9.40 mu/10(6) cells and 10.17 +/- 6.18 mu/10(6) cells). In seminal plasma, uPA activity in patient group (2134 +/- 1581.3 IU/L) was also found significantly lower than that of normal group (3365 +/- 1859.5 IU/L). Positive correlations were observed between sperm motility and uPA quantities (r = 0.48, P < 0.005), as well as with uPA activities (r = 0.45, P < 0.005). Thus, it is inferred that membrane associated uPA on human spermatozoa may be related directly to sperm motility and fertility.

The amino-terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells: internalization is toxin mediated.

In contrast to two-chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome-inactivating protein) did not require plasminogen activator inhibitors to be internalized. To dissect this pathway, we constructed a chimera consisting of the amino-terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP-3). The chimeric ATF-SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome-inactivating activity. Besides being a potent inhibitor of protein synthesis in cell-free assays, ATF-SAP was specifically cytotoxic toward cells expressing human uPAR. Competition experiments indicated that both the human uPAR and the LDL-related receptor protein are involved in mediating the cell killing ability of ATF-SAP. We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways. Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR-bound ligands through endocytic receptors.

Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis.

Expression/secretion vectors for the production of Fab' and single-chain (sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

156 Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single chain human tPA and vampire bat plasminogen activator

156 Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single chain human tPA and vampire bat plasminogen activator