Nucleic acid aptamers against proteases.

Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, protein-aptamer recognition, protease inhibition, and advantages of aptamers for pharmacological intervention with pathophysiological functions of proteases. Aptamers can be selected so that they bind their targets highly specifically and with affinities corresponding to KD values in the nM range. Aptamers can be selected so that they recognize their targets conformation-specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers.

Independent prognostic value of angiogenesis and the level of plasminogen activator inhibitor type 1 in breast cancer patients.

Tumour angiogenesis and the levels of plasminogen activator inhibitor type 1 (PAI-1) are both informative prognostic markers in breast cancer. In cell cultures and in animal model systems, PAI-1 has a proangiogenic effect. To evaluate the interrelationship of angiogenesis and the PAI-1 level in breast cancer, we have evaluated the prognostic value of those factors in a total of 228 patients with primary, unilateral, invasive breast cancer, evaluated at a median follow-up time of 12 years. Microvessels were immunohistochemically stained by antibodies against CD34 and quantitated by the Chalkley counting technique. The levels of PAI-1 and its target proteinase uPA in tumour extracts were analysed by ELISA. The Chalkley count was not correlated with the levels of uPA or PAI-1. High values of uPA, PAI-1, and Chalkley count were all significantly correlated with a shorter recurrence-free survival and overall survival. In the multivariate analysis, the uPA level did not show independent prognostic impact for any of the analysed end points. In contrast, the risk of recurrence was independently and significantly predicted by both the PAI-1 level and the Chalkley count, with a hazard ratio (95% CI) of 1.6 (1.01-2.69) and 1.4 (1.02-1.81), respectively. For overall survival, the Chalkley count, but not PAI-1, was of significant independent prognostic value. The risk of death was 1.7 (1.30-2.15) for Chalkley counts in the upper tertile compared to the lower one. We conclude that the PAI-1 level and the Chalkley count are independent prognostic markers for recurrence-free survival in patients with primary breast cancer, suggesting that the prognostic impact of PAI-1 is not only based on its involvement in angiogenesis.

Importance of the amino-acid composition of the shutter region of plasminogen activator inhibitor-1 for its transitions to latent and substrate forms.

The serpins are of general protein chemical interest due to their ability to undergo a large conformational change consisting of the insertion of the reactive centre loop (RCL), which becomes strand 4, into the central beta sheet A. To make space for the incoming RCL, the 'shutter region' opens by the beta strands 3A and 5A sliding apart over the underlying alpha helix B. Loop insertion occurs during the formation of complexes of serpins with their target serine proteinases and during latency transition. This type of loop insertion is unique to plasminogen activator inhibitor-1 (PAI-1). We report here that amino-acid substitutions in a buried cluster of three residues forming a hydrogen bonding network in the shutter region drastically accelerate PAI-1 latency transition; that the rate was in all cases normalized by the PAI-1 binding protein vitronectin; and that substitution of an adjacent beta strand 5A Lys residue, believed to anchor beta strand 5A to other secondary structural elements, had differential effects on the rates of latency transition in the absence and the presence of vitronectin, respectively. An overlapping, but not identical set of substitutions resulted in an increased tendency to substrate behaviour of PAI-1 at reaction with its target proteinases. These findings show that vitronectin regulates the movements of the RCL through conformational changes of the shutter region and beta strand 5A, are in agreement with RCL insertion proceeding by different routes during latency transition and complex formation, and contribute to the biochemical basis for the potential use of PAI-1 as a therapeutic target in cancer and cardiovascular diseases.

Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator: relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme.

We localized the epitopes for several murine mAbs to human urokinase-type plasminogen activator (uPA) by Ala scanning mutagenesis and related the localization to the effects of the mAbs on the molecular interactions of uPA. Several antibodies against the serine proteinase domain (SPD) were found to have overlapping epitopes composed of variable combinations of Arg178, Arg179, His180, Arg181, Tyr209, Lys211, and Asp214 in the so-called 37-loop and 60-loop, located near the active site and taking part in the binding of uPA to plasminogen activator inhibitor-1 (PAI-1). Besides inhibiting uPA-catalysed plasminogen activation, all antibodies to SPD strongly delayed the binding of uPA to PAI-1, decreasing the second-order rate constant 15- to 6500-fold. There was no correlation between the relative effects of the 37-loop and 60-loop substitutions on the second-order rate constant and on the binding of the antibodies, indicating that the antibodies did not delay complex formation by blocking residues of specific importance for the uPA-PAI-1 reaction, but rather by steric hindrance of the access of PAI-1 to the active site. The affinity of the SPD antibodies for the uPA-PAI-1 complex was only slightly lower than that for free uPA, indicating that the 37-loop and 60-loop are exposed in the complex. The epitopes for two antibodies to the kringle included Arg108, Arg109, and Arg110. The ability of these antibodies to block the binding of uPA to polyanions correlated with a reduced uPA-polyanion affinity after substitution of the three Arg residues.

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

A regulatory hydrophobic area in the flexible joint region of plasminogen activator inhibitor-1, defined with fluorescent activity-neutralizing ligands. Ligand-induced serpin polymerization.

We have characterized the neutralization of the inhibitory activity of the serpin plasminogen activator inhibitor-1 (PAI-1) by a number of structurally distinct organochemicals, including compounds with environment-sensitive spectroscopic properties. In contrast to latent and reactive center-cleaved PAI-1 and PAI-1 in complex with urokinase-type plasminogen activator (uPA), active PAI-1 strongly increased the fluorescence of the PAI-1-neutralizing compounds 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-bisnaphthyl-5,5'-disulfonic acid. The fluorescence increase could be competed by all tested nonfluorescent neutralizers, indicating that all neutralizers bind to a common hydrophobic area preferentially accessible in active PAI-1. Activity neutralization proceeded through two consecutive steps as follows: first step is conversion to forms displaying substrate behavior toward uPA, and second step is to forms inert to uPA. With some neutralizers, the second step was associated with PAI-1 polymerization. Vitronectin reduced the susceptibility to the neutralizers. Changes in sensitivity to activity neutralization by point mutations were compatible with the various neutralizers having overlapping, but not identical, binding sites in the region around alpha-helices D and E and beta-strand 1A, known to act as a flexible joint when beta-sheet A opens and the reactive center loop inserts as beta-strand 4A during reaction with target proteinases. The defined binding area may be a target for development of compounds for neutralizing PAI-1 in cancer and cardiovascular diseases.

A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.

Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type-1: implications for antibody-mediated PAI-1-neutralization and vitronectin-binding.

The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase-serpin complex implies insertion of the reactive centre loop of the serpin into the large central beta-sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including alpha-helix F. In order to elucidate this mechanism, we have used phage-display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against alpha-helix F and its flanking region in the serpin plasminogen activator inhibitor-1 (PAI-1). One of these is known to inhibit the reaction between PAI-1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI-1. When combined with the effects these antibodies have on PAI-1 activity, our epitope mapping points to the mobility of amino-acid residues in alpha-helix F and the loop connecting alpha-helix F and beta-strand 3A as being important for the inhibitory function of PAI-1. Although all antibodies reduced the affinity of PAI-1 for vitronectin, the potentiating effect of vitronectin on antibody-induced PAI-1 neutralization is based on formation of a ternary complex between antibody, PAI-1 and vitronectin, in which PAI-1 is maintained in a state behaving as a substrate for plasminogen activators. These results thus provide new details about serpin conformational changes and the regulation of PAI-1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI-1 in cancer and cardiovascular diseases.

The plasminogen activation system in tumor growth, invasion, and metastasis.

Generation of the serine proteinase plasmin from the extracellular zymogen plasminogen can be catalyzed by either of two other serine proteinases, the urokinase- and tissue-type plasminogen activators (uPA and tPA). The plasminogen activation system also includes the serpins PAI-1 and PAI-2, and the uPA receptor (uPAR). Many findings, gathered over several decades, strongly suggest an important and causal role for uPA-catalyzed plasmin generation in cancer cell invasion through the extracellular matrix. Recent evidence suggests that the uPA system is also involved in cancer cell-directed tissue remodeling. Moreover, the system also supports cell migration and invasion by plasmin-independent mechanisms, including multiple interactions between uPA, uPAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors, and growth factors. These interactions seem to allow temporal and spatial reorganizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. The increased knowledge about the plasminogen activation system may allow utilization of its components as targets for anti-invasive therapy.

Vitronectin and substitution of a beta-strand 5A lysine residue potentiate activity-neutralization of PA inhibitor-1 by monoclonal antibodies against alpha-helix F.

Some monoclonal antibodies against plasminogen activator inhibitor-1 (PAI-1) are able to inhibit its reaction with its target proteinases. We have characterized the effect on PAI-1 of two monoclonal antibodies, Mab-2 and Mab-6, with overlapping epitopes in a sequence encompassing beta-strand 1A, alpha-helix F, and the loop connecting alpha-helix F and beta-strand 3A (the hF/s3A loop). Mab-2 reduced the inhibitory activity of wild type PAI-1 and almost totally abolished the inhibitory activity of a PAI-1 variant harboring an Ala substitution of Lys 325 (335 in the alpha1-proteinase inhibitor template residue numbering) in beta-strand 5A. In both cases, the neutralizing effect of the antibody was strongly potentiated by vitronectin. Mab-6 had no effect on wild type PAI-1, but reduced the inhibitory activity of the K325A variant. The effect of Mab-6 was not potentiated by vitronectin. With both Mab-2 and Mab-6, the neutralization of PAI-1 activity was associated with PAI-1 substrate behaviour. Mab-2, but not Mab-6, prevented vitronectin from rescuing PAI-1 from cold-induced substrate behaviour. We propose that the antibodies act by weakening the anchoring of alpha-helix F to the adjacent structures, resulting in an increased flexibility of beta-strand 5A and the hF/s3A loop and a changed conformational response to the binding of vitronectin in the alpha-helix E region. The potentiating effect of vitronectin on neutralization of PAI-1 by antibodies is a novel concept in the development of compounds for neutralizing PAI-1 in vivo.

Engineering of conformations of plasminogen activator inhibitor-1. A crucial role of beta-strand 5A residues in the transition of active form to latent and substrate forms.

The serpin (serine proteinase inhibitor) family is of general protein chemical interest because of its ability to undergo large conformational changes, in which the surface-exposed reactive centre loop (RCL) is inserted as strand 4 in the large central beta-sheet A. Loop insertion is an integral part of the inhibitory mechanism and also takes place at conversion of serpins to the latent state, occurring spontaneously only in plasminogen activator inhibitor-1 (PAI-1). We have investigated the importance of beta-strand 5A residues for the activity and latency transition of PAI-1. An approximately fourfold increase in the rate of latency transition resulted from His-substitution of Gln324 (position 334 in the alpha(1)-proteinase inhibitor template numbering), which interacts with the underlying alpha-helix B. The side chains of Gln321 and Lys325 (template residues 331 and 335, respectively) form hydrogen bonds to the peptide backbone of a loop connecting alpha-helix F and beta-strand 3A. While substitution with Ala of Glu321 had only minor effects on the properties of PAI-1, substitution with Ala of Lys325 led to stabilization of the inhibitory activity at incubation conditions leading to conversion of wild-type PAI-1 to a substrate form, and to an anomalous reaction towards a monoclonal antibody, which induced a delay in the latency transition of the mutant, but not wild-type PAI-1. We conclude that the anchoring of beta-strand 5A plays a crucial role in loop insertion. These findings provide new information about the mechanism of an important example of protein conformational changes.

Solvent effects on activity and conformation of plasminogen activator inhibitor-1.

We have studied effects of the solvent composition on the activity and the conformation of human plasminogen activator inhibitor-1 (PAI-1) from HT-1080 fibrosarcoma cells. Non-ionic detergents, includine Triton X-100, reduced the inhibitory activity of PAI-1 more than 20-fold at 0 degrees C, but less than 2-fold at 37 degrees C, while glycerol partly prevented the detergent-induced activity-loss at 0 degrees C. The activity-loss was associated with an increase in PAI-1 substrate behaviour. Evaluating the PAI-1 conformation by proteolytic susceptibility of specific peptide bonds, we found that the V8-proteinase susceptibility of the Glu332-Ser333 (P17-P16) bond, part of the hinge between the reactive centre loop (RCL) and beta-strand 5A, and the endoproteinase Asp-N susceptibility of several bonds in the beta-strand 2A-alpha-helix E region were increased by detergents at both 0 and 37 degrees C. The susceptibility of the Gin321-Ala322 and the Lys325-Val326 bonds in beta-strand 5A to papain and trypsin, respectively, was increased by detergents at 0 degrees C, but not at 37 degrees C, showing a strict correlation between proteinase susceptibility of beta-strand 5A and activity-loss at 0 degrees C. Since the beta-strand 2A-alpha-helix E region also showed differential susceptibility to endoproteinase Asp-N in latent, active, and reactive centre-cleaved PAI-1, we propose that a detergent-induced conformational change of the beta-strand 2A-alpha-helix E region influences the movements of beta-sheet A, resulting in a cold-induced conformational change of beta-strand 5A and thereby an increased substrate behaviour at low temperatures. These results provide new information about the structural basis for serpin substrate behaviour.

Ligand binding properties of the very low density lipoprotein receptor. Absence of the third complement-type repeat encoded by exon 4 is associated with reduced binding of Mr 40,000 receptor-associated protein.

The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.

An ester bond linking a fragment of a serine proteinase to its serpin inhibitor.

Most known members of the serpin superfamily are serine proteinase inhibitors. Serpins are therefore important regulators of blood coagulation, complement activation, fibrinolysis, and turnover of extracellular matrix. Serpins form SDS-resistant complexes of 1:1 stoichiometry with their target proteinases by reaction of their P1-P1' peptide bond with the active site of the proteinases. The nature of the interactions responsible for the high stability of the complexes is a controversial issue. We subjected the complex between the serine proteinase urokinase-type plasminogen activator (uPA) and the serpin plasminogen activator inhibitor-1 (PAI-1) to proteolytic digestion under nondenaturing conditions. The complex could be degraded to a fragment containing two disulfide-linked peptides from uPA, one of which included the active site Ser, while PAI-1 was left undegraded. By further proteolytic digestion after denaturation and reduction, it was also possible to degrade the PAI-1 moiety, and we isolated a fragment containing 10 amino acids from uPA, encompassing the active site Ser, and 6 amino acids from PAI-1, including the P1 Arg. Characterization of the fragment gave results fully in agreement with the hypothesis that it contained an ester bond between the hydroxyl group of the active site Ser and the carboxyl group of the P1 Arg. These data for the first time provide direct evidence that serine proteinases are entrapped at an acyl intermediate stage in serine proteinase-serpin complexes.

Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer.

The uPA-mediated pathway of plasminogen activation is central to cancer metastasis. Whether uPA and PAI-1 are related to local recurrence, metastatic spread or both is not clear. We present a retrospective study of 429 primary breast cancer patients with a median follow-up of 5.1 years, in which the levels of uPA and PAI-1 in tumour extracts were analysed by means of an enzyme-linked immunosorbent assay. The median values of uPA and PAI-1, which were used as cut-off points, were 4.5 and 11.1 ng mg(-1) protein respectively. The levels of uPA and PAI-1 were correlated with tumour size, degree of anaplasia, steroid receptor status and number of positive nodes. Patients with high content of either uPA or PAI-1 had increased risk of relapse and death. We demonstrated an independent ability of PAI-1 to predict distant metastasis (relative risk 1.7, confidence limits 1.22 and 2.46) and that neither uPA nor PAI-1 provided any information regarding local recurrence.

Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor.

The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA.PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified alpha2MR/LRP and VLDLR respectively. alpha2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA.PAI-1 complex to both alpha2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by alpha-helix D and beta-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease.serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.

Specificity of serine proteinase/serpin complex binding to very-low-density lipoprotein receptor and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein.

Very-low-density lipoprotein receptor (VLDLR) and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein (alpha2MR/LRP) are multifunctional endocytosis receptors of the low-density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type-1 plasminogen-activator inhibitor (PAI-1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and alpha2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinase-type and tissue-type plasminogen activators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, alpha2-antiplasmin, alpha1-proteinase inhibitor, alpha1-antichymotrypsin, protease nexin-1, heparin cofactor II, and antithrombin III. Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor-associated protein (RAP)-sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS-1 cells, which have a high endogenous expression of alpha2MR/LRP. We found that the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrombin with PAI-1, protease nexin-1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/alpha2-antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and alpha2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and alpha2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.

Breast carcinoma epithelial cells express a very low-density lipoprotein receptor variant lacking the O-linked glycosylation domain encoded by exon 16, but with full binding activity for serine proteinase/serpin complexes and Mr-40,000 receptor-associated protein.

Very-low density lipoprotein receptor (VLDLR) belongs to the low-density lipoprotein receptor family of endocytosis receptors. It binds a variety of different ligands, including apolipoprotein E, Mr-40,000 receptor-associated-protein (RAP), and some serine proteinase/serpin complexes. We previously demonstrated the occurrence of two forms of VLDLR in SDS/PAGE, migrating with Mr 105,000 and Mr 130,000, respectively [Heegaard, C. W., Simonsen, A. C. W., Oka, K., Kjøller, L., Christensen, A., Madsen, B., Ellgaard, L., Chan, L. & Andreasen, P. A. (1995) J. Biol. Chem. 270, 20,855-20,869]. We now demonstrate that these two forms correspond to forms with the absence (type-II) and presence (type-I) of the O-linked glycosylation domain encoded by exon 16, respectively. We show that the two forms have the same binding affinity to RAP and serine proteinase/serpin complexes. Using reverse transcription and PCR, we demonstrate that the splice variation giving rise to the two forms is highly cell specific. In particular, we demonstrate that human breast carcinomas express predominantly or exclusively the variant lacking exon 16. By immunohistochemistry, we demonstrate that VLDLR is mainly expressed by the epithelial cancer cells in these carcinomas. The VLDLR variant expressed by epithelial cancer cells could function in the clearance of cell-surface-associated serine proteinase/serpin complexes in breast carcinomas.

Type-1 plasminogen-activator inhibitor -- conformational differences between latent, active, reactive-centre-cleaved and plasminogen-activator-complexed forms, as probed by proteolytic susceptibility.

We have analysed the susceptibility of latent, active, reactive-centre-cleaved and plasminogen-activator-complexed type-1 plasminogen-activator inhibitor (PAI-1) to the non-target proteinases trypsin, endoproteinase Asp-N, proteinase K and subtilisin. This analysis has allowed us to detect conformational differences between the different forms of PAI-1 outside the reactive-centre loop and beta-sheet A. Proteinase-hypersensitive sites were clustered in three regions. Firstly, susceptibility was observed in the region around alpha-helix E, beta-strand 1A, and the flanking loops, which are believed to form flexible joints during movements of beta-sheet A. Secondly, hypersensitive sites were observed in the loop between alpha-helix I and beta-strand 5A. Thirdly, the gate region, encompassing beta-strands 3C and 4C, was highly susceptible to trypsin in latent PAI-1, but not in the other conformations. The digestion patterns differed among all four forms of PAI-1, indicating that each represents a unique conformation. The differential proteolytic susceptibility of the flexible-joint region may be coupled to the differential affinity to vitronectin, binding in the same region. The analysis also allowed detection of conformational differences between reactive-centre-cleaved forms produced under different solvent conditions. The digestion pattern of plasminogen-activator-complexed PAI-1 was different from that of active PAI-1, but indistinguishable from that of one of the reactive-centre-cleaved forms, as the complexed and this particular cleaved PAI-1 were completely resistant to all the non-target proteinases tested. This observation is in agreement with the notion that complex formation involves reactive-centre cleavage and a large degree of insertion of the reactive-centre loop into beta-sheet A. Our analysis has allowed the identification of some flexible regions that appear to be implicated in the conformational changes during the movements of beta-sheet A and during the inhibitory reaction of serpins with their target proteinases.

Plasminogen activation system in human milk.

BACKGROUND: Plasmin is the major endogenous protease present in milk. The level of plasmin activity is controlled by the availability of the precursor plasminogen and by the levels of plasminogen activators and inhibitors. Recently, a differential distribution of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) has been demonstrated in bovine milk. To assess whether this distribution pattern is a general feature, the occurrence of components of the plasminogen activation system in different fractions of human milk was investigated. METHODS: Milk samples were separated into the following fractions; milk fat, skim milk, and milk cells by centrifugation. The different fractions were detected for the presence of plasminogen and plasminogen activators by immunoblotting and zymography. The distribution of t-PA and u-PA was investigated by ligand binding analysis. t-PA-catalyzed plasminogen activation was examined by a coupled chromogenic assay. RESULTS: A differential distribution of plasminogen, t-PA, and u-PA was found. Casein micelles were found to exhibit t-PA and plasminogen binding activity, whereas the u-PA receptor was identified as the u-PA binding component in the cell fraction. Furthermore, human casein enhanced t-PA-catalyzed plasminogen activation, comparable to the enhancing effect obtained with fibrinogen fragments. CONCLUSION: The finding of a differential distribution of u-PA and t-PA in milk suggests that the two activators may have different physiological functions, which involve protection against invading microorganisms and maintenance of patency and fluidity in the ducts of mammary gland, respectively.