Immunohistochemical localization of urokinase‐type plasminogen activator, type‐1 plasminogen‐activator inhibitor, urokinase receptor and α2‐macroglobulin receptor in human breast carcinomas

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We have analysed the susceptibility of latent, active, reactive-centre-cleaved and plasminogen-activator-complexed type-I plasniinogen-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 P-sheet A. Proteinase-hypersensitive sites were clustered in three regions. Firstly, susceptibility was observed in the region around a-helix E, p-strand IA, and the flanking loops, which are believed to form tlexible joints during movements of p-sheet A. Secondly, hypersensitive sites were observed in the loop between a-helix I and p-strand 5A. Thirdly, the gate region, encompassing ,&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 PAT-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 reactivecentre-cleaved forms produced under different solvent conditions. The digestion pattern of plasminogenactivator-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 /?-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 P-sheet A and during the inhibitory reaction of serpins with their target proteinases.

Section: Methodsmentioning

confidence: 83%

Type‐1 Plasminogen‐Activator Inhibitor

Egelund

Schousboe

Sottrup‐Jensen

et al. 1997

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“…Murine monoclonal anti‐(human PAI‐1) IgG from hybridoma clone 2 and monoclonal anti‐(human uPA) IgG from hybridoma clones 2 or 6 were described previously [25,26]. Subtilisin Carlsberg, trypsin, amiloride hydrochloride, diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride and dithioerythritol were purchased from Sigma (St Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

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

Egelund¹,

Petersen

Andreasen³

2001

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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 P 1 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 b-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.Keywords: plasminogen activator inhibitor type-1 (PAI-1); serine proteinase; serpin; urokinase-type plasminogen activator (uPA).The serpin superfamily comprises a wide variety of proteins, including many of the serine proteinase inhibitors in blood plasma. All serpins share the same overall fold, consisting of three b-sheets and nine a-helices. Serpins and proteinases form stable complexes by interaction of the active site of the proteinase with the reactive-centre peptide bond (P 1 ±P 1 H ) in the surface-exposed reactive-centre loop (RCL) of the serpin (reviewed in [1±5]). There is both biochemical and biophysical evidence that complexformation is associated with cleavage of the P 1 ±P 1 H peptide bond and covalent linkage of the carboxy group of the P 1 residue by an ester bond to the hydroxy group of the activesite serine residue of the proteinase, insertion of the N-terminal part of the RCL into b-sheet A, and translocation of the proteinase across the plane of b-sheet A towards the other pole of the molecule [6±14]. Enzymatic cleavage of peptide bonds by serine proteinases normally proceeds through an acylation step followed by a deacylation step. Thus, serpin inhibitors of serine proteinases are pseudo-substrates in the sense that the normal substrate pathway is not being completed, but stops at the acylenzyme intermediate stage. An important question concerning the mechanism for serpin inhibition is why the ester bond linking...

“…Antibodies. The following monoclonal antibodies against human proteins were used: anti-uPA IgG from hybridoma clone 6 (Christensen et al, 1996); anti-PAI-I IgG from hybridoma clone 2 (Nielsen et al, 1986); anti-tPA IgG (no. 3700, American Diagnostica, Greenwich, CT, USA) ; anti-thrombin IgG (EST 7, American Diagnostica) ; anti-antithrombin I11 IgG (no.…”

Section: Methodsmentioning

confidence: 99%

Specificity of Serine Proteinase/Serpin Complex Binding to Very‐Low‐Density Lipoprotein Receptor and α₂‐Macroglobulin Receptor/Low‐Density‐Lipoprotein‐Receptor‐Related Protein

Kasza

Petersen

Heegaard

et al. 1997

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Very-low-density lipoprotein receptor (VLDLR) and a,-macroglobulin receptorllow-density-lipoprotein-receptor-related protein (a,MFULRP) 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 a,MFULRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinasetype and tissue-type plasminogen aqtivators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, a,-antiplasmin, a,-proteinase inhibitor, a,-antichymotrypsin, protease nexin-1, heparin cofactor 11, and antithrombin 111. 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 a,MR/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-I, protein C inhibitor, and antithrombin 111, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/a,-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 a,MR/LRP are overlapping, but not identical. The results suggest that VLDLR and a,MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.Keywords: low-density-lipoprotein-receptor-related protein; plasminogen activator; serine proteinase; serpin ; very-low-density lipoprotein receptor.A variety of physiological events, like blood coagulation, complement activation, fibrinolysis, and turnover of extracellular matrix, implicate the function of extracellular serine proteinase enzyme systems. Regulation of extracellular proteolysis caCorrespondence to P.