scholarly journals Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

2011 ◽  
Vol 438 (1) ◽  
pp. 39-51 ◽  
Author(s):  
Kenneth A. Botkjaer ◽  
Sarah Fogh ◽  
Erin C. Bekes ◽  
Zhuo Chen ◽  
Grant E. Blouse ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Conformational Changes ◽  
Active Site ◽  
Protease Activity ◽  
Cultured Cells ◽  
Model Systems ◽  
Zymogen Activation ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator

2001 ◽  
Vol 268 (16) ◽  
pp. 4430-4439 ◽  
Author(s):  
Helle H. Petersen ◽  
Martin Hansen ◽  
Susanne L. Schousboe ◽  
Peter A. Andreasen
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

scholarly journals Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1522-1531 ◽  
Author(s):  
SE Lind ◽  
JR McDonagh ◽  
CJ Smith
Keyword(s):  
Plasminogen Activator ◽  
Active Site ◽  
Serine Proteases ◽  
Plasminogen Activators ◽  
Single Chain ◽  
Site Specific ◽  
Oxidative Inactivation ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Specific Oxidation

Abstract Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2′:6′,2″- terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.

A cyclic peptidylic inhibitor of murine urokinase-type plasminogen activator: changing species specificity by substitution of a single residue

2008 ◽  
Vol 412 (3) ◽  
pp. 447-457 ◽  
Author(s):  
Lisbeth M. Andersen ◽  
Troels Wind ◽  
Hanne D. Hansen ◽  
Peter A. Andreasen
Keyword(s):  
Plasminogen Activator ◽  
Serine Proteases ◽  
Enzyme Inhibitor ◽  
Competitive Inhibitor ◽  
Catalytic Triad ◽  
Model Systems ◽  
Binding Interaction ◽  
Alanine Scanning Mutagenesis ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

uPA (urokinase-type plasminogen activator) is a potential therapeutic target in a variety of pathological conditions, including cancer. In order to find new principles for inhibiting uPA in murine cancer models, we screened a phage-displayed peptide library with murine uPA as bait. We thereby isolated several murine uPA-binding peptide sequences, the predominant of which was the disulfide-bridged constrained sequence CPAYSRYLDC, which we will refer to as mupain-1. A chemically synthesized peptide corresponding to this sequence was found to be a competitive inhibitor of murine uPA, inhibiting its activity towards a low-molecular-mass chromogenic substrate as well as towards its natural substrate plasminogen. The Ki value for inhibition as well as the KD value for binding were approx. 400 nM. Among a variety of other murine and human serine proteases, including trypsin, mupain-1 was found to be highly selective for murine uPA and did not even measurably inhibit human uPA. The cyclic structure of mupain-1 was indispensable for binding. Alanine scanning mutagenesis identified Arg6 of mupain-1 as the P1 residue and indicated an extended binding interaction including the P5, P3, P2, P1 and P1′ residues of mupain-1 and the specificity pocket, the catalytic triad and amino acids 41, 99 and 192 located in and around the active site of murine uPA. Exchanging His99 of human uPA by a tyrosine residue, the corresponding residue in murine uPA, conferred mupain-1 susceptibility on to the latter. Peptide-derived inhibitors, such as mupain-1, may provide novel mechanistic information about enzyme–inhibitor interactions, provide alternative methodologies for designing effective protease inhibitors, and be used for target validation in murine model systems.

Monoclonal Antibodies to Human 54,000 Molecular Weight Plasminogen Activator Inhibitor from Fibrosarcoma Cells - Inhibitor Neutralization and One-Step Affinity Purification

1986 ◽  
Vol 55 (02) ◽  
pp. 206-212 ◽  
Author(s):  
L S Nielsen ◽  
P A Andreasen ◽  
J Grøndahi-Hansen ◽  
J Y Huang ◽  
P Kristensen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Umbilical Cord ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
One Step ◽  
Activator Inhibitor

SummaryMouse monoclonal antibodies were derived against a plasminogen activator inhibitor with a mol.wt. of ∼54,000 (54 K) from the human fibrosarcoma cell line HT-1080. Screening for hybrids producing antibodies directed against the inhibitor was performed with enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. Four clones of hybridomas producing IgG1 antibodies were further characterized. The inhibitor was purified ∼50-fold to homogeneity from conditioned cell culture fluid with a yield of ∼85% by a one-step procedure using Sepharose-conjugated monoclonal antibody. In the 125I-fibrin plate assay one of the antibodies neutralized the effect of the inhibitor on urokinase-type plasminogen activator. Two of the antibodies bound complexes between urokinase-type plasminogen activator and inhibitor while the remaining two antibodies did not. The antibodies could be used for immunocytochemical localization of the inhibitor in HT-1080 cells. All four antibodies cross-reacted with a plasminogen activator inhibitor derived from cultured human umbilical cord endothelial cells.

scholarly journals Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1522-1531
Author(s):  
SE Lind ◽  
JR McDonagh ◽  
CJ Smith
Keyword(s):  
Plasminogen Activator ◽  
Active Site ◽  
Serine Proteases ◽  
Plasminogen Activators ◽  
Single Chain ◽  
Site Specific ◽  
Oxidative Inactivation ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Specific Oxidation

Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2′:6′,2″- terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.

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