Definition of epitopes within plasminogen activator inhibitor type-1 (PAI-1) using multiple peptide synthesis

Perrie, A.-M.; MacGregor, Ian; Booth, Nuala A.

doi:10.1016/0268-9499(93)90134-h

Cited by 11 publications

(15 citation statements)

References 27 publications

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“…Concentration of pooled fractions was achieved on an Amicon ultrafiltration apparatus, using a YM2 membrane. membrane washed and blocked with 3% BSA before probing with radiolabelled tissue-type plasminogen activator, exactly as described previously [46].…”

Section: Bacterial Culturementioning

confidence: 99%

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Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Sancho

Tonge

Hockney

et al. 1994

European Journal of Biochemistry

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Plasminogen-activator inhibitor type 1 (PAI-l), the primary physiological inhibitor of tissue-type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high-level expression system that leads to the accumulation of PAI-1 at 30-50% total microbial protein. We have developed a single-step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI-Mitre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37°C with a half-life of approximately 48 h. At 20°C, PAI-1 activity was stable for a week and at 4°C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI-1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of highlevel expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI-1 in high yield.Activation of plasminogen to form the serine protease plasmin is central to the processes of fibrinolysis [l], cell migration, angiogenesis and tumour metastasis [2]. Activation of plasminogen is accomplished by either tissue-type or urokinase-type plasminogen activator. Regulation of the system occurs at several levels, including assembly of the components and stimulation of activation on fibrin [l] or at cell surfaces [3, 41. The inhibition of the active enzymes is an important element in the control of the plasminogedplasmin system. Plasminogen-activator inhibitor type 1 (PAI-1) is the main physiological inhibitor of both tissue-type plasminogen activator and urokinase-type plasminogen activator, with association rate constants of the order of 0.1 pM s- ' [5]. PAI-1 is present in a wide variety of tissues and cultured cells, where regulation of its expression by several stimuli has been observed [6]. It is present in the circulation both in cell-free plasma and in platelets, these two pools being clearly distinct [7]. The plasma concentration of PAI-1 is normally low, approximately 20 ng/ml, but higher levels are observed in a range of diseases, suggesting an association with thrombotic disorders [S]. The platelet pool of PAI-1 is released during aggregation, so that it protects the newly formed thrombus against premature dissolution. Platelet PAI-1 contributes to the resistance PAI-1 was identified as a member of the superfamily of serine protease inhibitors known as serpins, on the basis of its sequence [16-181. The serpins are the major class of inhibitors regulating the fibrinolytic, coagulation and complement cascades. They act as pseudosubstrates and form an SDS-stable covalent 1 : 1 complex with their target...

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Section: Bacterial Culturementioning

confidence: 99%

“…Proteins were transferred onto nitrocellulose and PAI-1 was detected as described previously [27], except that 0.6 mM 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine, 0.5 mM nitroblue tetrazolium in 200 mM Tris/HCl, pH 9.5, 10 mM MgCL was used as the substrate to detect alkaline phosphatase [46].…”

Section: Sds/page and Immunoblottingmentioning

confidence: 99%

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Sancho

Tonge

Hockney

et al. 1994

European Journal of Biochemistry

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“…None of the suggested epitopes were located at the hinge region of ␣-helix F, although the epitope of CLB-2C8 is located near this area (residues 128 -145) (39). Interestingly, other antibodies have been described with an epitope partially covering the same area (residues 125-132), that did not exert any inhibitory effect on PAI-1 (40). This further confirms that the functional properties of the currently described antibodies are due to the simultaneous interaction with residues Glu 128 -Arg 131 and with residue Lys 154 .…”

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confidence: 97%

Importance of the Hinge Region between α-Helix F and the Main Part of Serpins, Based upon Identification of the Epitope of Plasminogen Activator Inhibitor Type 1 Neutralizing Antibodies

Bijnens¹,

Gils²,

Knockaert³

et al. 2000

Journal of Biological Chemistry

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The serpin plasminogen activator inhibitor type 1 (PAI-1) is an important protein in the regulation of fibrinolysis and inhibits its target proteinases through formation of a covalent complex. In the present study, we have identified the epitope of two PAI-1 neutralizing monoclonal antibodies (MA-33H1F7 and MA-55F4C12). Based upon differential cross-reactivity data of these monoclonals with PAI-1 from different species and on a sequence alignment between these PAI-1s, combined with the three-dimensional structure, we predicted that the residues Glu Plasminogen activator inhibitor type 1 (PAI-1), 1 a member of the serine proteinase inhibitor (serpin) superfamily (1-4) is an important protein in the regulation of fibrinolysis. PAI-1 is the most important physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma (5).PAI-1 is unique among the serpins because of its functional and conformational flexibility. The active conformation of PAI-1 inhibits its target proteinases by the formation of a stable, inactive complex. After the formation of an initial, reversible Michaelis-like complex, the proteinase cleaves the active site of PAI-1 and forms a stable, covalent complex resulting in the inactivation of the proteinase (6, 7). The structure of a covalent complex between PAI-1 and t-PA in particular, or between a serpin and its target proteinase in general, is presently unknown. However, two different models have been proposed. According to one model, the proteinase moves after the initial attack to the opposite pole of the serpin, thereby resulting in a complete insertion of the N-terminal side of the reactive-site loop (7-9). Alternatively, it was hypothesized that movement of the proteinase following the initial proteinase/ serpin interaction is less extended, yielding a complex in which the N-terminal side of the reactive-site loop is only partially inserted (10, 11), accompanied by a distortion of the catalytic triad of the proteinase (6) and stabilized by multiple interactions between serpin and proteinase.Although PAI-1 is synthesized as an active molecule, it converts spontaneously to an inactive, latent form that can be partially reactivated by denaturing agents (12). In this latent conformation, the active site is inaccessible for the target proteinases as a result of the insertion of the N-terminal side of the reactive-site loop in ␤-sheet A of the PAI-1 molecule (13). In addition, a third conformation with substrate properties has been identified (14 -16). This form of PAI-1 reacts with t-PA or u-PA, resulting in a cleavage of the active site of PAI-1 but without the formation of a covalent complex (17).Previously, we have characterized a panel of monoclonal antibodies that neutralize PAI-1 activity by converting the active pathway into the non-inhibitory substrate pathway (18). For two of these antibodies, MA-55F4C12 and MA-33H1F7, the binding region was found to be located remote of the reactivesite loop, within a region comprising residues at positions 128 -156 (19). Within the three-...

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“…Using overlapping peptides, Perrie et al (44) demonstrated that ESPI-12 recognized an epitope spanning residues 342-349 of PAI-1 (P5-P3Ј), including the bait peptide bond, thereby explaining the inhibitory properties of the antibody through a direct interaction with the reactive center residue. The binding region of MAI-12 ( same as MA-7D4B7 (45)) has been suggested as being situated between amino acid residues 320 and 379 of PAI-1 (46) and is overlapping or possibly identical to that of ESPI-12 (47).…”

Section: Discussionmentioning

confidence: 99%

The Distal Hinge of the Reactive Site Loop and Its Proximity

Bijnens¹,

Gils²,

Stassen³

et al. 2001

Journal of Biological Chemistry

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The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.Plasminogen activator inhibitor type 1 (PAI-1), 1 a member of the serpin (serine proteinase inhibitor) superfamily (1-4), controls the plasminogen system at the level of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively). Because PAI-1 is the main physiological inhibitor of t-PA in plasma (5), increased levels of PAI-1 result in a hypofibrinolytic state and are correlated with various vascular disorders such as venous thrombo-embolism, coronary artery disease, myocardial infarction, and atherosclerosis (6 -9). The u-PA-inhibiting effect of PAI-1 has its main physiological implications in processes outside of the circulation (10, 11).PAI-1 is a unique serpin because of its functional and conformational flexibility (12). PAI-1, synthesized as an active molecule, converts spontaneously into a nonreactive, latent form, which can be partially reactivated by denaturing reagents (13). Additionally, a third distinct, noninhibitory form (substrate) is identified that is reactive toward its target proteinases without the formation of a stable complex (14 -16).Elucidation of the three-dimensional structure of active PAI-1 (17, 18) reveals that the N-terminal side of the reactive site loop (extended from P16 to P3Ј and including the bait peptide bond Arg 345 -Met 346 (P1-P1Ј)) is exposed and accessible for the target proteinase. The C-terminal side of the reactive site loop (P4Ј-P13Ј) forms strand s1C in ␤-sheet C.Conversion to the latent state implies the insertion of the N-terminal side of the reactive site loop into ␤-sheet A, the loss of strand s1C from ␤-sheet C, and the formation of an unusual extended loop by the C-terminal side of the reactive site loop, resulting in the distortion of the P1-P1Ј "bait" peptide bond (19). It is hypothesized that th...

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Definition of epitopes within plasminogen activator inhibitor type-1 (PAI-1) using multiple peptide synthesis

Cited by 11 publications

References 27 publications

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Importance of the Hinge Region between α-Helix F and the Main Part of Serpins, Based upon Identification of the Epitope of Plasminogen Activator Inhibitor Type 1 Neutralizing Antibodies

The Distal Hinge of the Reactive Site Loop and Its Proximity

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