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. 2006 Nov 1;399(3):387-96.
doi: 10.1042/BJ20060533.

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

Jan K Jensen  1 , Anders Malmendal, Birgit Schiøtt, Sune Skeldal, Katrine E Pedersen, Leyla Celik, Niels Chr Nielsen, Peter A Andreasen, Troels Wind
Affiliations

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

Jan K Jensen et al. Biochem J. .

Abstract

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA-PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA-PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction.

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Figures

Figure 1
Figure 1. Serpin structures
Ribbon diagrams of active PAI-114–1B (a stable active PAI-1 variant carrying four mutations [42], PDB code 1DVM [43]), latent PAI-1 (PDB code 1LJ5), and the complex between the serpin α1-proteinase inhibitor and the protease elastase (PDB code 2D26 [6]). For the serpins, the RCL is in red, the large serpin fragment is in orange, the small serpin fragment including hF is in blue and hD and hE from the flexible joint region are in green. Elastase is shown in purple.
Figure 2
Figure 2. Competition of paionin-1 phage binding in ELISAs
Binding of paionin-1 phage clone B to 10 nM PAI-1 from HT-1080 cells was detected in ELISA format 1 (see the Experimental section) in the presence of additions as indicated for each experiment. (A) Paionin-1: NH2-DVPCFGWCQDA-CO2H. Paionin-1, linear: NH2-DVPSFGWSQDA-CO2H. Bis-ANS and XR-5118: see [13]. (B) Effects from monoclonal anti-PAI-1 antibodies Mab-1, Mab-2 and Mab-6 and vitronectin on paionin-1 phage binding. Monoclonal anti-uPA Mab-2 was included as a control.
Figure 3
Figure 3. Structure of paionin-1 determined by NMR spectroscopy
(A) Overlay of the 15 low-energy structures in conformational group number 1, aligned according to the backbone of the CFGWC ring, shown in red. (B) The CFGWC ring of one representative structure from each of the nine conformational groups, each with a distinct colour, aligned according to the backbone.
Figure 4
Figure 4. Docking of paionin-1 into the structure of latent PAI-1 and residues important for paionin-1 binding in the flexible joint region
Latent PAI-1 shown as a ribbon diagram with the inserted RCL in red, the large serpin fragment in orange, the small serpin fragment including hF in blue and hD and hE from the flexible joint region in green. (A) The docked structure of paionin-1 from conformational group number 1 is shown as a stick model. (B) Residues which based on binding assays with mutated PAI-1 variants are implicated in paionin-1 binding are indicated.
Figure 5
Figure 5. Conductometric determination of the CMC for biotin–paionin-1
The electric conductance was monitored while titrating biotin–paionin-1 into water. The presented data have been corrected for the conductance resulting from adding equal volumes of buffer without peptide. The two broken lines represent linear regressions of data points below and above 100 μM biotin–paionin-1 respectively, crossing each other at 107 μM.
Figure 6
Figure 6. Localization of the paionin-1-binding site
(A) Binding between recombinant PAI-1, active, latent or in complex with uPA (complex), and paionin-1 phage in ELISA format 2 (see the Experimental section). Latent PAI-1 was prepared by incubating active PAI-1 at 37 °C for 16 h and uPA–PAI-1 complex was prepared by incubating active PAI-1 with a 2-fold molar excess of uPA at room temperature for 30 min. Results are means±S.D., representative of two independent experiments with similar results, each performed in triplicate. (B) Binding between recombinant human or murine PAI-1 and paionin-1 phage in ELISA format 3 (see the Experimental section). Results are means±S.D., representative of two independent experiments with similar results, each performed in triplicate. (C) Biotin–paionin-1 peptide binding to recombinant PAI-1 variants in ELISA format 4 (see the Experimental section). PAI-114–1B (active 14-1B) is a stable active PAI-1 variant carrying four mutations [42], other PAI-1 variants are indicated by their mutations. Except for active PAI-1 (active wild-type) and PAI-114–1B (active 14-1B), the tested variants were in their latent conformation. Signals were normalized to those obtained with latent PAI-1. Results are means±S.D. for three independent experiments. (D) SPR sensorgrams for association and dissociation of PAI-1 variants to immobilized biotin–paionin-1 with a C-terminal GAKK extension. Except for active (Act.), the variants were in their latent (Lat.) conformation. Protein (700 nM) was applied at a flow rate of 5 μl/min and the sensorgrams were corrected by subtraction of the signals from a reference cell.
Figure 7
Figure 7. Effects of paionin-1 on the binding of the uPA–PAI-1 complex to endocytosis receptors
(A) Effects of biotin–paionin-1 on the binding of 125I-uPA–PAI-1 (●) and 125I-RAP (○) to immobilized LRP-1A. Results are means±S.D. for at least two independent experiments. (B) Effects of biotin–paionin-1 on degradation of 125I-uPA–PAI-1 (●) and 125I-RAP (○) by U937 cells. results are means±S.D. for one experiment performed in duplicate, representative of three independent experiments.
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