Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C

Daniel M Dupont; Grant E Blouse; Martin Hansen; Lisa Mathiasen; Signe Kjelgaard; Jan Kristian Jensen; Anni Christensen; Ann Gils; Paul J Declerck; Peter A Andreasen; Troels Wind

doi:10.1074/jbc.M606851200

Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C

Daniel M Dupont, Grant E Blouse, Martin Hansen, Lisa Mathiasen, Signe Kjelgaard, Jan Kristian Jensen, Anni Christensen, Ann Gils, Paul J Declerck, Peter A Andreasen, Troels Wind

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

38 Citations (Scopus)

Abstract

Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule.

Original language	English
Journal	Journal of Biological Chemistry
Volume	281
Issue	47
Pages (from-to)	36071-81
Number of pages	11
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M606851200
Publication status	Published - 24 Nov 2006

Keywords

Animals
Arginine
Aspartic Acid
Binding Sites
Epitope Mapping
Humans
Kinetics
Mice
Plasminogen Activator Inhibitor 1
Protein Conformation
Protein Folding
Protein Structure, Secondary
Protein Structure, Tertiary
Spectrometry, Fluorescence
Vitronectin

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10.1074/jbc.M606851200

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@article{6588a0576d064566accd159295356b8e,

title = "Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C",

abstract = "Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule.",

keywords = "Animals, Arginine, Aspartic Acid, Binding Sites, Epitope Mapping, Humans, Kinetics, Mice, Plasminogen Activator Inhibitor 1, Protein Conformation, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Spectrometry, Fluorescence, Vitronectin",

author = "Dupont, {Daniel M} and Blouse, {Grant E} and Martin Hansen and Lisa Mathiasen and Signe Kjelgaard and Jensen, {Jan Kristian} and Anni Christensen and Ann Gils and Declerck, {Paul J} and Andreasen, {Peter A} and Troels Wind",

year = "2006",

month = nov,

day = "24",

doi = "10.1074/jbc.M606851200",

language = "English",

volume = "281",

pages = "36071--81",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology Inc.",

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Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C. / Dupont, Daniel M; Blouse, Grant E; Hansen, Martin et al.
In: Journal of Biological Chemistry, Vol. 281, No. 47, 24.11.2006, p. 36071-81.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C

AU - Dupont, Daniel M

AU - Blouse, Grant E

AU - Hansen, Martin

AU - Mathiasen, Lisa

AU - Kjelgaard, Signe

AU - Jensen, Jan Kristian

AU - Christensen, Anni

AU - Gils, Ann

AU - Declerck, Paul J

AU - Andreasen, Peter A

AU - Wind, Troels

PY - 2006/11/24

Y1 - 2006/11/24

N2 - Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule.

AB - Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule.

KW - Animals

KW - Arginine

KW - Aspartic Acid

KW - Binding Sites

KW - Epitope Mapping

KW - Humans

KW - Kinetics

KW - Mice

KW - Plasminogen Activator Inhibitor 1

KW - Protein Conformation

KW - Protein Folding

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Spectrometry, Fluorescence

KW - Vitronectin

U2 - 10.1074/jbc.M606851200

DO - 10.1074/jbc.M606851200

M3 - Journal article

C2 - 17018527

SN - 0021-9258

VL - 281

SP - 36071

EP - 36081

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 47

ER -

Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C

Abstract

Keywords

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