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The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity

BACKGROUND: Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been propo...

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Autores principales: Cochran, Blake J, Gunawardhana, Lakshitha P, Vine, Kara L, Lee, Jodi A, Lobov, Sergei, Ranson, Marie
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693429/
https://www.ncbi.nlm.nih.gov/pubmed/19442270
http://dx.doi.org/10.1186/1472-6750-9-43
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author Cochran, Blake J
Gunawardhana, Lakshitha P
Vine, Kara L
Lee, Jodi A
Lobov, Sergei
Ranson, Marie
author_facet Cochran, Blake J
Gunawardhana, Lakshitha P
Vine, Kara L
Lee, Jodi A
Lobov, Sergei
Ranson, Marie
author_sort Cochran, Blake J
collection PubMed
description BACKGROUND: Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use in vitro of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI-2 ΔCD-loop) for these purposes. RESULTS: We show that PAI-2 ΔCD-loop expressed and purified from the pQE9 vector system presents an easier purification target than the previously used pET15b system. Additionally, PAI-2 ΔCD-loop gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions. Importantly, absence of the CD-loop had no impact on the inhibition of both solution phase and cell surface uPA or on the clearance of receptor bound uPA from the cell surface. Furthermore, uPA:PAI-2 ΔCD-loop complexes had similar binding kinetics (K(D )~5 nM) with the endocytosis receptor Very Low Density Lipoprotein Receptor (VLDLR) to that previously published for uPA:PAI-2 complexes. CONCLUSION: We demonstrate that the CD-loop is redundant for the purposes of cellular uPA inhibition and cell surface clearance (endocytosis) and is thus suitable for the development of anti-uPA targeted cancer therapeutics.
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spelling pubmed-26934292009-06-08 The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity Cochran, Blake J Gunawardhana, Lakshitha P Vine, Kara L Lee, Jodi A Lobov, Sergei Ranson, Marie BMC Biotechnol Research Article BACKGROUND: Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use in vitro of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI-2 ΔCD-loop) for these purposes. RESULTS: We show that PAI-2 ΔCD-loop expressed and purified from the pQE9 vector system presents an easier purification target than the previously used pET15b system. Additionally, PAI-2 ΔCD-loop gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions. Importantly, absence of the CD-loop had no impact on the inhibition of both solution phase and cell surface uPA or on the clearance of receptor bound uPA from the cell surface. Furthermore, uPA:PAI-2 ΔCD-loop complexes had similar binding kinetics (K(D )~5 nM) with the endocytosis receptor Very Low Density Lipoprotein Receptor (VLDLR) to that previously published for uPA:PAI-2 complexes. CONCLUSION: We demonstrate that the CD-loop is redundant for the purposes of cellular uPA inhibition and cell surface clearance (endocytosis) and is thus suitable for the development of anti-uPA targeted cancer therapeutics. BioMed Central 2009-05-14 /pmc/articles/PMC2693429/ /pubmed/19442270 http://dx.doi.org/10.1186/1472-6750-9-43 Text en Copyright © 2009 Cochran et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Cochran, Blake J
Gunawardhana, Lakshitha P
Vine, Kara L
Lee, Jodi A
Lobov, Sergei
Ranson, Marie
The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity
title The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity
title_full The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity
title_fullStr The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity
title_full_unstemmed The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity
title_short The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity
title_sort cd-loop of pai-2 (serpinb2) is redundant in the targeting, inhibition and clearance of cell surface upa activity
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693429/
https://www.ncbi.nlm.nih.gov/pubmed/19442270
http://dx.doi.org/10.1186/1472-6750-9-43
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