Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates
Autor(a) principal: | |
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Data de Publicação: | 2011 |
Outros Autores: | , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/33903 http://dx.doi.org/10.1111/j.1742-4658.2011.08189.x |
Resumo: | Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. in addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. the Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by beta 2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and beta 2-tryptase. the resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [ where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1).s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma. |
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Korkmaz, BriceJegot, GwenhaelLau, Laurie C.Thorpe, MichaelPitois, ElodieJuliano, Luiz [UNIFESP]Walls, Andrew F.Hellman, LarsGauthier, FrancisUniv ToursSouthampton Gen HospUppsala UnivUniversidade Federal de São Paulo (UNIFESP)2016-01-24T14:17:01Z2016-01-24T14:17:01Z2011-08-01Febs Journal. Malden: Wiley-Blackwell, v. 278, n. 15, p. 2635-2646, 2011.1742-464Xhttp://repositorio.unifesp.br/handle/11600/33903http://dx.doi.org/10.1111/j.1742-4658.2011.08189.x10.1111/j.1742-4658.2011.08189.xWOS:000292933300003Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. in addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. the Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by beta 2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and beta 2-tryptase. the resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [ where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1).s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.Region CentreFonds Europeen de Developpement RegionalAgence Nationale pour la RechercheUniv Tours, INSERM, Unite Proteases & Vectorisat Pulm U618, F-37032 Tours, FranceSouthampton Gen Hosp, Sir Henry Wellcome Labs, Immunopharmacol Grp, Southampton, Hants, EnglandUppsala Univ, Biomed Ctr, Dept Cell & Mol Biol, Uppsala, SwedenUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilAgence Nationale pour la Recherche: ANR-07-PHYSIO-029-01Web of Science2635-2646engWiley-BlackwellFebs Journalhttp://olabout.wiley.com/WileyCDA/Section/id-406071.htmlinfo:eu-repo/semantics/openAccesscathepsin GchymaseFRET substratekineticsmast cellserine proteaseDiscriminating between the activities of human cathepsin G and chymase using fluorogenic substratesinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlereponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP11600/339032022-06-02 09:27:19.18metadata only accessoai:repositorio.unifesp.br:11600/33903Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652023-05-25T12:15:32.600050Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates |
title |
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates |
spellingShingle |
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates Korkmaz, Brice cathepsin G chymase FRET substrate kinetics mast cell serine protease |
title_short |
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates |
title_full |
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates |
title_fullStr |
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates |
title_full_unstemmed |
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates |
title_sort |
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates |
author |
Korkmaz, Brice |
author_facet |
Korkmaz, Brice Jegot, Gwenhael Lau, Laurie C. Thorpe, Michael Pitois, Elodie Juliano, Luiz [UNIFESP] Walls, Andrew F. Hellman, Lars Gauthier, Francis |
author_role |
author |
author2 |
Jegot, Gwenhael Lau, Laurie C. Thorpe, Michael Pitois, Elodie Juliano, Luiz [UNIFESP] Walls, Andrew F. Hellman, Lars Gauthier, Francis |
author2_role |
author author author author author author author author |
dc.contributor.institution.none.fl_str_mv |
Univ Tours Southampton Gen Hosp Uppsala Univ Universidade Federal de São Paulo (UNIFESP) |
dc.contributor.author.fl_str_mv |
Korkmaz, Brice Jegot, Gwenhael Lau, Laurie C. Thorpe, Michael Pitois, Elodie Juliano, Luiz [UNIFESP] Walls, Andrew F. Hellman, Lars Gauthier, Francis |
dc.subject.eng.fl_str_mv |
cathepsin G chymase FRET substrate kinetics mast cell serine protease |
topic |
cathepsin G chymase FRET substrate kinetics mast cell serine protease |
description |
Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. in addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. the Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by beta 2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and beta 2-tryptase. the resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [ where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1).s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma. |
publishDate |
2011 |
dc.date.issued.fl_str_mv |
2011-08-01 |
dc.date.accessioned.fl_str_mv |
2016-01-24T14:17:01Z |
dc.date.available.fl_str_mv |
2016-01-24T14:17:01Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
Febs Journal. Malden: Wiley-Blackwell, v. 278, n. 15, p. 2635-2646, 2011. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/33903 http://dx.doi.org/10.1111/j.1742-4658.2011.08189.x |
dc.identifier.issn.none.fl_str_mv |
1742-464X |
dc.identifier.doi.none.fl_str_mv |
10.1111/j.1742-4658.2011.08189.x |
dc.identifier.wos.none.fl_str_mv |
WOS:000292933300003 |
identifier_str_mv |
Febs Journal. Malden: Wiley-Blackwell, v. 278, n. 15, p. 2635-2646, 2011. 1742-464X 10.1111/j.1742-4658.2011.08189.x WOS:000292933300003 |
url |
http://repositorio.unifesp.br/handle/11600/33903 http://dx.doi.org/10.1111/j.1742-4658.2011.08189.x |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.none.fl_str_mv |
Febs Journal |
dc.rights.driver.fl_str_mv |
http://olabout.wiley.com/WileyCDA/Section/id-406071.html info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
http://olabout.wiley.com/WileyCDA/Section/id-406071.html |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
2635-2646 |
dc.publisher.none.fl_str_mv |
Wiley-Blackwell |
publisher.none.fl_str_mv |
Wiley-Blackwell |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
instname_str |
Universidade Federal de São Paulo (UNIFESP) |
instacron_str |
UNIFESP |
institution |
UNIFESP |
reponame_str |
Repositório Institucional da UNIFESP |
collection |
Repositório Institucional da UNIFESP |
repository.name.fl_str_mv |
Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP) |
repository.mail.fl_str_mv |
|
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1783460268738609152 |