Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates

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dc.contributor.authorKorkmaz, Brice
dc.contributor.authorJegot, Gwenhael
dc.contributor.authorLau, Laurie C.
dc.contributor.authorThorpe, Michael
dc.contributor.authorPitois, Elodie
dc.contributor.authorJuliano, Luiz [UNIFESP]
dc.contributor.authorWalls, Andrew F.
dc.contributor.authorHellman, Lars
dc.contributor.authorGauthier, Francis
dc.contributor.institutionUniv Tours
dc.contributor.institutionSouthampton Gen Hosp
dc.contributor.institutionUppsala Univ
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.date.accessioned2016-01-24T14:17:01Z
dc.date.available2016-01-24T14:17:01Z
dc.date.issued2011-08-01
dc.description.abstractCathepsin 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.en
dc.description.affiliationUniv Tours, INSERM, Unite Proteases & Vectorisat Pulm U618, F-37032 Tours, France
dc.description.affiliationSouthampton Gen Hosp, Sir Henry Wellcome Labs, Immunopharmacol Grp, Southampton, Hants, England
dc.description.affiliationUppsala Univ, Biomed Ctr, Dept Cell & Mol Biol, Uppsala, Sweden
dc.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, Brazil
dc.description.sourceWeb of Science
dc.description.sponsorshipRegion Centre
dc.description.sponsorshipFonds Europeen de Developpement Regional
dc.description.sponsorshipAgence Nationale pour la Recherche
dc.description.sponsorshipIDAgence Nationale pour la Recherche: ANR-07-PHYSIO-029-01
dc.format.extent2635-2646
dc.identifierhttp://dx.doi.org/10.1111/j.1742-4658.2011.08189.x
dc.identifier.citationFebs Journal. Malden: Wiley-Blackwell, v. 278, n. 15, p. 2635-2646, 2011.
dc.identifier.doi10.1111/j.1742-4658.2011.08189.x
dc.identifier.issn1742-464X
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/33903
dc.identifier.wosWOS:000292933300003
dc.language.isoeng
dc.publisherWiley-Blackwell
dc.relation.ispartofFebs Journal
dc.rightsinfo:eu-repo/semantics/openAccess
dc.rights.licensehttp://olabout.wiley.com/WileyCDA/Section/id-406071.html
dc.subjectcathepsin Gen
dc.subjectchymaseen
dc.subjectFRET substrateen
dc.subjectkineticsen
dc.subjectmast cellen
dc.subjectserine proteaseen
dc.titleDiscriminating between the activities of human cathepsin G and chymase using fluorogenic substratesen
dc.typeinfo:eu-repo/semantics/article
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