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

Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates
Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates
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 ?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 ?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.
cathepsin g, chymase, fret substrate, kinetics, mast cell, serine protease
1742-464X
2635-2646
Korkmaz, Brice
95c84e44-927d-4a5b-abf2-41b38071f124
Jégot, Gwenhael
4397adb7-3a1f-4c4b-be8d-89c7a724e783
Lau, Laurie C.
2af8045d-6162-4939-aba7-28dd2f60f6a8
Thorpe, Michael
3223b31d-e07f-4b4f-a98f-4a911c028bc2
Pitois, Elodie
a42d1a2a-9d0e-4635-8134-59a6fd4ab64a
Juliano, Luiz
c827f070-719e-4664-bf50-b0ef57fffed5
Walls, Andrew F.
aaa7e455-0562-4b4c-94f5-ec29c74b1bfe
Hellman, Lars
6c88ed4c-ec9b-4fe5-abd9-5f8154355e42
Gauthier, Francis
01110cac-1dab-4cd3-bd46-3dc9ada21670
Korkmaz, Brice
95c84e44-927d-4a5b-abf2-41b38071f124
Jégot, Gwenhael
4397adb7-3a1f-4c4b-be8d-89c7a724e783
Lau, Laurie C.
2af8045d-6162-4939-aba7-28dd2f60f6a8
Thorpe, Michael
3223b31d-e07f-4b4f-a98f-4a911c028bc2
Pitois, Elodie
a42d1a2a-9d0e-4635-8134-59a6fd4ab64a
Juliano, Luiz
c827f070-719e-4664-bf50-b0ef57fffed5
Walls, Andrew F.
aaa7e455-0562-4b4c-94f5-ec29c74b1bfe
Hellman, Lars
6c88ed4c-ec9b-4fe5-abd9-5f8154355e42
Gauthier, Francis
01110cac-1dab-4cd3-bd46-3dc9ada21670

Korkmaz, Brice, Jégot, Gwenhael, Lau, Laurie C., Thorpe, Michael, Pitois, Elodie, Juliano, Luiz, Walls, Andrew F., Hellman, Lars and Gauthier, Francis (2011) Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates. Febs Journal, 278 (15), 2635-2646. (doi:10.1111/j.1742-4658.2011.08189.x). (PMID:21599834)

Record type: Article

Abstract

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 ?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 ?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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e-pub ahead of print date: 20 May 2011
Published date: August 2011
Keywords: cathepsin g, chymase, fret substrate, kinetics, mast cell, serine protease
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 195717
URI: http://eprints.soton.ac.uk/id/eprint/195717
ISSN: 1742-464X
PURE UUID: 922d2495-653d-41e1-94da-49277ecf62dd
ORCID for Andrew F. Walls: ORCID iD orcid.org/0000-0003-4803-4595

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Date deposited: 25 Aug 2011 14:05
Last modified: 15 Mar 2024 02:39

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Contributors

Author: Brice Korkmaz
Author: Gwenhael Jégot
Author: Laurie C. Lau
Author: Michael Thorpe
Author: Elodie Pitois
Author: Luiz Juliano
Author: Andrew F. Walls ORCID iD
Author: Lars Hellman
Author: Francis Gauthier

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