Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates
Lau, Laurie C.
Juliano, Luiz [UNIFESP]
Walls, Andrew F.
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Cathepsin G (CG) (EC 188.8.131.52) and chymase (EC 184.108.40.206) 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 220.127.116.11). 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.
CitationFebs Journal. Malden: Wiley-Blackwell, v. 278, n. 15, p. 2635-2646, 2011.
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