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

摘要: Cathepsin G (CG) (EC 3.4.21.20) and chymase 3.4.21.39) are two closely-related chymotrypsin-like proteases that released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between substrate specificities, aiming better understand respective role during progression inflammatory diseases. In addition preference large aromatic residues at P1, both preferentially accommodate small hydrophilic S1' subsite. Despite significant structural differences in S2' subsite, prefer an acidic residue position. The Ala226/Glu substitution bottom CG S1 pocket, which allows but not a Lys is main difference, allowing discrimination activities these proteases. However, P1 accommodated much less efficiently than Phe, corresponding cleaved by β2-tryptase 3.4.21.59). optimized Lys-containing enhance sensitivity towards prevent cleavage β2-tryptase. resulting (ABZ-GIEPKSDPMPEQ-EDDnp) [where ABZ O-aminobenzoic acid EDDnp N-(2,4-dinitrophenyl)-ethylenediamine] was tryptase, with specificity constant 190 mM(-1)·s(-1). This quantification active or tissue extracts where it may be present together as we have shown using HMC-1 cell homogenate sputum sample patient severe asthma.