Probing cathepsin K activity with a selective substrate spanning its active site

• Autor(a) principal: Lecaille, Fabien
• Data de Publicação: 2003
• Outros Autores: Weidauer, Enrico, Juliano, Maria Aparecida [UNIFESP], Bromme, Dieter, Lalmanach, Gilles
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: http://dx.doi.org/10.1042/BJ20030468; http://repositorio.unifesp.br/handle/11600/27442
• topic: cathepsin; cysteine protease; fluorogenic substrate; trypanosome

The limited availability of highly selective cathepsin substrates seriously impairs studies designed to monitor individual cathepsin activities in biological samples. Among mammalian cysteine proteases, cathepsin K has a unique preference for a proline residue at P2, the primary determinant of its substrate specificity. Interestingly, congopain from Trypanosoma congolense also accommodates a proline residue in its S2 subsite. Analysis of a congopain model showed that amino acids forming its S2 subsite are identical with those of cathepsin K, except Leu(67) which is replaced by a tyrosine residue in cathepsin K. Furthermore, amino acid residues of the congopain S2' binding pocket, which accepts a proline residue, are strictly identical with those of cathepsin K. Abz-HPGGPQ-EDN(2)ph [where Abz represents o-aminobenzoic acid and EDN(2)ph (= EDDnp) represents N-(2,4-dinitrophenyl)ethylenediamine], a substrate initially developed for trypanosomal enzymes, was efficiently cleaved at the Gly-Gly bond by cathepsin K (k(cat)/K-m = 426 000 M-1 . s(-1)). On the other hand, Abz-HPGGPQ-EDN(2)ph was resistant to hydrolysis by cathepsins B, F, H, L, S and V (20 nM enzyme concentration) and the Y67L (Tyr(67) --> Leu)/L205A cathepsin K mutant (20 nM), but still acted as a competitive inhibitor. Taken together, the selectivity of Abz-HPGGPQ-EDN(2)ph to cathepsin K primarily depends on the S2 and S2' subsite specificities of cathepsin K and the ionization state of histidine at P3. Whereas Abz-HPGGPQ-EDN(2)ph was hydrolysed by wild-type mouse fibroblast lysates, its hydrolysis was completely abolished in the cathepsin K-deficient samples, indicating that Abz-HPGGPQ-EDN(2)ph can be used to monitor selectively cathepsin K activity in physiological fluids and cell lysates.