Active cathepsins B, H, K, L and S in human inflammatory bronchoalveolar lavage fluids

Abstract

Background information. Chronic inflammation and tissue remodelling result from an imbalance between proteolytic enzymes and their inhibitors in the lungs in favour of proteolysis. While many studies have examined serine proteases (e.g. cathepsin G and neutrophil elastase) and matrix metalloproteases, little is known about the role of papain-like CPs (cysteine proteases). The present study focuses on the thiol-dependent cathepsins (CPs) and their specific cystatin-like inhibitors [CPIs (CP inhibitors)] in human inflammatory BALFs (BAL fluids, where BAL stands for broncho-alveolar lavage).Results. Cathepsins B, K and S found were mostly zymogens, whereas cathepsins H and L were predominantly in their mature forms. Little immunoreactive cystatin C was found and the high-and low-molecular-mass ('weight') kininogens were extensively degraded. The BALF procathepsins B and L could be activated autocatalytically, indicating that alveolar fluid pro-CPs are reservoirs of mature enzymes. Hydrolysis patterns of 7-amino-4-methylcoumarin-derived peptide substrates showed that extracellular alveolar CPs remain proteolytically active, and that cathepsins B and L are the most abundant thiol-dependent endoproteases. The CP/CPI balance was significantly tipped in favour of cathepsins (3-or 5-fold), as confirmed by the extensive CP-dependent degradation of exogenous kininogens by BALFs. Conclusions.Although their importance for inflammation remains to be clarified, the presence of active cathepsins L, K and S suggests that they contribute to the extracellular breakdown of the extracellular matrix.

Regulation of TGF-β1-driven Differentiation of Human Lung Fibroblasts

“…1b). The protein expression outline of the secreted enzymes partly differed from those of bronchoalveolar lavage fluids (BALFs) from patients suffering from sarcoidosis or alveolar proteinosis (32), where mature Cat L was detected, as well from silicosis patients where Cats B and L were mainly observed as processed active forms (37). It also differs from those of sputum of cystic fibrosis patients, in which mature secreted Cat B (mainly as its double-chain form) was intensely revealed (38) as has been shown in emphysema BALFs (39).…”

“…Change in the Cathepsins/Cystatin C Balance during Fibroblast Differentiation-Given that the ratio cathepsins/inhibitors is disturbed in various lung diseases (22,32,37), we examined the balance of Cats and their natural inhibitors during fibroblast differentiation. The expression level of intracellular stefin B did not vary notably during differentiation; similarly, no significant change of the overall endopeptidase activity of Cats was detected in lysates (data not shown).…”

“…The fluorimetric assay (exc, 350 nm; em, 460 nm) was performed in 96-well plates (Nunc A/S, Roskilde, Denmark) with a Gemini spectrofluorimeter (Molecular Devices). Alternatively cathepsin inhibitory potential was determined as previously detailed (32), except that increasing volumes (0 -60 l) of culture media were incubated with E-64 titrated papain (5 nM) in the assay buffer at 30°C for 60 min. The residual papain activity was measured by monitoring the hydrolysis of Z-Phe-Arg-AMC (5 M), and the inhibitory capacity was deduced (33).…”

Cysteine Cathepsins S and L Modulate Anti-angiogenic Activities of Human Endostatin

“…Cells were further incubated with G10T peptide, its scrambled form (Sc-G10T), or a vehicle-only control. Total tubule branching was counted as described under "Experimental Procedures," and results were expressed as the average tube length per field of view (ϫ20 magnification) (16). **, p Ͻ 0.05.…”

“…Furthermore, proteolytic activities of cathepsins were measured at 37°C in both supernatants and cell lysates. Assays were performed in 0.1 M sodium acetate buffer, pH 5.5, 2 mM DTT, 2 mM EDTA, using Z-FR-AMC (50 mM) as a substrate for both cathepsins B and L and Z-LR-AMC (50 mM) as a preferred substrate for cathepsins S and K (Spectramax Gemini, Molecular Devices; excitation wavelength ϭ 350 nm, emission wavelength ϭ 460 nm) (16). Assay for specific cathepsin K activity was achieved by using Z-GPR-AMC (50 M) in the presence of CA-074.…”