Cathepsin K: a cysteine protease with unique kinin-degrading properties

Abstract

Taking into account a previous report of an unidentified enzyme from macrophages acting as a kininase, the ability of cysteine proteases to degrade kinins has been investigated. Wild-type fibroblast lysates from mice, by contrast with cathepsin K-deficient lysates, hydrolysed BK (bradykinin), and released two metabolites, BK-(1-4) and BK-(5-9). Cathepsin K, but not cathepsins B, H, L and S, cleaved kinins at the Gly4-Phe5 bond and the bradykinin-mimicking substrate Abz (o-aminobenzoic acid)-RPPGFSPFR-3-NO2-Tyr (3-nitrotyrosine) more efficiently (pH 6.0: kcat/K(m)=12500 mM(-1) x s(-1); pH 7.4: kcat/K(m)=6930 mM(-1) x s(-1)) than angiotensin-converting enzyme hydrolysed BK. Conversely Abz-RPPGFSPFR-3-NO2-Tyr was not cleaved by the Y67L (Tyr67-->Leu)/L205A (Leu205-->Ala) cathepsin K mutant, indicating that kinin degradation mostly depends on the S2 substrate specificity. Kininase activity was further evaluated on bronchial smooth muscles. BK, but not its metabolites BK(1-4) and BK(5-9), induced a dose-dependent contraction, which was abolished by Hoe140, a B2-type receptor antagonist. Cathepsin K impaired BK-dependent contraction of normal and chronic hypoxic rats, whereas cathepsins B and L did not. Taking together vasoactive properties of kinins and the potency of cathepsin K to modulate BK-dependent contraction of smooth muscles, the present data support the notion that cathepsin K may act as a kininase, a unique property among mammalian cysteine proteases.

Figure 1. Hydrolysis of BK by cathepsin K

Human cathepsin K was incubated with BK (enzyme/substrate molar ratio 1:2500) at 37 °C, in 0.1 M phosphate buffer, pH 6.0, containing 2 mM DTT and 1 mM EDTA for periods of 0–5 min (black bar). The same procedure was repeated in 0.1 M phosphate buffer, pH 7.4, containing 2 mM DTT and 1 mM EDTA (white bar). Hydrolysis products were further fractionated by reverse-phase HPLC (C₁₈ Brownlee ODS-032 column) (see the Experimental section for details). For the sake of clarity, the residual amount of BK was normalized.

Figure 2. Selective hydrolysis of BK by cathepsin K from skin fibroblasts

Fibroblast lysates from wild-type and cathepsin K-deficient (cat K^−/−) mice (C57BL/6J background) were prepared as described elsewhere [31]. After activation for 2 min in 0.1 M phosphate buffer, pH 6.0, containing 2 mM DTT and 0.01% Brij 35, and in presence of 1 mM EDTA, 0.04 mM pepstatin A and 0.4 mM PMSF, supernatants from fibroblast lysates (5 μg total protein/assay) were incubated with BK (0.16 μM) for 6 h at 30 °C. Native BK and/or its hydrolysis products were separated by reverse-phase HPLC, prior identification by N-terminal sequencing and ESI-Q-TOF MS (see the Experimental section). The major hydrolysis product, BK-(5–9), is identified by an arrow. (A) Wild-type lysates; (B) wild-type lysates+E-64 (10 μM); (C) cat K^−/− lysates; (D) cat K^−/− lysates+E-64 (10 μM). Abbreviation: mUA, milli-units of absorbance at 220 nm.

Figure 3. Effect of BK on bronchial smooth muscles from normal rat

(A) The dose–response curve was obtained by increasing concentrations of BK. After the initial administration of Ach (10⁻⁵ M), BK (10⁻¹² M–10⁻⁵ M) was added as described in the Experimental section. (B) Effect of antagonists on BK-mediated contraction. Rings of bronchial smooth muscles were pretreated in the presence of des-Arg¹⁰-Hoe140 (1 μM), a BK B₁ receptor antagonist (b, n=8), Hoe140 (1 μM), a BK B₂ receptor antagonist (c, n=4) or with both antagonists (d, n=7), prior to the addition of 0.5 μM BK. The responses were compared with the control condition (a, n=19). Antagonists were applied 20 min prior to the addition of BK. Results are means±S.E.M. Contractions are expressed as percentage of the Ach response.

Figure 4. Effect of cathepsins K, B, and L on BK-mediated transient contraction on normal bronchial smooth-muscle rings

Original representative traces showing the effect of cathepsins on BK-dependent contractions are reported: (A) E-64-treated cathepsin K (1 nM); (B) cathepsin K (1 nM); (C) cathepsin B (1 nM); and (D) cathepsin L (1 nM). BK (0.5 μM) was mixed with cathepsins (enzyme/substrate molar ratio 1:500) and immediately added (arrow) to the organ bath solution containing PSS.