The S2 subsites of cathepsins K and L and their contribution to collagen degradation

Abstract

The exchange of residues 67 and 205 of the S2 pocket of human cysteine cathepsins K and L induces a permutation of their substrate specificity toward fluorogenic peptide substrates. While the cathepsin L-like cathepsin K (Tyr67Leu/Leu205Ala) mutant has a marked preference for Phe, the Leu67Tyr/Ala205Leu cathepsin L variant shows an effective cathepsin K-like preference for Leu and Pro. A similar turnaround of inhibition was observed by using specific inhibitors of cathepsin K [1-(N-Benzyloxycarbonyl-leucyl)-5-(N-Boc-phenylalanyl-leucyl)carbohydrazide] and cathepsin L [N-(4-biphenylacetyl)-S-methylcysteine-(D)-Arg-Phe-beta-phenethylamide]. Molecular modeling studies indicated that mutations alter the character of both S2 and S3 subsites, while docking calculations were consistent with kinetics data. The cathepsin K-like cathepsin L was unable to mimic the collagen-degrading activity of cathepsin K against collagens I and II, DQ-collagens I and IV, and elastin-Congo Red. In summary, double mutations of the S2 pocket of cathepsins K (Y67L/L205A) and L (L67Y/A205L) induce a switch of their enzymatic specificity toward small selective inhibitors and peptidyl substrates, confirming the key role of residues 67 and 205. However, mutations in the S2 subsite pocket of cathepsin L alone without engineering of binding sites to chondroitin sulfate are not sufficient to generate a cathepsin K-like collagenase, emphasizing the pivotal role of the complex formation between glycosaminoglycans and cathepsin K for its unique collagenolytic activity.

Figure 1.

SDS-PAGE (12% Tris-glycine) of the purified Leu67Tyr/Ala205Leu cathepsin L mutant. (Lane 1) Crude supernatant fraction of the human Leu67Tyr/Ala205Leu mutant from P. pastoris; (lane 2) activation of the precursor mutant by treatment with pepsin; (lane 3) purified Leu67Tyr/Ala205Leu mutant after passage through the Butyl-Sepharose column; (lane 4) purified recombinant wild-type human cathepsin L. Molecular mass standards are reported in the left lane (Coomassie-blue staining).

Figure 2.

Binding mode of N-(4-biphenylacetyl)-S-methylcysteine-(D)-Arg-Phe-β-phenethylamide (Cat L inh. 7) with (A) cathepsin L and its Leu67Tyr/Ala205Leu variant, and with (B) cathepsin K and its Tyr67Leu/Leu205Ala variant. Wild-type (green) and mutant (yellow) binding modes are superimposed. (Dashed lines) The shortest distances between the P3 phenylethyl moiety (phenyl ring) and the S3 subsite, and the phenyl ring of Phe at P2 and the S2 subsite, respectively. The inhibitor is represented in stick mode and in ball-and-stick mode for the mutated protein side chains. Other selected nearby protein residues are show as thin lines.

Figure 3.

Collagenolytic and gelatinolytic activities of cathepsin K, cathepsin L, and Leu67Tyr/Ala205Leu cathepsin L mutant. (A) Cathepsins K, L, and L67Y/A205L cathepsin L mutant (600 nM) were incubated with skin collagen I (0.4 mg/mL) in 100 mM sodium acetate buffer (pH 5.5) containing 2 mM dithiothreitol and 2 mM EDTA, for 8 h at 28°C, in the presence or absence of 0.15% (w/v) C-4S. Samples were further analyzed by SDS-polyacrylamide electrophoresis using 4%–20% Tris/glycine gels (Coomassie-blue staining). Undigested bovine type I collagen was used as a standard. Molecular mass standards are indicated in the left lane. (B) Collagenolytic assays with soluble collagen II from articular joints (0.6 mg/mL).(C) Heat-denatured collagen I (gelatin; 0.4 mg/mL) was incubated with cathepsins K, L, and the two S2 mutants (10 nM), respectively, for 30 min at 28°C.

Figure 4.

Hydrolysis of Congo Red-labeled elastin and fluorescein-conjugated collagens. Bovine skin DQ-collagen I, human placenta DQ-collagen IV, and elastin-Congo Red were incubated with cathepsins K, L, and their respective S2 mutants as described in Materials and Methods. Collagenolytic activities were measured by monitoring the fluorescence release (excitation wavelength: 395 nm; emission wavelength: 415 nm). After removal of the uncleaved substrate, the elastinolytic activity was deduced from the absorbance of soluble released dye (λ = 490 nm). Proteolytic activities were expressed as normalized values (%), using wild-type cathepsin K as reference. The results (triplicate assays) are means ± SEM.