Collagens, which are predominantly located in the extracellular matrix (ECM), are built up of three polypeptide chains containing the repeating triplet sequence Gly-X-Y, where Pro is commonly found in the X position and Hyp (4-hydroxyproline) in the Y position. Collagen degradation is a key step in connective tissue/bone remodeling, angiogenesis, and organ development (Ortega et al. 2003). Fibrillar collagens I and II with their triple helical structural arrangement are highly resistant to general proteolysis and require specific proteinases for their degradation (Prockop and Kivirikko 1995). In contrast, type IV collagen, which is a major component of the basal lamina, is characterized by the presence of nonhelical domains. Among mammalian papain-like enzymes, cathepsin K, but not cathepsin L, possesses unique collagen I- and collagen II-degrading properties by forming a highly collagenolytic complex with bone and cartilage resident chondroitin-4-sulfate (C-4S) (Li et al. 2000, 2002, 2004). Cathepsin K with a double mutation in its S2 binding pocket (Y67L/L205A) lost its ability to accept Pro from the repeating triplet sequence Gly-X-Y at P2, leading to the full abolition of its collagenolytic activity (Lecaille et al. 2002b). Conversely, we assayed the L67Y/A205L cathepsin L mutant to address the effect of the exchange of residues 67 and 205 on its collagenolytic activity. Bovine type I collagen was incubated at 28°C in the presence or absence of C-4S with cathepsins L and K, and with L67Y/A205L cathepsin L (Fig. 3A). Coomassie-stained gels were evaluated by densitometry. As previously shown (Li et al. 2000, 2004), the stability of cathepsin K is dramatically improved in the presence of C-4S, resulting in a significantly increased collagenolytic activity. In contrast, cathepsin L displayed only a weak triple-helical collagenolytic activity that was suppressed after addition of 0.15% (w/v) C-4S, although cathepsin L does not form an active complex with C-4S as cathepsin K does (Li et al. 2004). Remaining amounts of the β11 band, and to a lesser extent β12 and γ bands, were faintly decreased in the presence of L67Y/A205L cathepsin L with respect to its wild-type form (Fig. 3A), according to densitometric analysis (data not shown). However, this minor difference did not reflect a significantly enhanced collagenase activity within the triple helix, but may indicate a small improvement of the overall activity of the cathepsin L mutant. While wild-type cathepsin K, in the presence of C-4S, fully degraded collagen II, as previously reported (Li et al. 2004), both wild-type and mutant cathepsins L did not reveal any proteolytic activities against bovine collagen II from cartilage (Fig. 3B), since no change in the relative amount of the α, β, and γ bands of collagen II was observed. On the other hand, cleavage of gelatin (denatured type I collagen) was similar for both wild-type and the S2 mutant of cathepsin L (Fig. 3C), which is in agreement with previous reports demonstrating that the gelatinolytic activities of cathepsins L and K are comparable (Lecaille et al. 2002b; Li et al. 2004). Elastinolytic and collagenolytic activities of the enzymes were further evaluated using Congo Red-labeled elastin and fluorescein-conjugated collagens. The Y67L/L205A cathepsin K mutant poorly degraded chromogenic elastin-Congo Red and fluorogenic DQ-collagens I and IV, thus comparing to wild-type cathepsin L (Fig. 4). In contrast, the L67Y/A205L cathepsin L mutant hydrolyzed DQ-collagen I and elastin-Congo Red somewhat more efficiently, although this activity remained weak. No significant improvement of its DQ-collagen IV degrading activity was observed. Taken together, present results indicate that the switch of the S2 subsite of cathepsin L into a S2 cathepsin K-like substrate specificity is insufficient to mimic the collagen-degrading properties of cathepsin K. Furthermore, they corroborate the hypothesis that only cathepsin K in conjunction with its complex formation with glycosaminoglycans is a potent collagenase; indeed, cathepsin L does not form a complex with glycosaminoglycans (Li et al. 2004).
Figure 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.
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Figure 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.
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In summary, substitutions of residues 67 and 205 induce an exchange of the respective S2 substrate specificity of cathepsins K and L toward small peptidyl substrates and inhibitors. Nevertheless, the S2 cathepsin K-like (L67Y/A205L) cathepsin L mutant is unable to generate a collagen-degrading activity similar to that of wild-type cathepsin K. The present data support our previous findings that the triple-helical collagenolytic activity of cathepsin K largely depends on its ability to form complexes with glycosaminoglycans (Li et al. 2004).