Human cathepsin L, a papain-like collagenase without proline specificity.

Several members of the papain-like peptidase family have the ability to degrade collagen molecules by cleaving within the triple helix region of this difficult substrate. A common denominator of these peptidases is their ability to cleave substrates with Pro in the P2 position. In humans, cathepsin K is the best-known papain-like collagenase. Here, we investigate the collagenolytic activity of human cathepsin L, which is closely related to cathepsin K. We show that, despite lacking proline specificity, cathepsin L efficiently cleaves type I collagen within the triple helix region and produces a cleavage pattern similar to that of cathepsin K. We demonstrate that both enzymes have similar affinities for type I collagen and are able to release proteolytic fragments from insoluble collagen. Moreover, cathepsin K is only approximately fourfold more potent than cathepsin L in releasing fragments from reconstituted fibrils of FITC-labeled collagen. Replacing active site residues of cathepsin L with those from cathepsin K introduces cathepsin K-like specificity towards synthetic substrates and increases the collagenolytic activity of cathepsin L. Replacing three residues in the S2 subsite is sufficient to produce a mutant with collagenolytic activity on par with human cathepsin K. These results provide a basis for engineering collagenolytic activity into non-collagenolytic papain-like scaffolds.