Rational design of human cathepsin L with efficient collagenolytic activity

Collagens are the main structural components of ECM and collagenolysis is vital for ECM remodelling, which is essential for normal organ homeostasis. Any dysregulation of this process can result in various pathological conditions. The degradation of the interstitial collagens is generally mediated by specific collagenases which are capable of cleaving at specific loci in the collagen triple helix. Lysosomal cysteine cathepsins have general housekeeping as well as some highly specialized functions. Amongst them, human cathepsin K is the only lysosomal cysteine protease which has potent collagenolytic activity against type I collagen. In this study, we have imparted collagenolytic property to another human cysteine protease, cathepsin L, by systematically engineering proline-specificity and GAG-binding surface in the protease. The designed proline-specific mutant shows high specificity for peptidyl substrate containing proline at P2 position but is incapable of cleaving collagen. However, when the proline-specific mutant is further engineered for GAG-binding surface, it can degrade type I collagen in presence of Chondroitin 4-Sulfate (C4-S). We also present high resolution crystal structures of these proline-specific (1.4 Å) and collagen-specific (1.8 Å) mutants. Finally docking studies with prolyl-peptidic substrate (Ala-Gly-Pro-Arg-Ala) at the active site and a GAG molecule (C4-S) at the GAG binding site enables us to identify key structural features responsible for collagenolytic activity of papain-like cysteine proteases. This study provides a rational approach to engineer an efficient collagenase from a human template enzyme which may have various noninvasive therapeutic implications with an expected additional advantage of immuno-compatiblity.