Human cathepsin L, a papain‐like collagenase without proline specificity

Abstract

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.

Cathepsin K modulates invasion, migration and adhesion of oral squamous cell carcinomas in vitro

“…Siponen et al () have recently shown by immunohistochemical analyses that CatK is expressed by melanocytes and macrophages, and sporadically in basal keratinocytes and endothelial cells, and that small amounts of CatK also exist extracellularly as well as intracellularly. Given that CatK has powerful collagenolytic activity, it is possible that it is mainly responsible for the degradation of extracellular matrix components such as collagens and elastin (Novinec and Lenarcic, ; Korenc et al , ). Another study showed that the expression levels of CatK in human dermal fibroblasts are upregulated by ultraviolet A radiation, suggesting that CatK may be involved in intracellular elastin degradation and contributes to solar elastosis (Xu et al , ).…”

The catalytic domain of cathepsin C (dipeptidyl-peptidase I) alone is a fully functional endoprotease

“…36 However, CatCΔEx does not cleave intact collagen ( Figure 4D), whereas cathepsin L, which lacks proline specificity does. 28 Therefore, the relation between collagenolytic activity and proline specificity is not straight forward. CatCΔEx has significantly lower kcat values for the hydrolysis of synthetic substrates than other cysteine cathepsins 35 and consequently kcat/Km values are 1 or 2 orders of magnitude lower in comparison to the dipeptidyl-peptidase activity of cathepsin C. 37 Nevertheless, kcat values in this order of magnitude are not unusual for proteolytic enzymes and similar values have recently been determined for the papain-like protease responsible for cell death in the plant N.benthamiana.…”

“…Nevertheless after the prolonged incubation (40 minutes) CatCΔEx was able to completely degrade gelatin into small fragments not observable on the SDS-PAGE (not shown). Native type I collagen, on the other hand, is a tough substrate for proteases that is successfully degraded by cathepsin K and in weaker manner by cathepsin L. 19,28 Samples of native type I collagen incubated with CatCΔEx showed no detectable collagenolytic activity of the protease ( Figure 4D).…”

Can Cysteine Protease Cross-Class Inhibitors Achieve Selectivity?

“…The interplay among human cathepsins in the physiopathology processes is observed for CatL, which plays a role in the collagenolytic activity, despite lacking proline specificity required for this activity. The CatL activity resulted in cleavage of type I collagen within the triple helix region of collagen-like CatK, even though CatL is 4-fold less potent …”