Sequential, but not Concurrent, Incubation of Cathepsin K and L with Type I Collagen Results in Extended Proteolysis

Degradation of extracellular matrix (ECM) during tendinopathy is, in part, mediated by the collagenolytic cathepsin K (catK) and cathepsin L (catL), with a temporal component to their activity. The objective of this study was to determine how catK and catL act in concert or in conflict to degrade collagen and tendon ECM during tissue degeneration. To do so, type I collagen gels or ECM extracted from apolipoprotein E deficient mouse Achilles tendons were incubated with catK and catL either concurrently or sequentially, incubating catK first, then catL after a delayed time period. Sequential incubation of catK then catL caused greater degradation of substrates over concurrent incubation, and of either cathepsin alone. Zymography showed there were reduced amounts of active enzymes when co-incubated, indicating that cannibalism, or protease-on-protease degradation between catK and catL was occurring, but incubation with ECM could distract from these interactions. CatK alone was sufficient to quickly degrade tendon ECM, but catL was not, requiring the presence of catK for degradation. Together, these data identify cooperative and conflicting actions of cathepsin mediated collagen matrix degradation by considering interactive effects of multiple proteases during tissue degeneration.