Mechanisms and kinetics of procathepsin D activation

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, wh ich resulted in a catalytically inactive enzyme form (D77A). The other carr ied a point mutation within the propeptide, which prevented activation by p rocessing at the 'autoproteolysis-site' (L26P). Neither mutant is capable o f processing itself to form pseudocathepsin D, and L26P is not able to proc ess D77A. Despite the inability of L26P to cleave either its own or a wild- type prosequence, it did exhibit activity against a synthetic peptide subst rate. The ability of intact precursor (zymogen) to cleave a peptide, but no t a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cle aving zymogen molecules in an intermolecular interaction. In addition, kine tic studies provide evidence for a two-phase mechanism for the conversion o f procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was obser ved that at least two additional proteinase activities, found in conditione d media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there a re likely to be multiple proteinases in the extracellular matrix that are c apable of activating procathepsin D, thereby triggering the second autocata lytic phase. This may also be important for solid tumors, where the presenc e of cathepsin D has been correlated with tumor growth and invasion.