Expression and characterization of cathepsin P

Area of Science:

• Biochemistry
• Molecular Biology
• Evolutionary Biology

Background:

• Rodent genomes possess unique Clan C1A proteases, including cathepsin P, exclusively expressed in the placenta.
• Cathepsin P was hypothesized to perform specialized functions related to cathepsin L in placental tissues.

Purpose of the Study:

• To characterize the biochemical properties and optimal activity conditions of cathepsin P.
• To investigate the evolutionary relationship and functional divergence of cathepsin P compared to cathepsin L.

Main Methods:

• Expression of inactive cathepsin P zymogen in Pichia pastoris.
• Enzymatic activation using various proteases (proteinase K, chymotrypsin, pancreatic elastase) and assessment of autoactivation.
• Determination of enzyme activity against peptide and protein substrates at different pH levels and in the presence of salts.

Main Results:

• Cathepsin P zymogen can be activated by external proteases and undergoes autoactivation at neutral pH.
• Activated cathepsin P exhibits optimal hydrolytic activity at neutral pH (6.5-7.5), with minimal activity at acidic pH.
• Activity is enhanced by salts like Na2SO4 and hyaluronate, and its properties differ significantly from cathepsin L.

Conclusions:

• Cathepsin P possesses unique biochemical properties distinct from cathepsin L, including neutral pH optimum and autoactivation.
• The evolutionary origin of cathepsin P likely resulted in a protease with a novel function, rather than a subfunction of cathepsin L.
• Cathepsin P may have evolved to fulfill a role performed by unrelated proteases in other mammalian species.