Potency and Selectivity of the Cathepsin L Propeptide as an Inhibitor of Cysteine Proteases

The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cerevisiae using a human procathepsin L/α-factor fusion construct containing a stop codon at position −1 (the C-terminal amino acid of the proregion). Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thr110Ala) expressed in Pichia pastoris, by small amounts of cathepsin L. The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC. The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide). The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with K i values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2. The K i for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 μM of the propeptide. The inhibitory activity was also found to be strongly pH-dependent. Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed K i values of 11 nM and 2900 nM, respectively, against cathepsin L. The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity.