Potency and Selectivity of the Cathepsin L Propeptide as an Inhibitor of Cysteine Proteases
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- 作者：Euridice Carmona ; É ; ric Dufour ; Cé ; line Plouffe ; Sachiko Takebe ; Patrizia Mason ; John S. Mort ; and Robert Mé ; nard
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：June 25, 1996
- 年：1996
- 卷：35
- 期：25
- 页码：8149 - 8157
- 全文大小：358K
- 年卷期：v.35,no.25(June 25, 1996)
- ISSN：1520-4995
文摘
The cathepsin L propeptide (phcl-2) was expressed inSaccharomyces cerevisiae using a humanprocathepsin L/
-factor fusion construct containing a stop codon atposition -1 (the C-terminal aminoacid of the proregion). Since the yield after purification wasvery low, the cathepsin L propeptide wasalso obtained by an alternate procedure through controlled processingof an inactive mutant of procathepsinL (Cys25Ser/Thr110Ala) expressed in Pichia pastoris, bysmall amounts of cathepsin L. The peptideresulting from the cleavage of the proenzyme (phcl-1) was then purifiedby HPLC. The purified propeptideswere characterized by N-terminal sequencing and mass spectrometry andcorrespond to incomplete formsof the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively,compared to 96 aa for the completecathepsin L propeptide). The two peptides were found to be potentand selective inhibitors of cathepsinL at pH 5.5, with Ki values of 0.088 nM forphcl-1 and 0.66 nM for phcl-2. The Ki forinhibition ofcathepsin S was much higher (44.6 nM with phcl-1), and no inhibition ofcathepsin B or papain could bedetected at up to 1 
M of the propeptide. The inhibitoryactivity was also found to be strongly pH-dependent. Two synthetic peptides of 75 and 44 aa corresponding toN-terminal truncated versions ofthe propeptide were also prepared by solid phase synthesis anddisplayed Ki values of 11 nM and2900nM, respectively, against cathepsin L. The data obtained for the 4propeptide derivatives of variouslengths indicate that the first 20 residues in the N-terminal region ofthe propeptide are more importantfor inhibition than the C-terminal region which contributes little tothe overall inhibitory activity.
-factor fusion construct containing a stop codon atposition -1 (the C-terminal aminoacid of the proregion). Since the yield after purification wasvery low, the cathepsin L propeptide wasalso obtained by an alternate procedure through controlled processingof an inactive mutant of procathepsinL (Cys25Ser/Thr110Ala) expressed in Pichia pastoris, bysmall amounts of cathepsin L. The peptideresulting from the cleavage of the proenzyme (phcl-1) was then purifiedby HPLC. The purified propeptideswere characterized by N-terminal sequencing and mass spectrometry andcorrespond to incomplete formsof the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively,compared to 96 aa for the completecathepsin L propeptide). The two peptides were found to be potentand selective inhibitors of cathepsinL at pH 5.5, with Ki values of 0.088 nM forphcl-1 and 0.66 nM for phcl-2. The Ki forinhibition ofcathepsin S was much higher (44.6 nM with phcl-1), and no inhibition ofcathepsin B or papain could bedetected at up to 1 M of the propeptide. The inhibitoryactivity was also found to be strongly pH-dependent. Two synthetic peptides of 75 and 44 aa corresponding toN-terminal truncated versions ofthe propeptide were also prepared by solid phase synthesis anddisplayed Ki values of 11 nM and2900nM, respectively, against cathepsin L. The data obtained for the 4propeptide derivatives of variouslengths indicate that the first 20 residues in the N-terminal region ofthe propeptide are more importantfor inhibition than the C-terminal region which contributes little tothe overall inhibitory activity.