Carmona E, Dufour E, Plouffe C, Takebe S, Mason P, Mort J S, Ménard R
Pharmaceutical Biotechnology Sector, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada.
Biochemistry. 1996 Jun 25;35(25):8149-57. doi: 10.1021/bi952736s.
The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cerevisiae using a human procathepsin L/alpha-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/Thrl10Ala) 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 Ki values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2. The Ki 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 microM 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 Ki 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.
使用一种人组织蛋白酶L/α-因子融合构建体在酿酒酵母中表达组织蛋白酶L前肽（phcl-2），该构建体在-1位置（前区域的C末端氨基酸）含有一个终止密码子。由于纯化后的产量非常低，组织蛋白酶L前肽也通过另一种方法获得，即通过少量组织蛋白酶L对毕赤酵母中表达的无活性组织蛋白酶L突变体（Cys25Ser/Thrl10Ala）进行可控加工。然后通过HPLC纯化由酶原（phcl-1）切割产生的肽。通过N端测序和质谱对纯化的前肽进行表征，它们对应于前区域的不完全形式（phcl-1和phcl-2分别为87和81个氨基酸，而完整的组织蛋白酶L前肽为96个氨基酸）。发现这两种肽在pH 5.5时是组织蛋白酶L的有效和选择性抑制剂，phcl-1的Ki值为0.088 nM，phcl-2的Ki值为0.66 nM。抑制组织蛋白酶S的Ki值要高得多（phcl-1为44.6 nM），在高达1 μM的前肽浓度下未检测到对组织蛋白酶B或木瓜蛋白酶的抑制作用。还发现抑制活性强烈依赖于pH。还通过固相合成制备了与前肽N端截短版本相对应的75和44个氨基酸的两种合成肽，它们对组织蛋白酶L的Ki值分别为11 nM和2900 nM。从各种长度的4种前肽衍生物获得的数据表明，前肽N端区域的前20个残基对抑制作用比C端区域更重要，C端区域对整体抑制活性贡献很小。
