Processing properties of recombinant human procathepsin L.
Human procathepsin L is highly expressed in mouse myeloma cells and processed into the mature enzyme under the acidic condition below pH 5.5. Different from the mature enzyme, it is stable at a neutral pH. To examine whether or not procathepsin L is autoprocessed intramolecularly, we constructed a mutant procathepsin L cDNA in which the codon for Cys138 proposed as the active site was mutated to encode Ser by PCR-mutagenesis. The mutant procathepsin L (C138S) was secreted into the culture medium from mouse myeloma cells expressing this mutant cDNA, but not processed into the mature form under the acidic condition. In addition, the mutant C138S was not processed by the incubation at 37 degrees C with wild-type procathepsin L or mature cathepsin L under the acidic condition. These findings showed that Cys138 is the active site of cathepsin L and that the autocatalytic processing occurs intramolecularly.
Nomura
T
T
Molecular Pharmacology Laboratory, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka, Japan.
Fujisawa
Y
Y
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases; D003553:Cystine; D017931:DNA Primers; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D009101:Multiple Myeloma; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D003553:Cystine; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C063905:procathepsin L; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse