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Processing properties of recombinant human procathepsin L
1Molecular Pharmacology Laboratory, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka, Japan.
Abstract:
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.
Insights
Human cathepsin L processing requires an acidic environment and involves an intramolecular mechanism. Researchers mutated a key cysteine residue (Cys138) to serine, preventing enzyme maturation and confirming its active site role.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Human procathepsin L is expressed in mouse myeloma cells and requires acidic conditions (pH < 5.5) for processing into mature cathepsin L.
- Mature cathepsin L exhibits stability at neutral pH, unlike its precursor form.
Purpose of the Study:
- To investigate the intramolecular autoprocessing mechanism of procathepsin L.
- To determine if Cys138 is the active site responsible for procathepsin L processing.
Main Methods:
- Constructed a mutant procathepsin L cDNA (C138S) using PCR-mutagenesis, replacing Cys138 with Ser.
- Expressed the mutant procathepsin L (C138S) in mouse myeloma cells.
- Incubated the mutant C138S with wild-type procathepsin L and mature cathepsin L under acidic conditions.
Main Results:
- Mutant procathepsin L (C138S) was secreted but not processed into the mature form, even under acidic conditions.
- The C138S mutant was not processed by wild-type procathepsin L or mature cathepsin L.
- Cys138 is essential for the active site function of cathepsin L.
Conclusions:
- Cys138 serves as the active site for cathepsin L.
- Procathepsin L undergoes intramolecular autocatalytic processing, dependent on the Cys138 active site.