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Related Experiment Videos

Processing properties of recombinant human procathepsin L

T Nomura1, Y Fujisawa

  • 1Molecular Pharmacology Laboratory, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka, Japan.

Biochemical and Biophysical Research Communications
|January 3, 1997
PubMed
Summary

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.

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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.

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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.