Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D.
Procathepsin D is the intracellular aspartyl protease precursor of cathepsin D, a major lysosomal enzyme. Procathepsin D is rapidly processed inside the cell, and, thus, examination of its proteolyic activation and structure has been difficult. To study this proenzyme, a nonglycosylated form of the human fibroblast procathepsin D was expressed in Escherichia coli, refold in vitro, and purified by affinity chromatography on pepstatinyl agarose. Sequence analysis of the refolded, autoactivated enzyme allowed determination of the autoproteolytic cleavage site. The sequence surrounding this cleavage site between residues LeuP26 and IleP27 (in the "pro" region) resembled the first cleavage site found during activation of other aspartyl proteases. Thus, the autoactivated procathepsin D is analogous to the pepsin activation intermediate, which has been termed pseudopepsin. The enzymatic activity, thermal and pH stability, and fluorescence spectra of pseudocathepsin D were compared to mature, predominantly two-chain, cathepsin D isolated from human placenta. The results indicated that pseudocathepsin D and mature enzyme have a similar Km toward a peptide substrate and cleave a protein substrate at identical sites. Temperature stability of the recombinant enzyme was similar to that of the tissue-derived enzyme. However, the recombinant enzyme had increased stability at low pH when compared to the glycosylated tissue-derived two-chain cathepsin D. Fluorescence spectra of the recombinant and tissue-derived enzymes were identical. Thus, the absence of asparagine-linked oligosaccharides and the presence of the remaining segment of propeptide did not significantly alter the structural and enzymatic properties of the enzyme.
Conner
G E
GE
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101.
Richo
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006868:Hydrolysis; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational
D004792:Enzyme Precursors; C058345:procathepsin D; D002402:Cathepsin D