Isolation and biochemical characterization of procathepsin E from human erythrocyte membranes.
Cathepsin E (CE) is an intracellular, nonlysosomal aspartic proteinase which consists of two identical subunits with a molecular weight of about 42 kDa (K. Yamamoto, M. Takeda, H. Yamamoto, M. Tatsumi, and Y. Kato, 1985, J. Biochem. 97, 821-830). In order to clarify its nature and proteolytic activation, the pro-CE and the mature enzyme were simultaneously purified from human erythrocyte membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reduced proenzyme showed a single protein band, exhibiting an apparent molecular weight of 46 kDa. The proenzyme was found to be catalytically inactive, but it was rapidly converted into the active enzyme after brief acid treatment at pH 4.0, which was accompanied by a reduction in molecular size to 43 kDa. The activated form of pro-CE was essentially identical with the mature enzyme in enzymatic properties such as specific activities toward synthetic and protein substrates, and susceptibilities to various protease inhibitors. The N-terminal amino acid sequence analysis revealed that pro-CE started with the third amino acid residue, Ser3, of the sequence of human gastric CE predicted from its cDNA sequence and that the autocatalytic cleavage occurred at the Met36-Ile37 and Phe39-Thr40 bonds to produce two mature isozymic forms. The mature enzyme purified from human erythrocyte membranes also showed two different N-terminal sequences identical with those of the in vitro-activated form of pro-CE. The proenzyme, as well as the mature enzyme from human and rat erythrocyte membranes, was shown to be as an endo-beta-N-acetylglucosaminidase H-resistant form, whereas CE from rat spleen was N-glycosylated with a high-mannose-type oligosaccharide chain, suggesting that the carbohydrate modification of this protein varies with the cell type or the cellular localization. These data also suggest that the proenzyme in human erythrocytes is processed from the high-mannose type to the complex/hybrid type during its biosynthesis at early stages of erythroid differentiation, which precedes the proteolytic activation.
Takeda-Ezaki
M
M
Department of Pharmacology, Kyushu University Faculty of Dentistry, Fukuoka, Japan.
Yamamoto
K
K
D000143:Acids / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002364:Caseins / Q000378:metabolism; D020059:Cathepsin E; D002403:Cathepsins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006454:Hemoglobins / Q000378:metabolism; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D000143:Acids; D002364:Caseins; D004792:Enzyme Precursors; D006454:Hemoglobins; D009842:Oligopeptides; D000255:Adenosine Triphosphate; D006596:Hexosaminidases; D002403:Cathepsins; C067581:procathepsin E; D020059:Cathepsin E