title: Role of N-glycosylation in cathepsin E. A comparative study of cathepsin E with distinct N-linked oligosaccharides and its nonglycosylated mutant.
abstract: Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N-glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N-glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex-type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high-mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.
authors.affiliation: Department of Pharmacology, Kyushu University Faculty of Dentistry, Fukuoka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Yasuda
authors.forename: S
authors.initials: S
authors.lastname: Ikeda
authors.forename: H
authors.initials: H
authors.lastname: Sakai
authors.forename: T
authors.initials: T
authors.lastname: Tsukuba
authors.forename: K
authors.initials: K
authors.lastname: Okamoto
authors.forename: K
authors.initials: K
authors.lastname: Nishishita
authors.forename: A
authors.initials: A
authors.lastname: Akamine
authors.forename: Y
authors.initials: Y
authors.lastname: Kato
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000581:Amidohydrolases / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002241:Carbohydrates / Q000494:pharmacology; D002384:Catalysis; D020059:Cathepsin E / Q000737:chemistry* / Q000378:metabolism; D056646:Cathepsin F; D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009439:Neuraminidase / Q000494:pharmacology; D009844:Oligosaccharides / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D017434:Protein Structure, Tertiary; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: D002241:Carbohydrates; D037102:Lectins; D009844:Oligosaccharides; D000255:Adenosine Triphosphate; D004247:DNA; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002403:Cathepsins; D056646:Cathepsin F; C534093:Ctsf protein, rat; D016282:Aspartic Acid Endopeptidases; D020059:Cathepsin E; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase