caa a22 4500 39755589X CHVBK 20180308164802.0 cr unu---uuuuu 161202e199601 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021197 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021197 Biochemical Properties of the Monomeric Mutant of Human Cathepsin E Expressed in Chinese Hamster Ovary Cells: Comparison with Dimeric Forms of the Natural and Recombinant Cathepsin E [Elektronische Daten] [Takayuki Tsukuba, Hideaki Sakai, Masayuki Yamada, Hidefumi Maeda, Hitoshi Hori, Takeshi Azuma, Akifumi Akamine, Kenji Yamamoto] Cathepsin E (CE) is the only known aspartic proteinase that exists as a homodimer consisting of two fully catalytically active monomers, which are covalently bound by a disulfide bond between two cysteine residues at the NH2-terminal region (Cyg43 in human pro-CE). To understand the physiological significance of the dimer formation, the monomeric mutant of human CE was constructed by site-directed mutagenesis (Cys43→Ser43) and expressed in Chinese hamster ovary (CHO) cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The cellular localization of the monomeric protein was compatible with that of the wild-type (dimeric form) of recombinant human CE expressed in the same cells. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannose-type oligosaccharide chain in the cells. In addition to the maximal activation at around pH 3.5, a substantial proportion of the monomeric pro-CE was converted to the mature form by incubation at pH 7 and 37°C for 5 min. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH 7. Although catalytic properties of the in vitro-activated monomeric CE appeared to be indistinguishable from those of the dimeric forms of natural and recombinant CE, the monomeric form was more unstable to pH and temperature changes than these dimeric forms. These results indicate that the dimerization of CE is not necessarily required for proper folding to express activity, correct intracellular localization and carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo. © by the Journal of Biochemistry Regular Papers nationallicence aspartic proteinase nationallicence cathepsin D nationallicence cathepsin E nationallicence monomer mutant nationallicence structural stabilization nationallicence Tsukuba Takayuki Departments of Pharmacology Kyushu University Faculty of Dentistry Higashi-ku, Fukuoka 812 aut Sakai Hideaki Department of Pharmacology, Nagasaki University School of Dentistry Nagasaki 852 aut Yamada Masayuki Biotech-nology Research Laboratory, Tosoh Co. Kanagawa 252 aut Maeda Hidefumi Conservative Dentistry II, Kyushu University Faculty of Dentistry Higashi-ku, Fukuoka 812 aut Hori Hitoshi Biotech-nology Research Laboratory, Tosoh Co. Kanagawa 252 aut Azuma Takeshi Second Department of Internal Medicine, Fukui Medical School Fukui 910-11 aut Akamine Akifumi Conservative Dentistry II, Kyushu University Faculty of Dentistry Higashi-ku, Fukuoka 812 aut Yamamoto Kenji Departments of Pharmacology Kyushu University Faculty of Dentistry Higashi-ku, Fukuoka 812 aut The Journal of Biochemistry Oxford University Press 119/1(1996-01), 126-134 0021-924X 119:1<126 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021197 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021197 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Tsukuba Takayuki Departments of Pharmacology Kyushu University Faculty of Dentistry Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Sakai Hideaki Department of Pharmacology, Nagasaki University School of Dentistry Nagasaki 852 aut NATIONALLICENCE 700 1- Yamada Masayuki Biotech-nology Research Laboratory, Tosoh Co. Kanagawa 252 aut NATIONALLICENCE 700 1- Maeda Hidefumi Conservative Dentistry II, Kyushu University Faculty of Dentistry Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Hori Hitoshi Biotech-nology Research Laboratory, Tosoh Co. Kanagawa 252 aut NATIONALLICENCE 700 1- Azuma Takeshi Second Department of Internal Medicine, Fukui Medical School Fukui 910-11 aut NATIONALLICENCE 700 1- Akamine Akifumi Conservative Dentistry II, Kyushu University Faculty of Dentistry Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Yamamoto Kenji Departments of Pharmacology Kyushu University Faculty of Dentistry Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/1(1996-01), 126-134 0021-924X 119:1<126 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford