title: Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells.
abstract: The cDNA sequence encoding precursor forms of human cathepsin E (CE), an intracellular aspartic proteinase, was expressed in Chinese hamster ovary cells using an SV40 promotor-driven expression vector. By immunoelectron microscopic studies using an anti-human CE antibody and by Percoll density gradient fractionation, the expressed CE was found to be in two different intracellular fractions; the cytosolic compartment and the vacuolar system. The CEs in both the cytosolic and the vacuolar fractions were highly purified by a simple method involving Percoll density gradient fractionation, chromatography on concanavalin A-Sepharose, Mono Q, and TSK-GelG2000SW, and termed s-CE and v-CE, respectively. The v-CE was further separated into a major (v-CE1) and a minor (v-CE2) form by Mono Q chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the s-CE and v-CE1 consists of two polypeptides of 90 and 84 kDa, whereas v-CE2 is composed of 84- and 82-kDa polypeptides. The NH2-terminal amino acid sequence analyses showed that the 90- and 84-kDa proteins from both s-CE and v-CE started with Ser3 and Lys30 of the sequence of human gastric CE predicted from its cDNA sequence, respectively, and that the NH2 terminus of the 82-kDa protein of v-CE2 is the Ile37. Upon acid treatment at pH 3.5 and 37 degrees C for 5 min, the 90- and 84-kDa forms are rapidly converted to the 82-kDa form, indicating that the 90-, 84- and 82-kDa proteins are the pro-CE, the intermediate form, and the mature CE, respectively. All the forms of CE are N-glycosylated with high-mannose-type oligosaccharides. The catalytic properties of s-CE and v-CE are comparable to those of natural human CE. These results suggest that the recombinant CE is initially synthesized on membrane-bound ribosomes as a N-glycosylated preproenzyme and that, after cleavage of the signal segment, the 90-kDa proenzyme is proteolytically processed to the intermediate (84 kDa) and mature (82 kDa) forms by the transport system.
authors: Department of Pharmacology, Kyushu University Faculty of Dentistry, Fukuoka.
authors: T
authors: T
authors: Tsukuba
authors: H
authors: H
authors: Hori
authors: T
authors: T
authors: Azuma
authors: T
authors: T
authors: Takahashi
authors: R T
authors: RT
authors: Taggart
authors: A
authors: A
authors: Akamine
authors: M
authors: M
authors: Ezaki
authors: H
authors: H
authors: Nakanishi
authors: H
authors: H
authors: Sakai
authors: K
authors: K
authors: Yamamoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D020059:Cathepsin E; D002403:Cathepsins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D002403:Cathepsins; D020059:Cathepsin E