Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells

T. Tsukuba, H. Hori, T. Azuma, T. Takahashi, R. T. Taggart, A. Akamine, M. Ezaki, H. Nakanishi, H. Sakai, K. Yamamoto

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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 °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.

Original languageEnglish
Pages (from-to)7276-7282
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number10
Publication statusPublished - Jan 1 1993

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Cathepsin E
Cricetulus
Ovary
Cells
Fractionation
Chromatography
Complementary DNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tsukuba, T., Hori, H., Azuma, T., Takahashi, T., Taggart, R. T., Akamine, A., ... Yamamoto, K. (1993). Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells. Journal of Biological Chemistry, 268(10), 7276-7282.

Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells. / Tsukuba, T.; Hori, H.; Azuma, T.; Takahashi, T.; Taggart, R. T.; Akamine, A.; Ezaki, M.; Nakanishi, H.; Sakai, H.; Yamamoto, K.

In: Journal of Biological Chemistry, Vol. 268, No. 10, 01.01.1993, p. 7276-7282.

Research output: Contribution to journalArticle

Tsukuba, T, Hori, H, Azuma, T, Takahashi, T, Taggart, RT, Akamine, A, Ezaki, M, Nakanishi, H, Sakai, H & Yamamoto, K 1993, 'Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells', Journal of Biological Chemistry, vol. 268, no. 10, pp. 7276-7282.
Tsukuba T, Hori H, Azuma T, Takahashi T, Taggart RT, Akamine A et al. Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells. Journal of Biological Chemistry. 1993 Jan 1;268(10):7276-7282.
Tsukuba, T. ; Hori, H. ; Azuma, T. ; Takahashi, T. ; Taggart, R. T. ; Akamine, A. ; Ezaki, M. ; Nakanishi, H. ; Sakai, H. ; Yamamoto, K. / Isolation and characterization of recombinant human cathepsin E expressed in Chinese hamster ovary cells. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 10. pp. 7276-7282.
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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 °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.",
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AU - Akamine, A.

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