Functional analysis of the c-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a 'shuttle' peptide to help secretion

Takanori Muto, Michael D. Feese, Yoshihiro Shimada, Youko Kudou, Tomoyuki Okamoto, Tadashi Ozawa, Tomoyuki Tahara, Hideya Ohashi, Kinya Ogami, Takashi Kato, Hiroshi Miyazaki, Ryota Kuroki

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO has a unique C-terminal tail peptide of about 160 amino acids that consists mostly of hydrophilic residues and contains six N-linked sugar chains. In order to investigate the biological function of the C-terminal domain, two series of mutations were performed. One is systematic truncation from the C terminus. Another is elimination of N-glycosylation sites in the C-terminal domain by Asn to Gln mutations. After the mutant proteins were expressed by mammalian cells, it was found that the elimination of the N. linked sugar sites did not affect the biological activity, whereas truncation of the C-terminal domain resulted in elevation of in vitro activity up to 4-fold. The C-terminal peptide itself was found to inhibit the in vitro activity. Moreover, both the C-terminal truncation and the elimination of the N-glycosylation sites decreased the secretion level progressively down to 1/10 that of wild type, and the amount of the mutant left in the cell increased. The N-glycosylation in the C- terminal region was found to be important for secretion of TPO. Among six N- glycosylation sites in the C-terminal region, two locations, Asn-213 and Asn- 234, were found to be critical for secretion, and two other locations, Ash- 319 and Ash-327, did not affect the secretion.

Original languageEnglish
Pages (from-to)12090-12094
Number of pages5
JournalJournal of Biological Chemistry
Volume275
Issue number16
DOIs
Publication statusPublished - 2000 Apr 21
Externally publishedYes

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Glycosylation
Thrombopoietin
Functional analysis
Ashes
Thrombopoiesis
Peptides
Sugars
Mutation
Mutant Proteins
Bioactivity
Tail
Cells
Cytokines
Amino Acids
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional analysis of the c-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a 'shuttle' peptide to help secretion. / Muto, Takanori; Feese, Michael D.; Shimada, Yoshihiro; Kudou, Youko; Okamoto, Tomoyuki; Ozawa, Tadashi; Tahara, Tomoyuki; Ohashi, Hideya; Ogami, Kinya; Kato, Takashi; Miyazaki, Hiroshi; Kuroki, Ryota.

In: Journal of Biological Chemistry, Vol. 275, No. 16, 21.04.2000, p. 12090-12094.

Research output: Contribution to journalArticle

Muto, T, Feese, MD, Shimada, Y, Kudou, Y, Okamoto, T, Ozawa, T, Tahara, T, Ohashi, H, Ogami, K, Kato, T, Miyazaki, H & Kuroki, R 2000, 'Functional analysis of the c-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a 'shuttle' peptide to help secretion', Journal of Biological Chemistry, vol. 275, no. 16, pp. 12090-12094. https://doi.org/10.1074/jbc.275.16.12090
Muto, Takanori ; Feese, Michael D. ; Shimada, Yoshihiro ; Kudou, Youko ; Okamoto, Tomoyuki ; Ozawa, Tadashi ; Tahara, Tomoyuki ; Ohashi, Hideya ; Ogami, Kinya ; Kato, Takashi ; Miyazaki, Hiroshi ; Kuroki, Ryota. / Functional analysis of the c-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a 'shuttle' peptide to help secretion. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 16. pp. 12090-12094.
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