Functional analysis of the C-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a "shuttle" peptide to help secretion.
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, Asn-319 and Asn-327, did not affect the secretion.
Muto
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T
Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236, Japan.
Feese
M D
MD
Shimada
Y
Y
Kudou
Y
Y
Okamoto
T
T
Ozawa
T
T
Tahara
T
T
Ohashi
H
H
Ogami
K
K
Kato
T
T
Miyazaki
H
H
Kuroki
R
R
D000818:Animals; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D004797:Enzyme-Linked Immunosorbent Assay; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013926:Thrombopoietin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D011994:Recombinant Proteins; D005973:Glutamine; D001216:Asparagine; D013926:Thrombopoietin