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

• Published in: The Journal of biological chemistry 2000-04, Vol.275 (16), p.12090
• Main Authors: Muto, T, Feese, M D, Shimada, Y, Kudou, Y, Okamoto, T, Ozawa, T, Tahara, T, Ohashi, H, Ogami, K, Kato, T, Miyazaki, H, Kuroki, R
• Format: Article
• Language: English
• Subjects: Animals; Asparagine - chemistry; Asparagine - genetics; Blotting, Western; COS Cells; Enzyme-Linked Immunosorbent Assay; Glutamine - chemistry; Glutamine - genetics; Glycosylation; Humans; Mutagenesis, Site-Directed; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Structure-Activity Relationship; Thrombopoietin - chemistry; Thrombopoietin - genetics; Thrombopoietin - physiology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.275.16.12090

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.