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, Asn-319 and Asn-327, did not affect the secretion.
Highlights
Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis
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
What is the biological function of this unique C-terminal domain? It was speculated that the enhancement of the circulating half-life might be one function of this domain, as in the case of EPO (10)
Summary
Construction of C-Terminal Truncation or N-Glycosylation-defective Mutants of hTPO—The expression plasmid of wild type TPO, pHTF1, which included the full-length (amino acids 1–332) hTPO cDNA has been described previously (8). RNA was recovered from COS7 cells using Isogen (Nippon Gene, Tokyo) and subjected to reverse transcription, quantitative PCR using TaqMan Gold reverse transcription PCR kit (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. Electrophoresis and Western Blotting—The TPO mutant protein in the supernatant from COS7 cells was purified by immunoprecipitation with rabbit anti-hTPO polyclonal antibody (18) and separated by SDSpolyacrylamide gel electrophoresis with Multigel 10/20 or 15/25 (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) as described by Laemmli (19). To detect the mutant TPO remaining in the COS7 cells, total cellular protein was separated by SDS-polyacrylamide gel electrophoresis. In both cases, mutant TPO was detected by Western blot analysis. After 3 days of incubation, the proliferation of FDCP-hMPL5 cells was measured with the absorbance at 490 nm using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega Corp.)
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