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

J Biol Chem. 2000 Apr 21;275(16):12090-4. 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.

MeSH terms

  • 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*


  • Recombinant Proteins
  • Glutamine
  • Asparagine
  • Thrombopoietin