Protein characteristics of thrombopoietin.
Thrombopoietin (TPO) was purified from irradiated thrombocytopenic rat plasma. In the process of purification, some biochemical and biological characteristics were investigated. Rat plasma TPO was extremely hydrophobic and exhibited multiple peaks of activity on gel filtration. Both the low and high molecular weight fractions were separately subjected to further purification. Consequently, a rat TPO cDNA was cloned based on the amino acid sequences of purified rat plasma TPO. It revealed that each final purified rat plasma TPO was not a full-length form. In addition, rat hepatocytes and three rat hepatoma cell lines were found to produce rat TPO. Each native TPO derived from cultured cells was also partially purified, and hepatocyte-derived TPOs were shown to be heterogeneous in molecular weight. To study the structure of TPO, various recombinant TPO molecules were generated. Two disulfide bonds (Cys7-Cys151 and Cys29-Cys85) located in the N-terminal domain of TPO have an important effect on its biological activity. The human TPO muteins, sequentially deleted from the C-terminal, were expressed in COS-1 cells. TPO (1-151) was active, but TPO (1-150), which lacks Cys151, did not exhibit TPO activity. These findings indicate that the region essential for TPO activity is the N-terminal domain, which contains two disulfide bonds. Although the role(s) of the C-terminal domain is not clear at present, the potential N-glycosylation in the C-terminal domain is not directly required for exhibiting TPO activity.
Kato
T
T
Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Maebashi, Gunma, Japan.
D000818:Animals; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D051379:Mice; D009154:Mutation; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013926:Thrombopoietin / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004220:Disulfides; D011994:Recombinant Proteins; D013926:Thrombopoietin