Wada T, Nagata Y, Nagahisa H, Okutomi K, Ha S H, Ohnuki T, Kanaya T, Matsumura M, Todokoro K
Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan.
Biochem Biophys Res Commun. 1995 Aug 24;213(3):1091-8. doi: 10.1006/bbrc.1995.2239.
Thrombopoietin (Tpo) is a specific cytokine which regulates megakaryocyte differentiation and maturation. We isolated a truncated mouse Tpo cDNA, the product of which turned out to function neither as an active Tpo variant nor as an antagonist. To define the functional domains of the Tpo molecule further, various truncated and point-mutated Tpo molecules were prepared and their biological activity was assayed. It was found that deletion of the amino terminal side of a potential proteolytic cleavage site, Arg-Arg motif, caused complete loss of Tpo's activity, and that point-mutants lacking one of four conserved cysteine residues lost Tpo activity. We also noticed that Tpo activity was inhibited by the reducing agent. Thus, it was concluded that the amino terminal half of the Tpo is sufficient for Tpo activity, and that the cysteine residues, especially the last cysteine residue located two amino acids away from the Arg-Arg motif, are critical for this activity.
血小板生成素（Tpo）是一种调节巨核细胞分化和成熟的特异性细胞因子。我们分离出了截短的小鼠Tpo cDNA，结果表明其产物既不具有活性Tpo变体的功能，也不具有拮抗剂的功能。为了进一步确定Tpo分子的功能结构域，我们制备了各种截短和点突变的Tpo分子，并检测了它们的生物学活性。结果发现，潜在蛋白水解切割位点Arg-Arg模体的氨基末端缺失会导致Tpo活性完全丧失，而缺少四个保守半胱氨酸残基之一的点突变体也会丧失Tpo活性。我们还注意到还原剂会抑制Tpo活性。因此，可以得出结论，Tpo的氨基末端一半对于Tpo活性就足够了，并且半胱氨酸残基，尤其是距离Arg-Arg模体两个氨基酸的最后一个半胱氨酸残基，对于这种活性至关重要。
