Hoshi S, Yoshitomi H, Komatsu N, Yoshitake S, Okada M
Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan.
Biochem Biophys Res Commun. 1997 Feb 24;231(3):823-6. doi: 10.1006/bbrc.1997.6133.
Thrombopoietin (TPO) is a hemopoietic cytokine that specifically stimulates the growth and development of megakaryocytes. In addition to full-length TPO (corresponding to TPO-1), cDNAs of three truncated variants (TPO-2 to 4) have been isolated. However, whether or not these variants have biological activities has not been clarified presumably because of poor secretion into extracellular milieu. To examine the biological significance of the shortest variants, TPO-4, we prepared two fusion cDNAs of TPO-1 and TPO-4, each of which was combined with the finger-growth factor-kringle 1 (FGK1) domains of tissue plasminogen activator (tPA) as a tag and transfected them into baby hamster kidney (BHK) cells. An immunodetection assay for FGK1 antigen revealed that the amount of secreted TPO-4-FGK1 fusion protein was about 2% of that of TPO-1-FGK1. These fusion proteins after affinity purification had comparable activities on a molar basis in both megakaryocyte colony stimulating activity towards mouse bone marrow cells and growth promotion activity towards the TPO-responsive cell line UT-7/GM. These results indicate that the TPO-4 is biologically active in vitro, although its primary structure of the C-terminal half after the 111th amino acid residues is completely different from authentic TPO-1 due to frame shift.
血小板生成素（TPO）是一种造血细胞因子，可特异性刺激巨核细胞的生长和发育。除了全长TPO（对应于TPO-1）外，还分离出了三种截短变体（TPO-2至4）的cDNA。然而，这些变体是否具有生物学活性尚未明确，推测原因是其向细胞外环境的分泌较差。为了研究最短变体TPO-4的生物学意义，我们制备了TPO-1和TPO-4的两种融合cDNA，每种都与组织纤溶酶原激活物（tPA）的指状生长因子-kringle 1（FGK1）结构域结合作为标签，并将它们转染到幼仓鼠肾（BHK）细胞中。对FGK1抗原的免疫检测分析表明，分泌的TPO-4-FGK1融合蛋白的量约为TPO-1-FGK1的2%。这些亲和纯化后的融合蛋白在对小鼠骨髓细胞的巨核细胞集落刺激活性和对TPO反应性细胞系UT-7/GM的生长促进活性方面，在摩尔基础上具有相当的活性。这些结果表明，TPO-4在体外具有生物学活性，尽管由于移码，其第111个氨基酸残基之后的C末端一半的一级结构与天然TPO-1完全不同。
