Recny M A, Scoble H A, Kim Y
Genetics Institute, Inc., Cambridge, Massachusetts 02140.
J Biol Chem. 1987 Dec 15;262(35):17156-63.
Recombinant human erythropoietin (rhEPO) has been purified to apparent homogeneity from a Chinese hamster ovary cell line expressing a cDNA clone of the human gene. NH2-terminal sequencing of the recombinant hormone indicates that the 27-residue leader peptide is correctly and consistently cleaved during secretion of the recombinant protein into conditioned medium, yielding the mature NH2 terminus (Ala-Pro-Pro-Arg...). Analysis of the COOH terminus of rhEPO by peptide mapping and fast atom bombardment mass spectrometry (FABMS) demonstrates that the arginyl residue predicted to be at the COOH terminus (based on confirmation of both genomic and cDNA sequences) is completely missing from the purified protein. The truncated form of the recombinant hormone, designated des-Arg166 rhEPO, displays an in vivo specific activity of greater than 200,000 units/mg protein. Structural characterization of natural human urinary EPO (uEPO) by peptide mapping and FABMS reveals that the urinary hormone is also missing the COOH-terminal Arg166 amino acid residue, a modification that remained undetected until now. There is no evidence of further proteolytic processing at the COOH terminus beyond specific removal of the Arg166 amino acid residue in either rhEPO or uEPO. On the basis of the FABMS data, we propose that the physiologically active form of the hormone circulating in plasma and interacting with target cells in vivo is des-Arg166 EPO.
重组人促红细胞生成素（rhEPO）已从表达人基因cDNA克隆的中国仓鼠卵巢细胞系中纯化至表观均一。对重组激素的氨基末端测序表明，在重组蛋白分泌到条件培养基的过程中，27个氨基酸残基的前导肽被正确且一致地切割，产生成熟的氨基末端（丙氨酸-脯氨酸-脯氨酸-精氨酸……）。通过肽图分析和快原子轰击质谱法（FABMS）对rhEPO的羧基末端进行分析表明，预测位于羧基末端的精氨酰残基（基于基因组和cDNA序列的证实）在纯化的蛋白质中完全缺失。重组激素的截短形式，称为去-精氨酸166 rhEPO，其体内比活性大于200,000单位/毫克蛋白质。通过肽图分析和FABMS对天然人尿促红细胞生成素（uEPO）进行结构表征，结果显示尿激素也缺失羧基末端的精氨酸166氨基酸残基，这一修饰直到现在才被发现。在rhEPO或uEPO中，除了特异性去除精氨酸166氨基酸残基外，没有证据表明在羧基末端有进一步的蛋白水解加工。基于FABMS数据，我们提出在血浆中循环并在体内与靶细胞相互作用的激素的生理活性形式是去-精氨酸166 EPO。
