Wen D, Boissel J P, Tracy T E, Gruninger R H, Mulcahy L S, Czelusniak J, Goodman M, Bunn H F
Hematology/Oncology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
Blood. 1993 Sep 1;82(5):1507-16.
To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5' primers, we were able to successfully PCR-amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha-helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.
为了研究促红细胞生成素（Epo）的结构-功能关系，我们获得了编码多种哺乳动物成熟Epo蛋白的cDNA序列。第一组引物对应于小鼠和人类DNA之间的保守核苷酸序列，使我们能够通过聚合酶链反应（PCR）从仓鼠、猫、狮子、狗、马、绵羊、海豚和猪的基因组DNA中扩增内含子1/外显子2片段。对这些片段进行测序后，设计了第二代物种特异性引物。从贫血肾脏中提取RNA并进行逆转录。使用我们一系列物种特异性的5'引物，我们成功地从恒河猴、大鼠、绵羊、狗、猫和猪中PCR扩增出Epo cDNA。这些动物成熟Epo蛋白的推导氨基酸序列，与人类、食蟹猴和小鼠的已知序列相结合，显示出高度的同源性，这解释了在许多物种中观察到的生物学和免疫交叉反应性。人类Epo与猴Epo的同源性为91%，与猫和狗Epo的同源性为85%，与猪、绵羊、小鼠和大鼠Epo 的同源性为80%至82%。（1）连接NH2和COOH末端的二硫键；（2）N-糖基化位点；（3）预测的两亲性α-螺旋完全保守。相比之下，短二硫键（人类中的C29/C33）并非不变。在啮齿动物中，Cys33被脯氨酸取代。大多数氨基酸替换是保守的。C和D螺旋之间环的C末端部分变化最大，有几个氨基酸替换、缺失和/或插入。对内含子1/外显子2序列以及编码序列的最大简约性计算，使得能够构建与已知系统发育关系高度一致的进化树。
