Maki N, Sekiguchi F, Nishimaki J, Miwa K, Hayano T, Takahashi N, Suzuki M
Corporate Research and Development Laboratory, Saitama, Japan.
Proc Natl Acad Sci U S A. 1990 Jul;87(14):5440-3. doi: 10.1073/pnas.87.14.5440.
The recently discovered macrolide FK506 has been demonstrated to have potent immunosuppressive activity at concentrations 100-fold lower than cyclosporin A, a cyclic undecapeptide that is used to prevent rejection after transplantation of bone marrow and organs, such as kidney, heart, and liver. After the recent discovery that the cyclosporin A-binding protein cyclophilin is identical to peptidylprolyl cis-trans isomerase, a cellular binding protein for FK506 was found to be distinct from cyclophilin but to have the same enzymatic activity. In this study, we isolated a cDNA coding for FK506-binding protein (FKBP) from human peripheral blood T cells by using mixed 20-mer oligonucleotide probes synthesized on the basis of the sequence, Glu-Asp-Gly-Lys-Lys-Phe-Asp, reported for bovine FKBP. The DNA isolated contained an open reading frame encoding 108 amino acid residues. The first 40 residues of the deduced amino acid sequence were identical to those of the reported amino-terminal sequence of bovine FKBP, indicating that the DNA sequence isolated represents the gene coding for FKBP. Computer-assisted analysis of the deduced amino acid sequence indicates that FKBP exhibits no internal homology and does not have significant sequence similarity to any other amino acid sequences of known proteins, including cyclophilin. This result suggests that two catalytically similar proteins, cyclophilin and FKBP, evolved independently. In Northern blot analysis, mRNA species of approximately 1.8 kilobases that hybridized with human FKBP cDNA were detected in poly(A)+ RNAs from brain, lung, liver, and placental cells and leukocytes. Induction of Jurkat leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did not affect the level of FKBP mRNA. Southern blot analysis of human genomic DNA digested with different restriction enzymes suggests the existence of only a few copies of the DNA sequence encoding FKBP. This is in contrast to the result that as many as 20 copies of the cyclophilin gene and possible pseudogenes may be present in the mammalian genome.
最近发现的大环内酯类药物FK506已被证明在浓度比环孢素A低100倍时就具有强大的免疫抑制活性。环孢素A是一种环状十一肽，用于预防骨髓和器官（如肾脏、心脏和肝脏）移植后的排斥反应。在最近发现环孢素A结合蛋白亲环蛋白与肽基脯氨酰顺反异构酶相同时，人们发现FK506的一种细胞结合蛋白与亲环蛋白不同，但具有相同的酶活性。在本研究中，我们根据报道的牛FKBP序列Glu-Asp-Gly-Lys-Lys-Phe-Asp合成了混合的20聚体寡核苷酸探针，从人外周血T细胞中分离出编码FK506结合蛋白（FKBP）的cDNA。分离得到的DNA包含一个编码108个氨基酸残基的开放阅读框。推导的氨基酸序列的前40个残基与报道的牛FKBP的氨基末端序列相同，表明分离得到的DNA序列代表编码FKBP的基因。对推导的氨基酸序列进行计算机辅助分析表明，FKBP没有内部同源性，与任何已知蛋白质的氨基酸序列（包括亲环蛋白）也没有显著的序列相似性。这一结果表明，两种催化相似的蛋白质，亲环蛋白和FKBP，是独立进化的。在Northern印迹分析中，在来自脑、肺、肝、胎盘细胞和白细胞的聚腺苷酸加尾RNA中检测到与人类FKBP cDNA杂交的约1.8千碱基的mRNA种类。用佛波醇12-肉豆蔻酸酯13-乙酸酯和离子霉素诱导Jurkat白血病T细胞并不影响FKBP mRNA的水平。用不同限制性酶消化的人基因组DNA的Southern印迹分析表明，编码FKBP的DNA序列只有少数几个拷贝。这与哺乳动物基因组中可能存在多达20个亲环蛋白基因和假基因的结果形成对比。
