Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan.
Biochem Biophys Res Commun. 2012 Feb 24;418(4):736-41. doi: 10.1016/j.bbrc.2012.01.090. Epub 2012 Jan 28.
Human ABCB5, an ATP-binding cassette (ABC) transporter gene, has two major mRNA species. One transcript encodes an 812 amino acid polypeptide, ABCB5β, with a transmembrane domain and a nucleotide-binding domain. We isolated the cDNA of another ABCB5 mRNA that encodes a 1257 amino acid polypeptide. The translated ABCB5 protein is a full-sized ABC transporter that has an internally duplicated structure with two transmembrane domains and two nucleotide-binding domains. The 5' and 3' parts of the ABCB5 mRNA were expressed in the prostate and testis. HEK293 cells transfected with the ABCB5 cDNA expressed a 150-160kDa protein. The ABCB5 transfectants showed approximately 1.5-fold higher resistance to doxorubicin, and 2- to 3-fold higher resistance to paclitaxel and docetaxel. Cellular uptake of radiolabeled paclitaxel and docetaxel in the transfectants was lower than that in the parental HEK293 cells. Treatment of the transfectants with ABCB5-targeted siRNA lowered their resistance to docetaxel. Revertant cells that express a reduced amount of ABCB5 also showed a lowered level of docetaxel resistance. These results indicated that the expression of ABCB5 conferred resistance to taxanes and anthracyclines. Membrane vesicles prepared from ABCB5 baculovirus-infected Sf21 cells showed higher vanadate-sensitive ATPase activity than the Sf21 control vesicles. The k(m) and V(max) values of ATPase activity in the ABCB5 vesicles were 1.8mM and 65nmol/min/mg protein, respectively. ABCB5 ATPase activity was 1.25-fold higher in the presence of 100μM docetaxel than it was in the absence of docetaxel. These results indicates that the full-length ABCB5 protein has ATPase activity that is sensitive to docetaxel.
人 ABCB5，一种 ATP 结合盒（ABC）转运体基因，有两个主要的 mRNA 种。一种转录本编码一个 812 个氨基酸的多肽，ABCB5β，具有跨膜结构域和核苷酸结合结构域。我们分离了另一种 ABCB5 mRNA 的 cDNA，该 cDNA 编码一个 1257 个氨基酸的多肽。翻译的 ABCB5 蛋白是一个全尺寸的 ABC 转运体，具有内部重复结构，具有两个跨膜结构域和两个核苷酸结合结构域。ABCB5 mRNA 的 5'和 3'部分在前列腺和睾丸中表达。用 ABCB5 cDNA 转染的 HEK293 细胞表达了一个 150-160kDa 的蛋白质。ABCB5 转染细胞对阿霉素的耐药性增加了约 1.5 倍，对紫杉醇和多西紫杉醇的耐药性增加了 2-3 倍。转染细胞中放射性标记的紫杉醇和多西紫杉醇的细胞内摄取量低于亲本 HEK293 细胞。用 ABCB5 靶向 siRNA 处理转染细胞降低了它们对多西紫杉醇的耐药性。表达减少量 ABCB5 的回复细胞也表现出降低的多西紫杉醇耐药性。这些结果表明，ABCB5 的表达赋予了对紫杉烷类和蒽环类药物的耐药性。ABCB5 杆状病毒感染 Sf21 细胞制备的膜囊泡显示出比 Sf21 对照囊泡更高的钒酸盐敏感的 ATP 酶活性。ABCB5 囊泡中的 ATP 酶活性的 k(m)和 V(max)值分别为 1.8mM 和 65nmol/min/mg 蛋白。在存在 100μM 多西紫杉醇的情况下，ABCB5 ATP 酶活性比不存在多西紫杉醇时高 1.25 倍。这些结果表明全长 ABCB5 蛋白具有对多西紫杉醇敏感的 ATP 酶活性。
