摘要
目的研究人肝脏特异的有机阴离子转运蛋白基因OATP-C蛋白分子膜外区保守的490位阳离子赖氨酸对其有机阴离子底物摄取能力的影响。方法从人肝脏mRNA中克隆OATP-C野生型基因全长cDNA序列,通过定点突变将高度保守的第490位赖氨酸残基突变为中性氨基酸-苏氨酸,构建其C端的GFP融合蛋白的真核表达载体,转染HEK293细胞,观察突变体的细胞膜定位能力和底物摄取功能的变化。结果OATP-C野生型及突变体均能定位表达于HEK293细胞质膜;[3H]硫酸盐雌酮底物摄取实验显示,突变体5min底物摄取量较野生型下降了38.66%,浓度依赖的[3H]硫酸盐雌酮底物摄取动力学分析表证明,突变体Km和Vmax分别明显增高和降低。结论OATP-C蛋白膜外区第490位赖氨酸是关键的功能氨基酸,是其重要的结构功能单位。
Objective To investigate the alterations in uptake function of liver specific transporter OATP- C （organic anion transporting polypeptide C） by mutating positive charge consensus lysine K490T （Lys to Thr） localized on this polypeptide extracellular loop. Methods Wild type OATP-C full length cDNA was amplified from human liver mRNA by RT-PCR. Mutation of positive charge to neutral amino acid K490T （ Lys to Thr） was made by site-direct mutagenesis technique. The recombinant constructs of wild type and mutated OATP-C cDNA with GFP fusion gene at its C-terminal were generated by subcloning and transfected into HEK293 cells, then cell plasma membrane targeting expression and substrate uptake function were observed. Results Both the wild type and mutated OATP-C proteins were highly expressed on HEK293 cell plasma membrane. The uptake of [ 3H] estrone sulfat substrates of mutated OATP-C markedly reduced in 5 min by 38.66% as compared with wild type. Analysis of concentration-dependent uptake kinetics demonstrated that the mutants possessed a significantly higher Km value and lower Vmax value than those of wild type. Conclusion The conserved positive charge Lys-490 on extracellular loop of OATP-C may be a determinant amino acid in the uptake of organic anion substrates.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2007年第20期1925-1928,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30440015)
重庆市自然科学基金(CSTC-2005BB5043)
第三军医大学回国人员启动基金(XG200552)
西南医院归国人员启动基金(2005)~~
