Enhancement of polylysine-mediated transferrinfection by nuclear localization sequences: Polylysine does not function as a nuclear localization sequence
Enhancement of polylysine-mediated transferrinfection by nuclear localization sequences: Polylysine does not function as a nuclear localization sequence
复制标题
DOI：
10.1089/10430349950017699
复制
发表时间：
1999-07-01
影响因子：
4.2
通讯作者：
Jans, DA
中科院分区：
文献类型：
--
作者：
Chan, CK;Jans, DA
多聚赖氨酸（pLy）已成功用作受体介导的基因转移中的DNA载体，其转染增强作用被认为部分是由于pLy与猿猴病毒SV40大肿瘤抗原（T - ag）的核定位序列（NLS）相似，从而实现了有效的核靶向。在本研究中，我们测试了携带共价连接包含T - ag NLS的肽段的pLy（pLyP101衍生物）是否能够增强转铁蛋白 - pLy介导的转染（“转铁蛋白感染”）。与pLy本身或携带交联的T - ag NLS突变肽段的pLy衍生物（pLyP101T）不同，pLyP101显著增强了表达β - 半乳糖苷酶的报告质粒的转铁蛋白感染。其原因是pLyP101 - 质粒DNA复合物能够被NLS结合的输入蛋白亚基高亲和力识别，而pLyP101T - 和pLy - 质粒复合物则不能。共聚焦激光扫描显微镜用于确定在复合质粒存在的情况下荧光标记的pLyP101和pLyP101T的核输入动力学，结果表明pLyP101复合物而不是pLyP101T复合物在细胞核中迅速积累。我们得出结论，pLy本身不具有NLS的功能，添加能够与细胞核输入机制相互作用的外源性NLS可以通过增强pLy - DNA复合物的核靶向作用来提高转铁蛋白感染。
Polylysine (pLy) has been used successfully as a DNA carrier in receptor-mediated gene transfer, enhancement of transfection having been proposed to be in part through efficient nuclear targeting stemming from the resemblance of ply to the nuclear localization sequence (NLS) from simian virus SV40 large tumor antigen (T-ag), In this study we test whether ply carrying covalently attached peptides comprising the T-ag NLS (the pLyP101 derivative) can enhance transferrin-ply-mediated transfection ("transferrinfection"). Unlike ply itself or a ply derivative (pLyP101T) carrying cross-linked T-ag NLS mutant peptides, pLyP101 significantly enhanced transferrinfection of a beta-galactosidase-expressing reporter plasmid. The basis of this was shown to be the ability of the pLyP101-plasmid DNA complex to be recognized with high affinity by the NLS-binding importin subunits, in contrast to pLyP101T- and ply-plasmid complexes. Confocal laser scanning microscopy was used to determine the nuclear import kinetics of fluorescently labeled pLyP101 and pLyP101T in the presence of complexed plasmid, indicating that pLyP101 and not pLyP101T complexes accumulated rapidly in the nucleus. We conclude that ply itself does not function as an NLS and that the addition of exogenous NLSs conferring interaction with the cellular nuclear import machinery can increase transferrinfection by enhancing the nuclear targeting of pLy-DNA complexes.