Facchini L M, Lingwood C A
Division of Infection, Immunity, Injury and Repair, Research Institute, University of Toronto, Toronto, Canada.
Exp Cell Res. 2001 Sep 10;269(1):117-29. doi: 10.1006/excr.2001.5297.
Inefficient nuclear incorporation of foreign DNA remains a critical roadblock in the development of effective nonviral gene delivery systems. DNA delivered by traditional protocols remains within endosomal/lysosomal vesicles, or is rapidly degraded in the cytoplasm. Verotoxin I (VT), an AB(5) subunit toxin produced by enterohaemorrhagic Escherichia coli, binds to the cell surface glycolipid, globotriaosylceramide (Gb(3)) and is internalized into preendosomes. VT is then retrograde transported to the Golgi, endoplasmic reticulum (ER), and nucleus of highly VT-sensitive cells. We have utilized this nuclear targeting of VT to design a unique delivery system which transports exogenous DNA via vesicular traffic to the nucleus. The nontoxic VT binding subunit (VTB) was fused to the lambda Cro DNA-binding repressor, generating a 14-kDa VTB-Cro chimera. VTB-Cro binds specifically via the Cro domain to a 25-bp DNA fragment containing the consensus Cro operator. VTB-Cro demonstrates simultaneous specific binding to Gb(3). Treatment of Vero cells with fluorescent-labeled Cro operator DNA in the presence of VTB-Cro, results in DNA internalization to the Golgi, ER, and nucleus, whereas fluorescent DNA alone is incorporated poorly and randomly within the cytoplasm. VTB-Cro mediated nuclear DNA transport is prevented by brefeldin A, consistent with Golgi/ER intracellular routing. Pretreatment with filipin had no effect, indicating that caveoli are not involved. This novel VTB-Cro shuttle protein may find practical applications in the fields of intracellular targeting, gene delivery, and gene therapy.
外源DNA的低效核内掺入仍然是有效非病毒基因递送系统发展的关键障碍。传统方法递送的DNA仍保留在内体/溶酶体囊泡中，或在细胞质中迅速降解。志贺毒素I（VT）是由肠出血性大肠杆菌产生的一种AB(5)亚基毒素，它与细胞表面糖脂三己糖神经酰胺（Gb(3)）结合并内化到前内体中。然后，VT逆行转运至高尔基体、内质网（ER）以及对VT高度敏感细胞的细胞核。我们利用VT的这种核靶向作用设计了一种独特的递送系统，该系统通过囊泡运输将外源DNA转运至细胞核。将无毒的VT结合亚基（VTB）与λ Cro DNA结合阻遏物融合，产生了一个14 kDa的VTB-Cro嵌合体。VTB-Cro通过Cro结构域特异性结合到一个含有共有Cro操纵子的25 bp DNA片段上。VTB-Cro同时还能特异性结合Gb(3)。在VTB-Cro存在的情况下，用荧光标记的Cro操纵子DNA处理Vero细胞，会导致DNA内化到高尔基体、内质网和细胞核中，而单独的荧光DNA在细胞质中的掺入较差且随机。布雷菲德菌素A可阻止VTB-Cro介导的核DNA运输，这与高尔基体/内质网的细胞内路径一致。用制霉菌素预处理没有效果，表明小窝不参与其中。这种新型的VTB-Cro穿梭蛋白可能在细胞内靶向、基因递送和基因治疗领域找到实际应用。
