Preparation of potent cytotoxic ribonucleases by cationization: Enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups
Preparation of potent cytotoxic ribonucleases by cationization: Enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups
复制标题
DOI：
10.1021/bi010248g
复制
发表时间：
2001-06-26
期刊：
影响因子：
2.9
通讯作者：
Yamada, H
中科院分区：
文献类型：
--
作者：
Futami, J;Maeda, T;Yamada, H
通过碳二亚胺反应，分别用乙二胺（将羧酸根阴离子的负电荷转化为正电荷）、2-氨基乙醇（消除负电荷）和牛磺酸（保持负电荷）酰胺化牛核糖核酸酶A的羧基。人RNase 1也用乙二胺修饰。令人惊讶的是，修饰的RNA酶对3 T3-SV-40细胞都具有细胞毒性，尽管它们的核糖核酸溶解活性降低。然而，它们的酶活性并没有完全消除过量的胞质RNA酶抑制剂（RI）的存在下。对于无细胞毒性的天然RNase A和RNase 1，它们被RI完全灭活。更有趣的是，在细胞毒性RNase衍生物中。细胞毒性与净正电荷密切相关。RNase 1和RNase A修饰乙二胺比天然存在的细胞毒性牛精液RNase更具有细胞毒性。使用荧光标记的RNA酶衍生物的实验表明，阳离子越多的RNA酶越有效地吸附到细胞上。因此，羧基修饰可改变RNase与RI的互补性，从而赋予RNase细胞毒性;阳离子化可提高RNase的细胞摄取效率，从而增强其细胞毒性。细胞外的人酶如RNase 1可以通过阳离子化有效地内化到细胞中的发现表明，阳离子化是将蛋白质有效递送到细胞中的简单策略，并且可能开辟新疗法的开发方式。
Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives. cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.