Preparation of Potent Cytotoxic Ribonucleases by Cationization: Enhanced Cellular Uptake and Decreased Interaction with Ribonuclease Inhibitor by Chemical Modification of Carboxyl Groups
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- 作者：Junichiro Futami ; Takashi Maeda ; Midori Kitazoe ; Emiko Nukui ; Hiroko Tada ; Masaharu Seno ; Megumi Kosaka ; and Hidenori Yamada
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：June 26, 2001
- 年：2001
- 卷：40
- 期：25
- 页码：7518 - 7524
- 全文大小：203K
- 年卷期：v.40,no.25(June 26, 2001)
- ISSN：1520-4995
文摘
Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negativecharges 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 modifiedwith ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despitetheir decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminatedby the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 whichwere not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNasederivatives, cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modifiedwith ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. Anexperiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNaseswere more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groupscould change complementarity of RNase to RI and as a result endow RNase cytotoxicity and thatcationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. Thefinding that an extracellular human enzyme such as RNase 1 could be effectively internalized into thecell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein intocells and may open the way of the development of new therapeutics.