Preparation of potent cytotoxic ribonucleases by cationization: enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups.
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.
Futami
J
J
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama 700-8530, Japan.
Maeda
T
T
Kitazoe
M
M
Nukui
E
E
Tada
H
H
Seno
M
M
Kosaka
M
M
Yamada
H
H
D016475:3T3 Cells / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D002412:Cations / Q000378:metabolism*; D002417:Cattle; D002461:Cell Line, Transformed / Q000187:drug effects / Q000201:enzymology; D003432:Cross-Linking Reagents / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D010926:Placental Hormones / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D012235:Rhodamines / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*
D002264:Carboxylic Acids; D002412:Cations; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010926:Placental Hormones; D012235:Rhodamines; C022052:placental ribonuclease inhibitor; C027755:rhodamine isothiocyanate; D012259:Ribonuclease, Pancreatic; D005022:Ethyldimethylaminopropyl Carbodiimide