Preparation of potent cytotoxic ribonucleases by cationization: Enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups

Junichiro Futami, T. Maeda, M. Kitazoe, E. Nukui, Hiroko Tada, Masaharu Seno, Megumi Kosaka, H. Yamada

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Abstract

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.

Original languageEnglish
Pages (from-to)7518-7524
Number of pages7
JournalBiochemistry
Volume40
Issue number25
Publication statusPublished - Jun 26 2001

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Chemical modification
Ribonucleases
ethylenediamine
Pancreatic Ribonuclease
Cytotoxicity
Derivatives
Carbodiimides
Ethanolamine
Taurine
Anions

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Preparation of potent cytotoxic ribonucleases by cationization: Enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups",
abstract = "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.",
author = "Junichiro Futami and T. Maeda and M. Kitazoe and E. Nukui and Hiroko Tada and Masaharu Seno and Megumi Kosaka and H. Yamada",
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T1 - Preparation of potent cytotoxic ribonucleases by cationization

T2 - Enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups

AU - Futami, Junichiro

AU - Maeda, T.

AU - Kitazoe, M.

AU - Nukui, E.

AU - Tada, Hiroko

AU - Seno, Masaharu

AU - Kosaka, Megumi

AU - Yamada, H.

PY - 2001/6/26

Y1 - 2001/6/26

N2 - 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.

AB - 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.

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