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J. Biol. Chem., Vol. 278, Issue 7, 4813-4820, February 14, 2003

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The Cytotoxic Activity of Ribosome-inactivating Protein Saporin-6 Is Attributed to Its rRNA N-Glycosidase and Internucleosomal DNA Fragmentation Activities^*

Shveta Bagga, Divya Seth, and Janendra K. Batra^§

From the Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India

Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr⁷², Tyr¹²⁰, Glu¹⁷⁶, Arg¹⁷⁹, and Trp²⁰⁸, and two invariant residues, Tyr¹⁶ and Arg²⁴, proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr¹⁶, Tyr⁷², and Arg¹⁷⁹ are absolutely critical; Tyr¹²⁰ and Glu¹⁷⁶ can be partially dispensed with, whereas Trp²⁰⁸ and Arg²⁴ do not appear to be involved in this activity. The residues Tyr⁷², Tyr¹²⁰, Glu¹⁷⁶, Arg¹⁷⁹, and Trp²⁰⁸ were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr¹⁶ and Arg²⁴ do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.

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