The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities.
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, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), 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 Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) 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.
Bagga
Shveta
S
Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India.
Seth
Divya
D
Batra
Janendra K
JK
D004249:DNA Damage / Q000187:drug effects*; D006801:Humans; D000922:Immunotoxins / Q000378:metabolism / Q000494:pharmacology*; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology*; D010940:Plant Proteins / Q000378:metabolism / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012339:RNA, Ribosomal, 28S / Q000037:antagonists & inhibitors; D054788:Ribosome Inactivating Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D029750:Saponaria; D000076985:Saporins; D020298:U937 Cells
D000922:Immunotoxins; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D012339:RNA, Ribosomal, 28S; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins; D000076985:Saporins