Ribonuclease A variants with potent cytotoxic activity.
Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.
Departments of Biochemistry and Chemistry, University of Wisconsin, Madison, WI 53706, USA.
P A
PA
Leland
L W
LW
Schultz
B M
BM
Kim
R T
RT
Raines
D000818:Animals; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic