Ribonuclease A variants with potent cytotoxic activity

Abstract

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.

Figure 1

Molecular interactions between pRI (red) and RNase A (blue). This figure was created with the programs molscript (20) and raster3d (21) by using atomic coordinates derived by x-ray diffraction analysis (17). (A) Three-dimensional structure of the crystalline pRI⋅RNase A complex. The indicated residues were replaced in RNase A variants. (B) Contacts between RI and RNase A near Gly-88 of RNase A. The views in A and B are from opposite sides of the pRI⋅RNase A complex. Trp-259 of pRI corresponds to Trp-264 of hRI.

Figure 2

Agarose gel-based assay of ribonuclease inhibition by hRI. Inhibition was assessed by visualizing the ribonuclease-catalyzed degradation of 16S- and 23S-rRNA in the absence or presence of excess hRI. − designates experiments that lacked ribonuclease or hRI (or both). (A) Inhibition of a ribonuclease (0 or 10 ng) by wild-type hRI (0, 20, or 40 units). (B) Inhibition of RNase A varying at residue 88 (0 or 10 ng) by wild-type hRI or W264A hRI (0 or 20 units).

Figure 3

Effect of ribonucleases on the proliferation in culture of K-562 cells. Proliferation was measured by incorporation of [methyl-³H]thymidine into cellular DNA after a 44-h incubation with the ribonucleases. Values reported are the mean from three cultures and are expressed as a percentage of the control, which is the mean from cultures lacking exogenous ribonuclease. The standard error of each value is <11%.