Mechanism of ribonuclease cytotoxicity.

Bovine seminal ribonuclease (BS-RNase), a dimeric homolog of bovine pancreatic ribonuclease A (RNase A), is toxic to mammalian cells. In contrast to dimeric BS-RNase, a monomeric BS-RNase and RNase A are not cytotoxic and are bound tightly by cytosolic ribonuclease inhibitor. To elucidate the mechanism of ribonuclease cytotoxicity, we constructed a series of hybrid and semisynthetic enzymes and examined their properties. In five hybrid enzymes, divergent residues in BS-RNase were replaced with the analogous residues of RNase A so as to diminish an interaction with a putative cellular receptor. In a semisynthetic enzyme, the disulfide bonds that cross-link the monomeric subunits of dimeric BS-RNase were replaced with thioether bonds, which can withstand the reducing environment of the cytosol. Each hybrid and semisynthetic enzyme had ribonucleolytic and cytotoxic activities comparable with those of wild-type BS-RNase. These results suggest that dimeric BS-RNase (pI = 10.3) enters cells by adsorptive rather than receptor-mediated endocytosis and then evades cytosolic ribonuclease inhibitor so as to degrade cellular RNA. This mechanism accounts for the need for a cytosolic ribonuclease inhibitor and for the cytotoxicity of other homologs of RNase A.