Abstract
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.
Highlights
Bovine seminal ribonuclease (EC 3.1.27.5, BS-RNase)1 is a cytotoxic protein
We demonstrated that the ribonucleolytic activity of BS-RNase is necessary for its cytotoxic activity (Kim et al, 1995a)
The cytosol of cells is a reducing environment, which is a hazard for disulfide bonds such as the two that cross-link the monomers of dimeric BS-RNase
Summary
Bovine seminal ribonuclease (EC 3.1.27.5, BS-RNase) is a cytotoxic protein. In various assays, this cytotoxicity can manifest itself as an immunosuppressive, antitumor, embryotoxic,. Noncovalent interactions in the MxM form enable it to remain dimeric and retain its lethal enzymatic activity in the absence of its intersubunit disulfide bonds. These data lead us to propose that BS-RNase evolved its MxM form to evade RI and thereby retain its enzymatic activity in vivo (Kim, 1994; Kim et al, 1995b). We test this hypothesis with a semisynthetic dimer of BS-RNase that is cross-linked by thioether (C–S–C) rather than disulfide (S–S) bonds. Since thioether bonds are not cleaved by thiols, this semisynthetic enzyme will remain dimeric in the reducing environment of the cytosol
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