Ribonuclease A variants with potent cytotoxic activity

Abstract

Bovine pancreatic ribonuclease A (RNase A; EC 3.1.27.5) catalyzes the cleavage of RNA (1). Select homologs of RNase A use this activity to effect diverse biological phenomena. Angiogenin (ANG) promotes the growth of new blood vessels (2). Bovine seminal ribonuclease (BS-RNase) displays immunosuppressive, embryotoxic, aspermatogenic, and antitumor activities (3). Onconase (ONC), a ribonuclease from the oocytes and early embryos of the Northern leopard frog (Rana pipiens), is a potent toxin to several transformed cell lines (4). Significantly, the biological activities of ANG, BS-RNase, and ONC depend on their ribonucleolytic activity. Though an efficient catalyst of RNA cleavage, RNase A does not display any pronounced biological action.

Members of the RNase A superfamily have divergent amino acid sequences but similar tertiary structures. The amino acid sequences of BS-RNase A and RNase A, which are from the same species, are 80% identical (5). ONC and human ANG, however, share only 30% and 33% amino acid identity, respectively, with RNase A (6, 7). The majority of nonconserved residues are in surface loops, which appear to play a significant role in the special biological activities of ANG, BS-RNase, and ONC (8, 9). The three-dimensional structure of the pancreatic-type ribonucleases is comprised of a central four-stranded antiparallel β-sheet, flanked by two α-helices. The active site is located in a cleft defined by a third N-terminal α-helix and one edge of the β-sheet. This structure is stabilized by three or four disulfide bonds.

Ribonuclease inhibitor (RI) is a 50-kDa protein that constitutes ≈0.01% of the cytosolic protein in mammalian cells (10–12). Mammalian RIs are highly conserved. For example, porcine RI (pRI) and human RI (hRI) share 77% amino acid sequence identity, with no insertions or deletions except for a four-residue extension on the N terminus of hRI. Both inhibitors contain 15 leucine-rich, β–α repeat units arranged symmetrically in a horseshoe. The β-strands form a solvent-exposed β-sheet that defines the inner circumference of RI. The α-helices define the outer surface of the inhibitor (13). RI forms a 1:1, noncovalent complex with target ribonucleases, including RNase A (14, 15) and ANG (14). Values of the inhibition constant (K_i) are in the fM range. The unusually low K_i values are a consequence of an association rate constant (k_on) close to the diffusion limit and a dissociation rate constant (k_off) near 10⁷⋅s¹ (14–16).

Atomic details of RI–ribonuclease interactions are apparent from the three-dimensional structures of the crystalline pRI⋅RNase A and hRI⋅ANG complexes (17–19). To a first approximation, the two structures are similar. In both, one-third of the ribonuclease rests within the RI horseshoe and the remainder extends out of the plane defined by the inhibitor (Fig. ​(Fig.11A). This arrangement centers each enzyme’s active site on the C terminus of the inhibitor. The intermolecular contacts in the RI⋅ribonuclease complexes differ significantly, but this heterogenety is caused largely by the low sequence identity between RNase A and ANG.

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.

The inhibitory activity of RI is manifested in the cytosol. This location provides the reducing environment that is necessary to maintain RI activity. Mammalian RIs contain 30 or 32 reduced cysteine residues (22). Oxidation of a single cysteine residue causes rapid oxidation of the remaining cysteine residues and consequent inactivation of RI (23). All known RI ligands, including RNase A, are secreted ribonucleases. This observation and the cytosolic localization of RI supports the hypothesis that the inhibitor functions to preserve the integrity of cellular RNA should a secretory ribonuclease reach the cytosol (11, 24).

The protection offered to cells by RI is limited. The cytotoxic activities of BS-RNase and ONC appear to be a consequence of their abilities to escape inactivation by RI. BS-RNase is isolated as a homodimer, covalently linked by two disulfide bonds. As a dimer, BS-RNase is not inhibited by RI and is cytotoxic. RI becomes a potent inhibitor when BS-RNase is reduced to its monomeric form (8, 25–27). Though monomeric, ONC also evades tight binding by RI (28). ONC retains the elements of tertiary structure that characterize pancreatic-type ribonucleases, but its surface loops are truncated severely compared with their counterparts in RNase A and ANG. Further, the majority of RNase A and ANG residues that contact RI are replaced by dissimilar residues in ONC.

We have directly investigated the relationship between RI inhibition of ribonucleases and ribonuclease cytotoxicity. Specifically, we reasoned that RNase A could be endowed with a cytotoxic activity by specifically decreasing its susceptibility to inactivation by RI. We created two RI-evasive RNase A variants by incorporating amino acid residues that introduce steric and electrostatic strain into the RI⋅RNase A complex. As anticipated, these variants are toxic to a transformed cell line. Our data indicate that ribonuclease cytotoxicity is a direct consequence of an enzyme’s ability to overcome inhibition by RI.

Not determined.

Acknowledgments

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