Tandemization Endows Bovine Pancreatic Ribonuclease with Cytotoxic Activity

• Published in: Journal of molecular biology Vol. 358; no. 5; pp. 1305 - 1313
• Main Authors: Leich, Franziska, Köditz, Jens, Ulbrich-Hofman, Renate, Arnold, Ulrich
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 19.05.2006
• Subjects: Animals; Cattle; Cell Death - drug effects; Circular Dichroism; cytotoxicity; Cytotoxins - chemistry; Cytotoxins - genetics; Cytotoxins - metabolism; Dimerization; Enzyme Inhibitors - metabolism; Enzyme Stability; Humans; In Vitro Techniques; K562 Cells; Kinetics; Models, Molecular; Protein Conformation; Protein Engineering; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - toxicity; ribonuclease A; Ribonuclease, Pancreatic - chemistry; Ribonuclease, Pancreatic - genetics; Ribonuclease, Pancreatic - metabolism; Ribonuclease, Pancreatic - toxicity; tandem enzyme; Thermodynamics; enzyme stability; protein engineering; ONC; RI; GdnHCl; RNase A; ribonuclease A; cytotoxicity; tandem enzyme; 6-FAM-dArU(dA) 2-6-TAMRA
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2006.03.007

Due to their ability to degrade RNA, selected members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol of target cells, where they degrade cellular RNA and cause cell death. The cytotoxic activity of most RNases, however, is abolished by the cytosolic ribonuclease inhibitor (RI). Consequently, the development of RNase derivatives with the ability to evade RI binding is a desirable goal. In this study, tandem enzymes consisting of two RNase A units that are bound covalently via a peptide linker were generated by gene duplication. As deduced from the crystal structure of the RNase A·RI complex, one RNase A unit of the tandem enzyme can still be bound by RI. The other unit, however, should remain unbound because of steric hindrance. This free RNase A unit is expected to maintain its activity and to act as a cytotoxic agent. The study of the influence of the linker sequence on the conformation and stability of these constructs revealed that tandemization has only minor effects on the activity and stability of the constructs in comparison to monomeric RNase A. Relative activity was decreased by 10–50% and the melting temperature was decreased by less than 2.5 K. Furthermore, the cytotoxic potency of the RNase A tandem enzymes was investigated. Despite an in vitro inhibition by RI, tandemization was found to endow RNase A with remarkable cytotoxic activity. While monomeric RNase A is not cytotoxic, IC 50 values of the RNase A tandem variants decreased to 70.3–12.9 μM. These findings might establish the development of a new class of chemotherapeutic agents based on pancreatic ribonucleases.