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Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction

Nature Biotechnology volume 17, pages 265–270 (1999)Cite this article

Abstract

Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucle- ases—pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)—to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89→Cys) and a mutant EDN (Thr87→Cys). The transferrin–rhRNase(Gly89→Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.

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Figure 1: Representation of the structure of the 1:1 complex between porcine RI and RNase A generated with TURBO FRODO (A. Roussel, A.-G. Inisan & C. Cambillau, AFMB and Bio Graphics, Marseille, France).
Figure 2: List of various constructs of rhRNase(G89C) and rhEDN(T87C) conjugates.
Figure 3: SDS-PAGE of rhRNase(Gly89C→Cys) conjugates on 10–20% gradient gel.
Figure 4: Enzyme activity and RI resistance of RNase conjugates.
Figure 5: Cellular protein synthesis inhibition by RNase conjugates.

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Acknowledgements

We thank Pat Johnson and Joan Barrick for technical assistance, and JoAnn Castelli for reading the manuscript. Certain commercial equipment, instruments, and materials are identified in this paper in order to specify the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.

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Author notes

  1. Jane E. Ladner

    Present address: Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, Rockville, 20850, MD

  2. Cynthia Sung

    Present address: Drug Delivery and Kinetics Resource, Bioengineering and Physical Science Program, Office of Research Services, National Institutes of Health, Bethesda, 20892, MD

  3. Shailendra K. Saxena

    Present address: Alfacell Corporation, Bloomfield, NJ, 07003

  4. Ester Boix

    Present address: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK

  5. Veena M. Vasandani

    Present address: Department of Otolaryngology, Head and Neck Surgery, University of Virginia School of Medicine, Charlottesville, VA, 22908

Authors and Affiliations

  1. Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health , Bethesda, 20892, MD

    Motoshi Suzuki, Shailendra K. Saxena, Ester Boix, Robert J. Prill, Veena M. Vasandani & Richard J. Youle

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Correspondence to Richard J. Youle.

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Suzuki, M., Saxena, S., Boix, E. et al. Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction. Nat Biotechnol 17, 265–270 (1999). https://doi.org/10.1038/7010

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