Bovine Seminal Ribonuclease and Its Special Features: When Two is Better Than One

  • Delia Picone
  • Antonello Merlino
  • Roberta Spadaccini
Chapter

Abstract

Bovine Seminal Ribonuclease (BS-RNase) is a structurally well-characterized protein which has been isolated in Naples in the sixties. It is a homodimer whose subunits are linked by two disulfide bridges and exists as an equilibrium mixture of two isomers, with and without swapping of the N-termini. The protein has an enzymatic activity very similar to that of the well-known Ribonuclease A, but it also displays a potent antitumor activity. We describe our current understanding of how BS-RNase exerts its cytotoxicity against malignant cells, which is strictly related to the RNA hydrolysis occurring in the host cytosol. Structural and biochemical data collected on the BS-RNase isoforms and on some mutants suggest that the swapped form is responsible for the citotoxicity, mainly because its compact 3D structure allows the protein to evade the RNase inhibitor, a protein acting as a cell sentry against exogenous ribonucleases. Structural comparisons among dimeric ribonucleases and site-directed mutagenesis studies suggest that only a few residues are critical to stabilize this compact structure even in the cytosol, where the reducing environment promotes the selective cleavage of the interchain disulfides. However, further engineering studies are needed to develop new potential anticancer drugs based on BS-RNase and its derivatives.

Keywords

Antitumor Activity Cytotoxic Activity Seminal Plasma Quaternary Structure High Substrate Concentration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    D’Alessio G (2009) It takes two to flirt with a dimeric RNase. Biopolymers 91:989–994CrossRefPubMedGoogle Scholar
  2. 2.
    Di Donato A, D’Alessio G (1981) Heterogeneity of bovine seminal ribonuclease. Biochemistry 20:7232–7237CrossRefPubMedGoogle Scholar
  3. 3.
    Di Donato A, Galletti P, D’Alessio G (1986) Selective deamidation and enzymatic methylation of seminal ribonuclease. Biochemistry 25:8361–8368CrossRefPubMedGoogle Scholar
  4. 4.
    Piccoli R, Tamburrini M, Piccialli G, Di Donato A, Parente A, D’Alessio G (1992) The dual-mode quaternary structure of seminal RNase. Proc Natl Acad Sci U S A 89:1870–1874CrossRefPubMedGoogle Scholar
  5. 5.
    Haigis MC, Kurten EL, Raines RT (2003) Ribonuclease inhibitor as an intracellular sentry. Nucleic Acids Res 31:1024–1032CrossRefPubMedGoogle Scholar
  6. 6.
    Piccoli R, Di Donato A, D’Alessio G (1988) Co-operativity in seminal ribonuclease function kinetic studies. Biochem J 253:329–336PubMedGoogle Scholar
  7. 7.
    Matousek J (1973) The effect of bovine seminal ribonuclease (AS RNase) on cells of crocker tumour in mice. Experientia 29:858–859CrossRefPubMedGoogle Scholar
  8. 8.
    Fang EF, Ng TB (1815) Ribonucleases of different origins with a wide spectrum of medicinal applications. Biochim Biophys Acta 2011:65–74Google Scholar
  9. 9.
    Di Donato A, Cafaro V, Romeo I, D’Alessio G (1995) Hints on the evolutionary design of a dimeric RNase with special bioactions. Protein Sci 4:1470–1477CrossRefPubMedGoogle Scholar
  10. 10.
    Ercole C, Lopez-Alonso JP, Font J, Ribo M, Vilanova M, Picone D et al (2011) Crowding agents and osmolytes provide insight into the formation and dissociation of RNase A oligomers. Arch Biochem Biophys 506:123–129CrossRefPubMedGoogle Scholar
  11. 11.
    Bracale A, Spalletti-Cernia D, Mastronicola M, Castaldi F, Mannucci R, Nitsch L et al (2002) Essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuclease. Biochem J 362:553–560CrossRefPubMedGoogle Scholar
  12. 12.
    Piccoli R, Vescia S, Bridges SH, D’Alessio G (1990) The antitumor action of seminal ribonuclease tested with the plasmacytoma spleen colonization assay. Ital J Biochem 39:242–249PubMedGoogle Scholar
  13. 13.
    Mastronicola MR, Piccoli R, D’Alessio G (1995) Key extracellular and intracellular steps in the antitumor action of seminal ribonuclease. Eur J Biochem 230:242–249CrossRefPubMedGoogle Scholar
  14. 14.
    Mancheno JM, Gasset M, Onaderra M, Gavilanes JG, D’Alessio G (1994) Bovine seminal ribonuclease destabilizes negatively charged membranes. Biochem Biophys Res Commun 199:119–124CrossRefPubMedGoogle Scholar
  15. 15.
    D’Errico G, Ercole C, Lista M, Pizzo E, Falanga A, Galdiero S et al (1808) Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease. Biochim Biophys Acta 2011:3007–3015Google Scholar
  16. 16.
    Notomista E, Mancheno JM, Crescenzi O, Di Donato A, Gavilanes J, D’Alessio G (2006) The role of electrostatic interactions in the antitumor activity of dimeric RNases. FEBS J 273:3687–3697CrossRefPubMedGoogle Scholar
  17. 17.
    Futami J, Maeda T, Kitazoe M, Nukui E, Tada H, Seno M et al (2001) Preparation of potent cytotoxic ribonucleases by cationization: enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups. Biochemistry 40:7518–7524CrossRefPubMedGoogle Scholar
  18. 18.
    Kim JS, Soucek J, Matousek J, Raines RT (1995) Structural basis for the biological activities of bovine seminal ribonuclease. J Biol Chem 270:10525–10530CrossRefPubMedGoogle Scholar
  19. 19.
    Capasso S, Giordano F, Mattia CA, Mazzarella L, Zagari A (1983) Refinement of the structure of bovine seminal ribonuclease. Biopolymers 22:327–332CrossRefPubMedGoogle Scholar
  20. 20.
    Schlunegger MP, Bennett MJ, Eisenberg D (1997) Oligomer formation by 3D domain swapping: a model for protein assembly and misassembly. Adv Protein Chem 50:61–122CrossRefPubMedGoogle Scholar
  21. 21.
    Mazzarella L, Capasso S, Demasi D, Di Lorenzo G, Mattia CA, Zagari A (1993) Bovine seminal ribonuclease: structure at 1.9 A resolution. Acta Crystallogr D Biol Crystallogr 49:389–402CrossRefPubMedGoogle Scholar
  22. 22.
    Vitagliano L, Adinolfi S, Riccio A, Sica F, Zagari A, Mazzarella L (1998) Binding of a substrate analog to a domain swapping protein: X-ray structure of the complex of bovine seminal ribonuclease with uridylyl(2’,5’)adenosine. Protein Sci 7:1691–1699CrossRefPubMedGoogle Scholar
  23. 23.
    Vitagliano L, Adinolfi S, Sica F, Merlino A, Zagari A, Mazzarella L (1999) A potential allosteric subsite generated by domain swapping in bovine seminal ribonuclease. J Mol Biol 293:569–577CrossRefPubMedGoogle Scholar
  24. 24.
    Vitagliano L, Merlino A, Zagari A, Mazzarella L (2000) Productive and nonproductive binding to ribonuclease A: X-ray structure of two complexes with uridylyl(2’,5’)guanosine. Protein Sci 9:1217–1225CrossRefPubMedGoogle Scholar
  25. 25.
    Di Donato A, Piccoli R, D’Alessio G (1987) Co-operativity in seminal ribonuclease function: binding studies. Biochem J 241:435–440PubMedGoogle Scholar
  26. 26.
    Merlino A, Vitagliano L, Sica F, Zagari A, Mazzarella L (2004) Population shift vs induced fit: the case of bovine seminal ribonuclease swapping dimer. Biopolymers 73:689–695CrossRefPubMedGoogle Scholar
  27. 27.
    Ma B, Nussinov R (2010) Enzyme dynamics point to stepwise conformational selection in catalysis. Curr Opin Chem Biol 14:652–659CrossRefPubMedGoogle Scholar
  28. 28.
    Dossi K, Tsirkone VG, Hayes JM, Matousek J, Pouckova P, Soucek J et al (2009) Mapping the ribonucleolytic active site of bovine seminal ribonuclease. The binding of pyrimidinyl phosphonucleotide inhibitors. Eur J Med Chem 44:4496–4508CrossRefPubMedGoogle Scholar
  29. 29.
    Berisio R, Sica F, De Lorenzo C, Di Fiore A, Piccoli R, Zagari A et al (2003) Crystal structure of the dimeric unswapped form of bovine seminal ribonuclease. FEBS Lett 554:105–110CrossRefPubMedGoogle Scholar
  30. 30.
    D’Alessio G, Di Donato A, Piccoli R, Russo N (2001) Seminal ribonuclease: preparation of natural and recombinant enzyme, quaternary isoforms, isoenzymes, monomeric forms; assay for selective cytotoxicity of the enzyme. Methods Enzymol 341:248–263CrossRefPubMedGoogle Scholar
  31. 31.
    Sica F, Di Fiore A, Zagari A, Mazzarella L (2003) The unswapped chain of bovine seminal ribonuclease: crystal structure of the free and liganded monomeric derivative. Proteins 52:263–271CrossRefPubMedGoogle Scholar
  32. 32.
    D’Ursi A, Oschkinat H, Cieslar C, Picone D, D’Alessio G, Amodeo P et al (1995) Assignment and secondary-structure determination of monomeric bovine seminal ribonuclease employing computer-assisted evaluation of homonuclear three-dimensional 1H-NMR spectra. Eur J Biochem 229:494–502CrossRefPubMedGoogle Scholar
  33. 33.
    Avitabile F, Alfano C, Spadaccini R, Crescenzi O, D’Ursi AM, D’Alessio G et al (2003) The swapping of terminal arms in ribonucleases: comparison of the solution structure of monomeric bovine seminal and pancreatic ribonucleases. Biochemistry 42:8704–8711CrossRefPubMedGoogle Scholar
  34. 34.
    Spadaccini R, Ercole C, Gentile MA, Sanfelice D, Boelens R, Wechselberger R et al (2012) NMR studies on structure and dynamics of the monomeric derivative of BS-RNase: new insights for 3D domain swapping. PLoS ONE 7:e29076CrossRefPubMedGoogle Scholar
  35. 35.
    Liu Y, Gotte G, Libonati M, Eisenberg D (2001) A domain-swapped RNase A dimer with implications for amyloid formation. Nat Struct Biol 8:211–214CrossRefPubMedGoogle Scholar
  36. 36.
    Merlino A, Picone D, Ercole C, Balsamo A, Sica F (2012) Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease. Biochimie 94:1108–1118CrossRefPubMedGoogle Scholar
  37. 37.
    Sica F, Di Fiore A, Merlino A, Mazzarella L (2004) Structure and stability of the non-covalent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ribonuclease protein inhibitor. J Biol Chem 279:36753–36760CrossRefPubMedGoogle Scholar
  38. 38.
    Ercole C, Avitabile F, Del Vecchio P, Crescenzi O, Tancredi T, Picone D (2003) Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase. Eur J Biochem 270:4729–4735CrossRefPubMedGoogle Scholar
  39. 39.
    Picone D, Di Fiore A, Ercole C, Franzese M, Sica F, Tomaselli S et al (2005) The role of the hinge loop in domain swapping. The special case of bovine seminal ribonuclease. J Biol Chem 280:13771–13778CrossRefPubMedGoogle Scholar
  40. 40.
    Merlino A, Ercole C, Picone D, Pizzo E, Mazzarella L, Sica F (2008) The buried diversity of bovine seminal ribonuclease: shape and cytotoxicity of the swapped non-covalent form of the enzyme. J Mol Biol 376:427–437CrossRefPubMedGoogle Scholar
  41. 41.
    Ercole C, Spadaccini R, Alfano C, Tancredi T, Picone D (2007) A new mutant of bovine seminal ribonuclease with a reversed swapping propensity. Biochemistry 46:2227–2232CrossRefPubMedGoogle Scholar
  42. 42.
    Giancola C, Ercole C, Fotticchia I, Spadaccini R, Pizzo E, D’Alessio G et al (2011) Structure-cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants. FEBS J 278:111–122CrossRefPubMedGoogle Scholar
  43. 43.
    Cafaro V, Bracale A, Di Maro A, Sorrentino S, D’Alessio G, Di Donato A (1998) New muteins of RNase A with enhanced antitumor action. FEBS Lett 437:149–152CrossRefPubMedGoogle Scholar
  44. 44.
    Ercole C, Colamarino RA, Pizzo E, Fogolari F, Spadaccini R, Picone D (2009) Comparison of the structural and functional properties of RNase A and BS-RNase: a stepwise mutagenesis approach. Biopolymers 91:1009–1017CrossRefPubMedGoogle Scholar
  45. 45.
    Merlino A, Russo Krauss I, Perillo M, Mattia CA, Ercole C, Picone D et al (2009) Toward an antitumor form of bovine pancreatic ribonuclease: the crystal structure of three noncovalent dimeric mutants. Biopolymers 91:1029–1037CrossRefPubMedGoogle Scholar
  46. 46.
    Gotte G, Helmy AM, Ercole C et al (2012) Double domain swapping in bovine seminal RNase: formation of distinct N- and C-swapped tetramers and multimers with increasing biological activities PLoS One 7:e46804Google Scholar
  47. 47.
    Ellis GA, Hornung ML, Raines RT (2011) Potentiation of ribonuclease cytotoxicity by a poly(amidoamine) dendrimer. Bioorg Med Chem Lett 21:2756–2758CrossRefPubMedGoogle Scholar
  48. 48.
    Soucek J, Raines RT, Haugg M, Raillard-Yoon SA, Benner SA (1999) Structural changes to ribonuclease A and their effects on biological activity. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 123:103–111CrossRefPubMedGoogle Scholar
  49. 49.
    Russo A, Antignani A, Giancola C, D’Alessio G (2002) Engineering the refolding pathway and the quaternary structure of seminal ribonuclease by newly introduced disulfide bridges. J Biol Chem 277:48643–48649CrossRefPubMedGoogle Scholar
  50. 50.
    Lee JE, Raines RT (2005) Cytotoxicity of bovine seminal ribonuclease: monomer versus dimer. Biochemistry 44:15760–15767CrossRefPubMedGoogle Scholar
  51. 51.
    Antignani A, Naddeo M, Cubellis MV, Russo A, D’Alessio G (2001) Antitumor action of seminal ribonuclease, its dimeric structure, and its resistance to the cytosolic ribonuclease inhibitor. Biochemistry 40:3492–3496CrossRefPubMedGoogle Scholar
  52. 52.
    Kim JS, Soucek J, Matousek J, Raines RT (1995) Catalytic activity of bovine seminal ribonuclease is essential for its immunosuppressive and other biological activities. Biochem J 308(Pt 2):547–550PubMedGoogle Scholar
  53. 53.
    Opitz JG, Ciglic MI, Haugg M, Trautwein-Fritz K, Raillard SA, Jermann TM et al (1998) Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA. Biochemistry 37:4023–4033CrossRefPubMedGoogle Scholar
  54. 54.
    Ciglic MI, Jackson PJ, Raillard SA, Haugg M, Jermann TM, Opitz JG et al (1998) Origin of dimeric structure in the ribonuclease superfamily. Biochemistry 37:4008–4022CrossRefPubMedGoogle Scholar
  55. 55.
    Soucek J, Marinov I, Benes J, Hilgert I, Matousek J, Raines RT (1996) Immunosuppressive activity of bovine seminal ribonuclease and its mode of action. Immunobiology 195:271–285CrossRefPubMedGoogle Scholar
  56. 56.
    Slavik T, Matousek J, Fulka J, Raines RT (2000) Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation. J Exp Zool 287:394–399CrossRefPubMedGoogle Scholar
  57. 57.
    Bracale A, Castaldi F, Nitsch L, D’Alessio G (2003) A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action. Eur J Biochem 270:1980–1987CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Delia Picone
    • 1
  • Antonello Merlino
    • 1
    • 2
  • Roberta Spadaccini
    • 3
  1. 1.Department of Chemical SciencesUniversity of Naples Federico IINaplesItaly
  2. 2.Institute of Biostructure e Bioimages, CNRNaplesItaly
  3. 3.Department of Biological, Geological and Environmental SciencesUniversity of SannioBeneventoItaly

Personalised recommendations