Double domain swapping in bovine seminal RNase: formation of distinct N- and C-swapped tetramers and multimers with increasing biological activities

PLoS One. 2012;7(10):e46804. doi: 10.1371/journal.pone.0046804. Epub 2012 Oct 11.

Abstract

Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M = M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini.When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities.Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers.Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetic Acid / chemistry
  • Animals
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / metabolism
  • Antineoplastic Agents / pharmacology
  • Cattle
  • Cell Survival / drug effects
  • Cells, Cultured
  • Chromatography, Gel
  • Cross-Linking Reagents / chemistry
  • Dinitrofluorobenzene / analogs & derivatives
  • Dinitrofluorobenzene / chemistry
  • Disulfides / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Endoribonucleases / chemistry*
  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism
  • Enzyme Stability
  • Kinetics
  • Male
  • Models, Molecular*
  • Mutation
  • Poly A-U / metabolism
  • Protein Multimerization*
  • Protein Structure, Tertiary*
  • Substrate Specificity
  • Temperature

Substances

  • Antineoplastic Agents
  • Cross-Linking Reagents
  • Disulfides
  • Poly A-U
  • Dinitrofluorobenzene
  • Endoribonucleases
  • ribonuclease SPL
  • 1,5-difluoro-2,4-dinitrobenzene
  • Acetic Acid

Grants and funding

This work has been supported by the grants “Gottex60%-08” and “FR-GOTTEG-09”, funded by the Italian Ministero Istruzione Università e Ricerca (MIUR), and by the University of Verona, Italy. “CTQ2010-21567-C02-02” from the Spanish Ministerio de Ciencia e Innovación (MICINN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.