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"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B. L., et al. (2007) Proteins 66, 183-195], were further characterized and tested for their possible in vitro cytotoxic activity. Results obtained are the following. Besides dimers, also trimers and higher oligomers could be identified among the products of the covalently linking reaction, and the "zero-length" dimers prepared by us appear not to be a unique species. The product was indeed heterogeneous, and results obtained with two RNase A mutants, E9A and K66A, indicated that amino and carboxyl groups others than those belonging to Lys66 and Glu9 are involved in the amide bond. As for their functional properties, the "zero-length" dimers degrade poly(A).poly(U) (dsRNA) with an activity that increases with the increase of the oligomer's basicity and yeast RNA (ssRNA) with an activity that instead decreases with the increase of oligomer's basicity, which is in agreement with previous data. No cytotoxicity of the RNase A "zero-length" dimers could be evidenced in assays performed with various tumor cells lines; the dimers, instead, become cytotoxic if cationized by conjugation with polyethylenimine (PEI) [Futami et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, PEI derivatives of RNase A "zero-length" dimers and PEI derivatives of native RNase A resulted to be equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, the acquired cytotoxicity does not seem to be specific for tumor cells: PEI-cationized native RNase A was also cytotoxic toward human monocytes.

RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDC-treated RNase A monomer were found to carry an increased number of positive charges (about 6 ± 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH(2) monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.

Polyspermine-ribonuclease A (PS-RNase A) and polyspermine-dimeric ribonuclease A (PS-dimeric RNase A) were prepared by cross-linking ribonuclease A or its covalently linked dimer to polyspermine (PS) using dimethyl suberimidate. The two RNase A derivatives were tested for a possible antitumor action. The in vitro and in vivo cytotoxic activity of PS-RNase A, although strong, is not higher than that known for free polyspermine. PS-dimeric RNase A, which was characterized by mass spectroscopy, titration of free amine groups, and enzymatic assays, proved instead to be a definitely more efficient antitumor agent, both in vitro and in vivo. This result could tentatively be explained in view of the importance of positive charges for ribonuclease activity, considering the higher basicity of PS-dimeric RNase A compared to that of PS-(monomeric)RNase A. It must be also taken into account that the dimeric RNase A moiety of PS-dimeric RNase A could evade the cytoplasmic ribonuclease inhibitor, which instead could trap the monomeric RNase A moiety of the other derivative. The two RNase A derivatives degrade poly(A).poly(U) under conditions where native RNase A is inactive. The results of this work demonstrate once again the importance of positive charges for the functions of mammalian pancreatic type ribonucleases in general, in particular for RNase A derivatives, and the potential therapeutic use of the ribonuclease A derivatives.

Do the polarities of the N-terminus or the apolarity of the C-terminus of bovine RNase A influence the relative yields of its two 3D domain-swapped dimeric conformers, the N-dimer and C-dimer? We have addressed this question by substituting Ala-4 or Ala-5 with serine (A4S and A5S mutants) or Ser-123 with alanine (S123A mutant) through site-directed mutagenesis. Both the polarity of the N-terminus and the apolarity of the C-terminus of RNase A were, therefore, increased. CD spectra revealed no significant differences between the secondary structures of the mutants and native RNase A. According to thermal denaturation analyses, the A4S and A5S mutants are less stable, and the S123A mutant is more stable than wild type RNase A. By subjecting the mutants under mild or drastic denaturing conditions, side-by-side with native and recombinant RNase A, to a thermally induced oligomerization procedure, the following results were obtained. (i) The N-terminal mutants showed a higher propensity, with respect to the native protein, to form N-dimers under mild unfolding conditions. (ii) The C-terminal mutant showed a higher propensity to form the C-dimer under severely unfolding conditions. These results are discussed in light of the relative stabilities of the various RNase A species under different environmental conditions, and we conclude that the hydrophilic or hydrophobic character of the RNase N-terminus or C-terminus can be an important variable governing the oligomerization of RNase A and possibly other proteins through the 3D domain-swapping mechanism.