Bovine pancreatic ribonuclease A forms 3D domain-swapped oligomers by lyophilization from 40% acetic acid solutions or if subjected to various thermally-induced denaturation procedures. Considering that the intrinsic swapping propensity of bovine seminal RNase, the only member of the pancreatic-type RNase super-family that is dimeric in nature, is decreased from 70 to 30% if Arg80 is substituted by Ser (the corresponding residue in native RNase A), we introduced the opposite mutation in position 80 of the pancreatic enzyme. Our aim was to detect if the RNase A tendency to aggregate through domain swapping could increase. Aggregation of the S80R-RNase A mutant was induced either through the 'classic' acetic acid lyophilization, or through a thermally-induced method. The results indicate that the S80R mutant aggregates to a higher extent than the native protein, and that the increase occurs especially through N-terminal swapping. Additional investigations on the dimeric and multimeric species formed indicate that the S80R mutation increases their stability against regression to monomer, and does not significantly change their structural and functional features.

"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.

After a short introduction with some examples of cytotoxic ribonucleases, the importance of natural or artificial dimerization (oligomerization) as a way for a ribonuclease to acquire novel functional properties has been pointed out. In particular, the role of the three dimensional domain swapping mechanism in bovine pancreatic ribonuclease A oligomerization, as well as its impact for the acquisition of novel biological functions (among which a remarkable antitumor action) by the enzyme protein in oligomeric form have been discussed. Finally, the structural and functional features that could explain why oligomeric ribonuclease A becomes able to display a cytotoxic activity, and the possible use and limits of the three dimensional domain-swapped oligomers of ribonuclease A as anticancer therapeutic agents have been described and discussed.

A significant aspermatogenic activity, ascertained by microscopic studies of seminiferous tubules and interstitial tissue, and by the observation of the entrance of immunity and fibrocytic cells in mice injected with polyspermine (PS) or polyspermine conjugated to monomeric or dimeric RNase A (PS-RNase A or PS-dimeric RNase A, respectively), was found either in mice injected or in non-injected testes. Polyspermine and its complexes with RNase A destroyed all spermatogenic and intertestitial tissue, including Leydic cells, as well as their ability to secrete testosterone. The total loss of spermatogenic activity in injected testes is irreversible because spermatogonia cells also were destroyed. The injection of PS into both mice testes determined the total degeneration of testicle tissue in 50% of injected testes. The second half of testes was also partly degenerated, and if they were re-injected, almost all testes were fully destroyed. PS-dimeric RNase A injected once into both testicles produced a stronger degeneration and also the interruption of testosterone secretion in comparison with the effects due to injection of mice with PS or PS-RNase A. In all mice treated with these substances, as well as in rats in which PS was injected twice into their testes, we detected polymorfonucleates, monocytes, plasma cells, lymphocytes and fibrocytic cells. Antibodies against PS, PS-RNase A or PS-dimeric RNase A did not influence the aspermatogenic activity. Animals in which a repeated intra-peritoneal injection was carried out did not lose body mass and remained in good condition, with the exception of mice injected with spermine.

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.

Previously we have shown that monomeric RNase A has no significant biological activity, whereas its oligomers (dimer to tetramer) prepared by lyophilizing from 50% acetic acid solutions, show remarkable aspermatogenic and antitumor activities. Furthermore, conjugates prepared by chemical binding of native RNase A to polyethylene glycol (PEG) have shown a significant aspermatogenic and antitumor activities. In this work we show that the chemical conjugation of PEG to the RNase A C-dimer, and to the two RNase A trimers (NC-trimer and C- trimer) decreases the aspermatogenic activity of the oligomers while increasing their inhibitory activity on the growth of the human UB900518 amelanotic melanoma transplanted in athymic nude mice. Moreover, the PEG-conjugated RNaseA oligomers are devoid, like the free oligomers, of any embryotoxic activity.

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.

By lyophilizing RNase A from 40% acetic acid solutions, two dimeric aggregates, the “minor” and “major” dimers (named here N-dimer and C-dimer, respectively), form by 3D domain swapping at a ratio of 1:4. Trimeric and tetrameric aggregates are also obtained. The two dimers and the higher oligomers also form without a lyophilization step. By keeping RNase A dissolved at a high concentration (generally 200 mg/ml) in various media at temperatures ranging from 23 to 70 °C for times varying from a few minutes to 2 h, various oligomers, in particular the two dimeric conformers, formed in quite different amounts, often inverting their relative quantities depending on the more or less severe unfolding conditions. When unfolding mainly concerned the N terminus of the protein, richer in hydrophilic residues, the N-dimer, formed by 3D domain swapping of the N-terminal α-helix of each monomer, prevailed over the C-dimer. Under more vigorous denaturing conditions, where also the C terminus of RNase A, richer in hydrophobic amino acids, unfolded, the C-dimer, formed by 3D domain swapping of the C-terminal β-strand, prevailed over the other, possibly because of the induction to aggregation promoted by the hydrophobic residues present in the C termini of the two monomers.

By lyophilizing RNase A from 40% acetic acid solutions, two dimeric aggregates, the "minor" and "major" dimers (named here N-dimer and C-dimer, respectively), form by 3D domain swapping at a ratio of 1:4. Trimeric and tetrameric aggregates are also obtained. The two dimers and the higher oligomers also form without a lyophilization step. By keeping RNase A dissolved at a high concentration (generally 200 mg/ml) in various media at temperatures ranging from 23 to 70 degrees C for times varying from a few minutes to 2 h, various oligomers, in particular the two dimeric conformers, formed in quite different amounts, often inverting their relative quantities depending on the more or less severe unfolding conditions. When unfolding mainly concerned the N terminus of the protein, richer in hydrophilic residues, the N-dimer, formed by 3D domain swapping of the N-terminal alpha-helix of each monomer, prevailed over the C-dimer. Under more vigorous denaturing conditions, where also the C terminus of RNase A, richer in hydrophobic amino acids, unfolded, the C-dimer, formed by 3D domain swapping of the C-terminal beta-strand, prevailed over the other, possibly because of the induction to aggregation promoted by the hydrophobic residues present in the C termini of the two monomers.