SEC chromatograms of K113N BS-RNase aggregates obtained by lyophilising the protein from a 40% (v/v) acetic acid solution.

<p>(<b>A</b>), Superdex 75 chromatogram of the mutant (blue) overlapped with a chromatogram of wt BS-RNase (red). (<b>B</b>), SEC of K113N BS-TT₁ and TT₂ gathered together immediately after their elution from the aggregates mixture (day 0-blue curve) and re-cromatographed after storage in 0.2 M NaPi, pH 6.7, for one, two, or three days (1-red, 2-dark green, 3-pink curves, respectively).</p

10% SDS-PAGE analysis of the crosslinking reactions of BS-RNase tetramers.

<p>(<b>A</b>), (<b>B</b>) DVS reaction time-course of TT₁ and TT₂, respectively. 8 µg of each aliquot-sample was electrophoresed after blocking the reaction with 0.2 M β-mercaptoethanol, final concentration. (<b>C</b>), (<b>D</b>) DFDNB reaction of BS-RNase tetramers and RNase A dimers analyzed under reducing and non-reducing conditions, respectively. Samples were concentrated to 1 mg/ml, and 10 µg of each were analyzed.</p

Double Domain Swapping in Bovine Seminal RNase: Formation of Distinct N- and C-swapped Tetramers and Multimers with Increasing Biological Activities

<div><p>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.</p> <p>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.</p> <p>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.</p> <p>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.</p> </div

SEC chromatograms and PAGE under non denaturing conditions of BS-RNase aggregates obtained by lyophilising the protein from a 40% (v/v) acetic acid solution.

<p>(<b>A</b>) SEC pattern obtained with a Sephadex G100 column. Elution with ammonium acetate 0.1 M, pH 5.65, flow rate of 0.4 ml/min. (<b>B</b>) SEC chromatogram of BS-RNase multimers superimposed with that of RNase A oligomers: both patterns were obtained with a Superdex 75 10/300 GL column. Elution with 0.2 M NaPi, pH 6.7, flow rate 0.1 ml/min. (<b>C</b>) Enlarged Superdex 75 SEC pattern of BS-RNase aggregates; in the inset, 7.5% non denaturing PAGE of the two BS-tetramers, run-time 110 min. (<b>D</b>) Additional purification of the two BS-RNase tetramers: their mixture was concentrated to 25 µl in 0.4 M NaPi, and re-chromatographed in the Superdex 75 column equilibrated with the same buffer (dashed+dotted line). Then, TT₁ and TT₂ fractions were further purified: once for TT₁, continuous line; twice for TT₂, dotted and dashed lines, respectively. In the right part of the panel are reported the models of two N-swapped BS-RNase tetramers proposed by Adinolfi <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Adinolfi1" target="_blank">[13]</a>: they cannot be associated to both tetramers. The various BS-RNase species are: D, native dimer; TT₁ and TT₂, two tetrameric conformers, H (1 and 2), hexamers; L.O., larger oligomers. Concerning RNase A, grey italics labels: <i>M</i>, native monomer, <i>N_D</i>, N-terminal-swapped dimer, <i>C_D</i>, C-terminal-swapped dimer; <i>T</i>, trimers; <i>NCN_TT</i>: double N+C-swapped tetramer; <i>CNC_TT</i>: double C+N-swapped tetramer; <i>P*</i>: pentamers; <i>H*</i>: hexamers. The asterisk* is present to mention that P and H positions are derived from data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Gotte1" target="_blank">[21]</a>.</p

Quantification and structural features of BS-RNase oligomers.

a<p>Calculated from DLS analysis.</p>b<p>The elution volumes of the BS-tetramers derive from their additional SEC purification with 0.4 M NaPi as eluent (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone-0046804-g001" target="_blank">Figure 1D</a>).</p>c<p>L.O.: mixture of BS-RNase octamers and larger oligomers.</p

SEC profiles of wt and K113N BS-RNase aggregates obtained through thermal treatment.

<p>The various environmental conditions applied in aqueous solvents <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Gotte2" target="_blank">[35]</a> are indicated in each of the <b>A–D</b> panels, in which the profiles obtained with the two BS-RNase variants (blue and red curves) are compared with the corresponding RNase A chromatograms (dotted black curves). Oligomers were obtained as follows: tubes containing 2.5–3.0 µl (0.5 mg) of each solution were put for 60 min in a thermostatically controlled bath at one of the temperatures indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Gotte2" target="_blank">[35]</a>. Then, 200 µl of 0.2 M NaPi, pH 6.7, heated to the same temperature of incubations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Gotte2" target="_blank">[35]</a>, were added. Each sample was transferred to an ice-cold bath for 5 min, then injected onto a gel filtration Superdex 75 HR10/30 column. The different oligomers formed are labeled and correspond to the ones prepared by the lyophilization procedure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone-0046804-g001" target="_blank">Figure 1</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Crestfield1" target="_blank">[11]</a>. GDMCl, guanidine hydrochloride; EtOH, ethanol.</p

Molecular docking models of <i>N</i>C<i>N</i>-swapped BS-RNase and RNase A.

<p>(<b>A</b>), <i>left panel</i>, “quasi-linear” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Cozza1" target="_blank">[40] </a><i>N</i>C<i>N</i>_TT model: the stabilizing intermolecular H-bonds between the two central subunits are indicated with orange circles; <i>right panel</i>, the same structure rotated 90° around the x-axis. (<b>B</b>) modeled structure of RNase A <i>N</i>C<i>N</i>_TT<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Cozza1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#pone.0046804-Liu5" target="_blank">[41]</a> reported for comparison.</p

Enzymatic and cytotoxic properties of BS-RNase oligomers.

a<p>IC₅₀ mean values (± S.D.) from three independent experiments on VIT1 cells after 72 h.</p>b<p>folds vs the IC₅₀ value of native BS-RNase dimer D.</p>c<p>L.O.: mixture of BS-RNase hexamers, octamers, and larger oligomers.</p

Action of BS-RNase oligomers on the proliferation of mesenchimal VIT1 cells.

<p>(<b>A</b>) wt, and (<b>B</b>) K113N BS-RNase. Cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 10% FBS, and 50 µg/ml gentamicin sulphate. After addition of the BS-RNase species, 10 to 240 µg/ml of BS-RNase (native) dimer, or of 10 to 80 µg/ml of BS TT_1/C, TT_2/N, or a mixture of larger oligomers (L.O.), cells were incubated for 72 h at 37°C with 5% (v/v) CO₂. At the end of the treatments cells were stained with a Crystal Violet solution and the survival was measured, and compared to the control lacking any RNase species, as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046804#s3" target="_blank">Materials and Methods</a>. Experiments were performed in triplicate; the S.D. are comprised between 4.5 and 6.0% for wt (<b>A</b>), and between 5.1 and 7.7% for K113N BS-RNase (<b>B</b>).</p