Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase Carmine Ercole 1 , Francesca Avitabile 1 , Pompea Del Vecchio 1 , Orlando Crescenzi 1 , Teodorico Tancredi 2 and Delia Picone 1 1 Dipartimento di Chimica, Universita ` di Napoli Federico II, Italy; 2 Istituto Chimica Biomolecolare del CNR, Napoli, Italy Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1–20 residues, and M¼M, without exchange. As a consequence, the hinge region 16–22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M¼M is about 7 : 3. Kinetic analysis of the swapping pro- cess for a recombinant sample shows that it folds mainly in the M¼M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS- RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation sig- nificantly affects both the dimerization and swapping pro- cesses but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues. Keywords: bovine seminal ribonuclease; domain swapping; proline; ribonuclease A; site-directed mutagenesis. Bovine seminal ribonuclease (BS-RNase), the only dimeric protein in the pancreatic-type ribonuclease family, is characterized in solution by an equilibrium between two different structures [1]: in the form dubbed MxM, the N-terminal arms are exchanged, or swapped, between the two identical subunits, whereas in the form indicated as M¼M no swapping occurs. In the native protein, the equilibrium ratio between MxM and M¼M is about 7 : 3. The two identical subunits are linked through two disulfide bridges between Cys31 and 32 of one subunit with Cys32¢ and 31¢, respectively, of the partner subunit. Each subunit has 83% of the amino-acid sequence identical with that of bovine pancreatic RNase A. In particular, both enzymes exhibit active sites constituted by identical amino-acid residues in the same sequence position. Beside ribonuclease activity, BS-RNase is endowed with several additional biological activities, such as allostery [2], cytotoxicity toward malignant cells [3], immunosuppression and antispermato- genesis [4]. Domain swapping in BS-RNase was found to be determinant for all of these activities, which may suggest a physiological role for this structural peculiarity. A folded and stable monomeric derivative of BS-RNase can be obtained by selective reduction of the dimeric protein with a moderate excess of dithiothreitol, and stabilized by either alkylation of the exposed thiol groups [5] or reaction with glutathione [6]. All monomeric derivatives of BS-RNase are catalytically more active than the native dimeric enzyme, but they do not exhibit any allosteric property and have nodetectable ÔspecialÕ biological action [7]. In a recent paper, we reported an NMR characterization of the N67D variant of monomeric BS-RNase [8], hence- forth called mBS. The mutation avoids sample heterogen- eity arising from the spontaneous deamidation of Asn67 [9], but it does not affect enzymatic activity. Comparison of the solution structures, as well as specific NMR relaxation experiments, indicated that the hinge region 16–22 is much more flexible in mBS than in RNase A. However, this region shows the greatest sequence difference from RN- ase A: GNSPSSS in BS-RNase vs. STSAASS in RNase A. As a consequence of its flexibility, the structure of this segment is not well defined in the solution structure of mBS (Fig. 1A). Moreover, owing to extensive overlap of diag- nostic signals, we could not unequivocally assign trans isomerism to Pro19. Mutagenic studies have shown that Pro19 and Leu28, which in BS-RNase makes a hydrophobic contact at the interface between the two subunits (Fig. 1B), are two crucial residues in inducing dimerization and swapping N-terminal arms in RNase A variants [10,11]. As the first step of a study aimed to investigate, through a Correspondence to D. Picone, Dipartimento di Chimica, Universita ` di Napoli Federico II, Via Cintia, 80126, Napoli, Italy. Fax: + 39 081 674409, Tel.: + 39 081 674406, E-mail: picone@chemistry.unina.it Abbreviations: BS-RNase, bovine seminal ribonuclease; mBS, mono- meric N67D BS-RNase; RNase A, bovine pancreatic ribonuclease; DVS, divinyl sulfone. (Received 1 August 2003, revised 2 October 2003, accepted 7 October 2003) Eur. J. Biochem. 270, 4729–4735 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03872.x systematic mutagenic approach, the role played by the hinge and interface regions in the swapping process, we prepared BS-RNase variants by replacing Pro19 and Leu28 with the corresponding residues from RNase A. Here we report a characterization of monomeric P19A, L28Q and P19A/ L28Q variants of mBS carried out by 2D NMR, CD and differential scanning microcalorimetry, and an investigation of the kinetics of swapping of all variant dimers in comparison with that of the parent protein. Materials and methods Construction of mBS mutants Site-directed mutagenesis was performed by a megaprimer PCR method [12] to produce the mutants coding for P19A- mBS, L28Q-mBS and P19A/L28Q-mBS, starting from the pET-22b(+) plasmid cDNA coding for the wild-type enzyme which already carries the N67D mutation, to avoid sample heterogeneity by spontaneous deamidation at the Asn67 site [8]. PCR amplification was performed with an Eppendorf Mastercycler amplifier. The forward flanking primer sequence used in these experiments, 5¢-GAGTGCGGCC GCAAGCTTGGGCTG-3¢, had an estimated T m of 82 °C. The reverse flanking primer sequence, 5¢-ATATACA TATGAAAGAAAG-3¢, had a calculated T m of 42 °C. The mutagenic primers for each variant are: P19A (5¢-AGA GCTGCT AGCAGAGTTG-3¢) and L28Q (5¢-CACAT CATC CTGGTTGCAA-3¢) (nucleotides that represent mutations are underlined). For the mutant P19A/L28Q- mBS the mutagenic primer L28Q was used starting from the pET-22b(+) plasmid cDNA coding for the mutant P19A- mBS. The amplified, mutated genes were separated, excised, and purified from the agarose gel followed by cloning into pET-22b(+) between the HindIII and NdeIsites. Insertion of the correct mutations was confirmed by DNA sequencing. Recovery of proteins All the proteins were expressed in Escherichia coli and purified in monomeric form, with Cys31 and 32 linked to two glutathione molecules, as described previously [13]. Monomers with Cys31 and 32 in the reduced form were obtained by selective reduction of the mixed disulfide bridges with a 5 : 1 molar excess of dithiothreitol for 20 min at room temperature in 0.1 M Tris/acetate buffer, pH 8.4. The samples were either carboxyamidomethylated with iodoacetamide [5], to obtain the monomeric proteins used for CD and microcalorimetric analysis, or dialyzed against 0.1 M Tris/acetate, pH 8.4, for 20 h at 4 °C, to obtain dimers. The last step of the purification procedure was always a gel filtration on Sephadex G-75 to separate monomers from dimers. All dimerization steps were performed at 4 °C. Recombinant RNase A was obtained and purified as described previously [8]. Protein homogeneity was verified by SDS/PAGE and MALDI-TOF MS, registered at the Sezione di Fig. 1. Ribbon representation of the solution structure of mBS-RNase (A), as derived from heteronuclear NMR data (pdb accession code 1WQW), and the X-ray structure of the MxM form of BS-RNase (B; pdb accession code 1BSR). Pro19 and Leu28 are highlighted. The figure was drawn with MOLMOL software [25]. 4730 C. Ercole et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Spettrometria di Massa of the CIMCF, Universita ` degli Studi di Napoli Federico II. Protein concentration was measured by UV spectrophotometry. Kinetics of interconversion of dimeric forms To follow the interconversion kinetics, dimer samples were incubated at 37 °C. At given times, aliquots were with- drawn, the interchain disulfide bridges were selectively reduced as described above [1], and the mixture was chromatographed on an analytical Superdex 75 HR 10/30 column. The amount of MxM and M¼M was evaluated quantitatively by integrating the peaks of dimer and monomer, respectively. Assessing the extent of the N-terminal swap at equilibrium Cross-linking experiments were performed using divinyl sulfone (DVS) as a 10% solution in ethanol. The protein (20 lg) in sodium acetate buffer (100 m M ,pH5,100lL) and DVS (1 lL of the 10% solution) was incubated at 30 °C [11]; this is  1000-fold excess of sulfone over each subunit of the protein. Aliquots were withdrawn over a period of 96 h, quenched with 2-mercaptoethanol (final concentration 200 m M ), incubated for 15–30 min at room temperature, and loaded on gels for reducing SDS/PAGE. The ratio of monomer to cross-linked dimer was estimated qualitatively by Coomassie blue staining. NMR NMR measurements were performed on Bruker DRX400 and DRX500 spectrometers. All spectra were collected using the standard Bruker pulse sequence library. Protein concentration was 2 m M in 95% H 2 O/5% D 2 O, pH 5.65. CD The CD spectra were recorded with a Jasco J-715 spectro- polarimeter equipped with a Peltier-type temperature con- trol system (model PTC-348WI). The instrument was calibrated with an aqueous solution of D -10-camphorsulf- onic acid at 290 nm [14]. Molar ellipticity per mean residue, [h] in degreesÆcm 2 Ædmol )1 , was calculated from the equation [h] ¼ h obs mrw/10lC,whereh obs is the ellipticity measured in degrees, mrw is the mean residue molecular mass (117 Da [5]), C is the protein concentration in gÆL )1 ,andl is the optical path length of the cell in cm. A 0.1-cm path length cell and a protein concentration of  0.3 mgÆmL )1 in 10 m M sodium acetate buffer, pH 5.0, were used. CD spectra were recorded at 25 °C with a time constant of 16 s, a 2-nm band width, and a scan rate of 5 nmÆmin )1 ; they were signal- averaged over at least five scans, and baseline-corrected by subtracting the buffer spectrum. Thermal unfolding curves were recorded in the temperature scan mode at 222 nm from 25 °Cupto85°C with a scan rate of 0.5 KÆmin )1 . Scanning calorimetry Calorimetric measurements were performed on a second- generation Setaram Micro-DSC. A scanning rate of 0.5 °CÆmin )1 was chosen for all experiments. The raw data were converted into an apparent molar heat capacity taking into account the instrument calibration curve and the buffer–buffer scanning curve, and by dividing each data point by the scan rate and the protein molar concentration in the sample cell. Finally, the excess molar heat capacity function, <DCp>, was obtained after baseline subtraction, assuming as reference the heat capacity of the native state [15]. Results Recombinant mBS and its P19A, L28Q and P19A/L28Q variants (P19A-mBS, L28Q-mBS and P19A/L28Q-mBS, respectively), all with Cys31 and 32 linked to two glutathi- one molecules, were obtained in pure form with a yield of about 15 mgÆper L culture. Each of these variants retains a catalytic activity against yeast RNA comparable with that of parent mBS, indicating that a native conformation is present. A further indication of the similarity of their global fold to that of the parent protein is provided by the 1D 1 H-NMR spectra (data not shown), which display all the characteristic signals in almost identical positions. The similarity was confirmed by CD measurements (Fig. 2). The estimation of secondary-structure content, performed by the neural network-based procedure implemented in the program K 2 D [16,17], yielded very similar values for all the protein samples (28% a-helix, 36% b-sheet and 40% random coil); these values are also in good agreement with the secondary structure derived from the NMR structure of mBS [8]. To allow a more accurate evaluation of the effect of single point mutations on the solution structure of monomeric derivatives, we analysed the 2D NMR spectra of the different variants of mBS. Figure 3 shows the expanded regions of TOCSY spectra of L28Q-mBS (panel L28Q), P19A/L28Q-mBS (panel PALQ) and P19A-mBS (panel P19A), in comparison with the same region of the parent mBS (panel mBS). The new signal at 8.40–1.40 p.p.m., which appears in the spectra of P19A-mBS and Fig. 2. Far-UV spectra of mBS-RNase (solid curve), P19A-mBS-RNase (dashed curve), L28Q-mBS-RNase (dotted/dashed curve) and P19A/ L28Q-mBS-RNase (dotted curve) in 10 m M sodium acetate buffer, pH 5.0, 25 °C. The horizontal dotted line indicates the zero value of the ellipticity. Ó FEBS 2003 Structural properties of BS-RNase variants (Eur. J. Biochem. 270) 4731 P19A/L28Q-mBS, has been attributed to Ala19 in spite of the difference with the assignment made for RNase A in very similar experimental conditions [18]. Moreover, in the spectra of L28Q-mBS and P19A/L28Q-mBS, the correla- tions that belong to Leu28 are missing, and are replaced by a set of new signals tentatively attributed to Gln28 (data not shown). Apart from these expected differences, the high similarity among the spectra of the variants and the parent protein provides additional, strong evidence of an essentially identical fold of all variant proteins. However, the NH-C b H 3 cross-peak of Ala19 is significantly broader than other cross-peaks; this may reflect an equilibrium between different conformations of the 16–22 hinge region, occurring at a rate comparable to the NMR chemical shift time scale. A thermodynamic characterization of all monomeric proteins was performed by differential scanning calori- metry. The results, shown in Table 1, agreed with the temperature-induced unfolding curves obtained by CD measurements (data not shown). No sizeable differences were found among the monomeric variants tested, and only for P19A-mBS a significant thermal stabilization occurs. The introduction of Ala for Pro19 makes mBS more similar to RNase A, which has a thermal stability higher than that of mBS. However, P19A/L28Q-mBS has the same thermal stability of the parent protein, in spite of greater similarity to RNase A. To directly characterize the process of exchange of the N-termini, we prepared dimers of all variants. The mono- meric proteins were submitted to mild reduction, which selectively removes the glutathione molecules linked to Cys31 and 32, followed by air oxidation and gel filtration on Sephadex G-75 to obtain the corresponding dimers. We obtained 80% of dimer for both BS-RNase and P19A-BS- RNase, whereas in the case of L28Q and P19A/L28Q variants the yield of dimer was significantly lower,  50%. The recombinant dimers were obtained predominantly in their nonswapped forms; the extent of exchange of the N-termini, as evaluated by selective reduction of the interchain disulfide bridges followed by gel filtration (vide supra), was initially  15%. The dimers were then incubated at 37 °C for a week, to allow equilibration between the interconverting forms. A qualitative evaluation of the extent of swapping at equilibrium was performed by cross-linking experiments with DVS, followed by SDS/PAGE analysis under reducing conditions. DVS covalently joins the two His residues of the active site (His12 and His119) [11], which belong to the same subunit in M¼ M and to different subunits in MxM. Thus, upon reduction and denaturation, either a product of molecular mass 27 000 Da (from MxM) or a product of molecular mass 13 500 Da (from M¼M) is obtained. Figure 4 depicts the time-dependent course of this reaction in the equilibrium mixtures of dimers. After 24 h of reaction, all proteins show almost the same relative proportion of exchanging and nonexchanging forms. However, after 48 h, a slight prevalence of the MxM form can be seen in the BS-RNase and P19A variant, whereas the L28Q and P19A/L28Q variants still contain comparable amounts of MxM and M¼M. This becomes even more Fig. 3. Regions of 500 MHz 2D TOCSY spectra of L28Q-mBS-RNase (panel L28Q), P19A/L28Q-mBS-RNase (panel PALQ), P19A-mBS-RNase (panel P19A), and mBS-RNase (panel mBS). The N 1 H/b-methyl cross-peak of Ala19 is boxed in the P19A and PALQ panels. Table 1. Thermodynamic parameters of thermal denaturation of mBS, P19A-mBS, L28Q-mBS, P19A/L28Q-mBS and RNase A. Estimated error on Td, DdH and DdCp was 0.2 °C, 5% and 10%, respectively. Td(°C) DdH(Td) (kJÆmol )1 ) DdCp (kJÆmol )1 ÆK )1 ) mBS 53.4 370 4.8 P19A-mBS 55.0 383 4.6 L28Q-mBS 53.0 360 7.5 P19A/L28Q-mBS 53.0 387 5.0 RNase A 58.8 423 4.1 4732 C. Ercole et al.(Eur. J. Biochem. 270) Ó FEBS 2003 evident when the reaction time is extended to 96 h: the approximate 1 : 1 ratio for L28Q and P19A/L28Q variants did not change, whereas a clear prevalence of the exchanged form is present in native BS-RNase and P19A variant. A more detailed comparison of the swapping process among the different BS-RNase variants has been performed by a different method, based on the observation that, after selective reduction of the interchain disulfide bridges, the MxM form retains a dimeric structure, whereas the M¼M form dissociates into two monomers upon gel filtration [1]. We observed (Fig. 5A) that, at 37 °C, the dislocation of the N-terminal arms for the recombinant BS-RNase is very similar to that reported for the native enzyme [1], reaching the same 7 : 3 equilibrium in  5 days. This result also provides indirect evidence of correct pairing of the inter- chain disulfide bridges in the recombinant sample [11]. The kinetics of the N-terminal arms swapping in the P19A-BS- RNase is comparable to that of BS-RNase (Fig. 5B). Interestingly, the amount of the MxM form at the plateau as evaluated by integrating the peaks obtained on gel filtration is 70%, i.e. in complete agreement with that obtained for the native enzyme and also in agreement with the qualitative estimation based on the DVS reaction. In contrast, Fig. 5C,D clearly shows that replacement of Leu28 with Gln affects in a significant way the kinetics and equilibrium of swapping in both the L28Q-BS-RNase and P19A/L28Q-BS-RNase variants. Furthermore, the plateau corresponds to 50% of MxM for both proteins, in agreement with the DVS cross-linking results. As reported previously [1], at 4 °C the interconversion of the native protein was obviously slowed down. For instance, after 500 h only 25% of MxM is present in BS-RNase, and 20% in P19A-BS-RNase, i.e. the process is still far from equilibrium. To assess this hypothesis, we monitored the reverse process, namely the interconversion of MxM into M¼M, for P19A-BS-RNase. To isolate the MxM dimer, the equilibrium mixture, containing both forms in the usual 7 : 3 ratio, was reduced selectively with dithiothreitol and gel filtered. The fractions containing the reduced dimer were pooled and air oxidized. The amount of the MxM form in the isolated sample at this stage was 85%. Aliquots were then separately incubated at 4 °Cand37°C, and the extent of the swapping was assessed as previously described. Figure 6 shows the amount of the MxM form present as a function of incubation time. P19A-BS-RNase behaves like the native sample [1], reaching the typical equilibrium mixture in  180 h at 37 °C, whereas at 4 °C the process seems kinetically frozen, and the content of MxM is constant over several weeks. Discussion In this work, we have investigated the effects of mutation of two paradigmatic residues involved in the dislocation of BS-RNase N-terminal arms, i.e. Pro19 and Leu28, located in the hinge peptide and in the intersubunit interface, respectively. CD and NMR spectra suggest a close similarity in the global fold and in the structural properties of all monomeric variants to those of the parent protein. These results are in agreement with the solution structure of mBS [8], which indicates that both residues are solvent exposed and make only a limited number of van der Waals interactions with the rest of the protein. The only difference related to the substitution of Pro with Ala is a slight increase in the thermal stability, which becomes more similar to that of RNase A. This result is in agreement with theoretical calculations [19], and CD and calorimetric studies on the A19P-RNase A variant [20], which both support a destabilizing effect resulting from the introduction of a Pro at position 19 in RNase A. Surprisingly, the substitution of Pro19 by Ala does not affect the dislocation of N-termini in BS-RNase. A Pro residue is often found in crucial regions of other domain swapped proteins [21], and cis–trans Pro isomerization has been regarded as a key event in protein folding (for a recent overview, see Wedemeyer et al. [22]). This observation led to the definition of hinge Pro residues as Ôquaternary structure helpersÕ. The similarity of kinetic behaviour between BS-RNase and P19A-BS-RNase seems to rule out the involvement of a cis–trans Pro19 isomerization as a crucial step in the swapping mechanism of BS-RNase. In the crystallographic structure of BS-RNase MxM form [23], the peptide bond between residues 18 and 19 has a trans conformation, and it seems reasonable to assume that the same holds for the P19A-BS-RNase MxM form. If Pro19 were cis in the M¼M form, the P19A variant would hardly be expected to display such similar thermodynamic and kinetic behaviour in the interconversion process, as a cis peptide bond is significantly less stable for Ala than for Pro. In other words, in the P19A M¼M form, Ala19 would either be forced into an unfavourable cis conformation or would have already assumed the intrinsically preferred trans conformation; in both cases, an effect on the swapping process would be expected. Even from a structural point of view, the role of Pro19 is still ambiguous. All structural studies on BS-RNase report poor definition of the hinge region around Pro19, and this abnormality has often been taken as an indication of high Fig. 4. SDS/PAGE analysis of the DVS cross- linking reaction of BS-RNase (lane 1), P19A- BS-RNase (lane 2), L28Q-BS-RNase (lane 3) and P19A/L28Q-BS-RNase (lane 4). Ó FEBS 2003 Structural properties of BS-RNase variants (Eur. J. Biochem. 270) 4733 intrinsic flexibility of this tract of the protein. Yet, in the crystallographic structure of a dimeric variant of human pancreatic ribonuclease, called PM8 [24], in which the N-terminal region and the whole hinge loop are identical with those of BS-RNase, the hinge assumes a 3 10 helix structure. This result suggests that the flexibility of the corresponding region in BS-RNase cannot be ascribed to the hinge loop itself, but probably arises from interactions with other parts of the molecule. Overall, our data indicate that Pro19 is not crucial in the swapping process, and suggest that the structural determi- nants for this process are located in regions different from the hinge loop 16–22. In the search for the region primarily involved in the swapping, we focused our attention on the interface between the subunits. In a previous study [8] we found that, in mBS, helix 2 (residues 24–32) is more disordered than in RNase A. This disorder may be attributed to the proximity of the hinge peptide (residues 16–22), or possibly to an unfavourable solvent exposure of the Leu28 side chain (residue 28 is Gln in RNase A). As far as the monomer is concerned, introduction of Gln for Leu at position 28 does not increase the stability or significantly affect the structure, in spite of the greater hydrophilicity and helical propensity of Gln. However, the presence of Leu28 in BS-RNase seems to favour the dimerization process: for both L28Q-BS- RNase and P19A/L28Q-BS-RNase, the yield of dimer obtained on oxidation of the monomeric, reduced protein is only 50%, which is significantly lower than for BS-RNase and P19A-BS-RNase (at least 80%). Interestingly, literature data for some variants of RNase A also report a higher yield of dimer when Gln28 is substituted with Leu11. The influence of the Leu side chain is also evident during the swapping process, and again the two mutants in which Leu28 had been substituted display closely matching kinetic behaviours and at equilibrium contain a smaller percentage of MxM (50%). Thus, several data indicate that Leu28 is crucial not only for the interaction between the subunits, but even for the swapping process. Finally, the close similarity between the swapping behaviour of the N-terminal arms in L28Q-BS-RNase and P19A/L28Q-BS-RNase confirms that Pro19 does not represent a key residue in this process. From a different perspective, our results provide further evidence of the evolutionary significance of the A19P and Q28L substitutions in the path leading from RNase A to Fig. 6. Kinetic analysis of the MxM to M=M conversion for P19A-BS- RNase. The percentage of MxM form as a function of the incubation time at 4 °C(r)and37°C(d) is reported. Fig. 5. Kinetic analysis of the M=M to MxM conversion for BS-RNase (A), P19A-BS-RNase (B), L28Q-BS-RNase (C) and P19A/L28Q-BS- RNase (D). The percentage of the MxM form as a function of the incubation time at 4 °C(r)and37°C(d) is reported for each dimeric protein. 4734 C. Ercole et al.(Eur. J. Biochem. 270) Ó FEBS 2003 BS-RNase. The former substitution destabilizes the mono- meric form, and the latter specifically favours the swapped dimer. Acknowledgements We thank the CIMCF of the University ÔFederico IIÕ of Naples where we acquired some of the NMR spectra. We are very grateful to Professor Giuseppe D’Alessio for critical reading of the manuscript. We also thank Professor Alberto Di Donato and Dr Valeria Cafaro for their kind hospitality and advice on protein expression and purification. Financial support was from CNR (Agenzia 2000) and Ministero dell’Istruzione e dell’Universita ` (Prin 2002). References 1. 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