Structural properties of the protein SV-IV Carlo Caporale 1 , Carla Caruso 1 , Giovanni Colonna 2,3 , Angelo Facchiano 4 , Pasquale Ferranti 4,5 , Gianfranco Mamone 4 , Gianluca Picariello 4 , Flavia Colonna 3 , Salvatore Metafora 6 and Paola Stiuso 2,3 1 Dipartimento di Agrobiologia ed Agrochimica, Universita ´ della Tuscia, Viterbo, Italy; 2 Dipartimento di Biochimica e Biofisica, Seconda University Napoli, Italy; 3 Centro di Ricerca Interdipartimentale di Scienze Computazionali e Biotecnologiche, Napoli, Italy; 4 Istituto di Scienze dell’Alimentazione, CNR, Roma, Italy; 5 Dipartimento di Scienza degli Alimenti, Universita ` degli Studi di Napoli ‘Federico II’, Italy; 6 Istituto Internazionale di Genetica e Biofisica, CNR, Napoli, Italy We have investigated the molecular mechanisms that produce different structural and functional behavior in the monomeric and trimeric forms of seminal vesicle protein no. 4, a protein with immunomodulatory, anti-inflamma- tory, and procoagulant activity secreted from the rat seminal vesicle epithelium. The monomeric and trimeric forms were characterized in solution by CD. Details of the self-association process and structural changes that accompany aggregation were investigated by different experimental approaches: trypsin proteolysis, sequence analysis, chemical modification, and computer modeling. The self-association process induces conformational change mainly in the 1–70 region, which appears to be without secondary structure in the monomer but contains a-helix in the trimer. In vivo, proteolysis of seminal vesicle protein no. 4 generates active peptides and this is affected by the monomer/trimer state, which is regulated by the concentration of the protein. The information obtained shows how conformational changes between the mono- meric and trimeric forms represent a crucial aspect of activity modulation. Keywords: monomer; proteolysis; seminal vesicle protein; SV-IV; trimer. SV-IV (seminal vesicle protein no. 4, according to its electrophoretic mobility in SDS/PAGE) is a basic (pI ¼ 8.9), thermostable, secretory protein of low M r (9758) secreted from the rat seminal vesicle epithelium under strict androgen transcriptional control [1–6]. SV-IV has been purified to homogeneity and characterized exten- sively [1–7]. We have demonstrated that this protein is a highly flexible molecule behaving in aqueous solution as a concentration-dependent self-associating system, with the degree of association (monomer « dimer « trimer equilibrium) related to its biological activity [7]. Its polypeptide sequence is 90 amino acids long and is encoded by a gene that has been isolated, sequenced, and expressed in Escherichia coli [8–11]. SV-IV possesses potent nonspecies-specific immunomodulatory, anti- inflammatory, and procoagulant activity [12–22]. We have demonstrated recently by electrospray MS that 10% of the native SV-IV molecules are phosphorylated in vitro by protein kinase C and that this modification involves only Ser58 [23]. Furthermore, we have unam- biguously demonstrated that a Tyr36-linked phosphate group is present in 14% of all native SV-IV molecules [24]. SV-IV possesses a marked ability to inhibit both in vivo and in vitro phospholipase A 2 activity and the platelet- activating factor biosynthetic pathway [13–15]. The native protein, transformed by transglutaminase (EC 2.3.2.13) into a complex polymer, binds to the surface of epididymal spermatozoa, greatly decreasing their strong immunogenic- ity [25,26]. Although many studies have been devoted to the functional aspects of this protein, very little is known about its structural properties and conformational behavior in aqueous solutions. Recent studies have shown that its biological activities are modulated by molecular association of the protein [7]. In this paper, we characterize the solution structure of the monomeric and trimeric forms of SV-IV. Experimental CD spectra were deconvoluted into secon- dary-structural elements and compared with structural predictions. Finally, details of the self-association process and structural changes that accompany aggregation were investigated by different experimental approaches: trypsin proteolysis, sequence analysis, chemical modification, and computer modeling. Materials and methods The experiments were all repeated at least four times. Chemicals All chemicals were of reagent grade and purchased from BDH (Milan, Italy) or Sigma-Aldrich (Milan, Italy). HPLC-grade solvents and reagents were obtained from Carlo Erba (Milan, Italy). Endoproteinase Glu-C and trypsin (sequence-grade) were from Boehringer-Mann- heim. Correspondence to P. Stiuso, Dipartimento di Biochimica e Biofisica, Seconda Universita ` degli studi di Napoli, Via Costantinopoli 16, 80138-Napoli, Italy. Fax: + 39 81 5665869, E-mail: paola.stiuso@unina2.it Abbreviation: SV-IV, seminal vesicle protein no. 4. (Received 20 June 2003, revised 24 September 2003, accepted 14 November 2003) Eur. J. Biochem. 271, 263–271 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03925.x Purification of SV-IV SV-IV was purified to homogeneity from adult rat (Wistar– Fisher strain) seminal vesicle secretion by a previously published technique [1]. The purity of the protein was assessed by electrophoresis on 15% polyacrylamide gel in denaturing and non-denaturing conditions, analysis of amino-acid composition, fingerprint technique, and fast atom bombardment MS [3,22]. The preparations of SV-IV were completely free of lipopolysaccharide and tumor necrosis factor as determined by specific biological assays [27,28]. The concentration of the purified protein was evaluated by its molar absorption at 276 nm (4100 M )1 Æcm )1 ), calculated on the basis of the tyrosine and phenylalanine residues present in the polypeptide chain [7]. Spectral measurements CD measurements were performed at room temperature with a Jasco J-720 spectropolarimeter, using quartz cells withapathlengthof1cmand1mm.Meanresidue ellipticities were calculated from: ½h¼MRMh obs =cd where [h] is the mean residue ellipticity in degreesÆcm )2 Æ dmol )1 , h obs is the observed ellipticity, MRM is the mean residue molecular mass calculated from the sequence, d is the optical path length (cm), and c is the concentration in gÆmL )1 . The CD spectra were analyzed in the region between 200 and 250 nm to evaluate the amount of secondary structure by using the instrument computerized program. Spectroscopic analyses were always carried out on dialyzed samples. Concentration difference spectra The difference spectra were determined by comparison of the spectra measured with two different protein concen- trations in two different cells. The CD spectra were obtained at 25 °C, using two cells with different light-path lengths (L1 and L2) and filled with solutions of SV-IV in NaCl/P i (0.15 M NaCl in 0.05 M sodium phosphate buffer, pH 7.5). The SV-IV concentrations, C1 and C2, were chosen in such a way that C1 · L1 ¼ C2 · L2. In these conditions, equal numbers of molecules are expected to be in the light pathway at the two different concentrations used. Digestion of monomeric and trimeric SV-IV with trypsin First, 25 nmol monomeric (protein concentration 0.015 mgÆmL )1 ) and trimeric (protein concentration 1.0 mgÆmL )1 ) SV-IV were digested separately with trypsin (enzyme/ substrate, 1 : 50, w/w) at 37 °C in 0.1% ammonium bicarbonate buffer, pH 8.0. Aliquots of the incubation mixtures, corresponding to 5 nmol of the original protein, were then withdrawn at times ranging from 15 min to 12 h and freeze-dried. The digests were then dissolved in 0.2 mL aqueous 0.1% trifluoroacetic acid and resolved by RP- HPLC on a l-Bondapak C 18 column. Eluent A was aqueous 0.1% trifluoroacetic acid and eluent B was 0.07% trifluoroacetic acid in acetonitrile. The elution was per- formed at a flow rate of 1 mLÆmin )1 using the following program: 10 min 5% B followed by a two-step linear gradient from 5% to 18% B over 50 min and from 18% to 28% B over 70 min. Peaks were collected manually and freeze-dried. HPLC procedures were carried out on a Beckman GOLD apparatus equipped with a variable- wavelength monitor (model 166). The l-Bondapak C 18 column (0.39 · 30 cm) was from Waters-Millipore (Mil- ford, MA, USA). Sequence analyses The purified tryptic peptides of monomeric and trimeric SV-IV were dissolved in aqueous 0.1% trifluoroacetic acid (30–60 lL); aliquots (200–500 pmol) were submitted to sequence analysis using a pulsed liquid-phase automatic sequencer (model 477A) equipped on-line with phenyl- thiohydantoin amino acid analyzer (model 120A). Relevant reagents were from Perkin Elmer/Applied Biosystems. Samples were loaded on to a trifluoroacetic acid-treated glass-fiber filter, coated with polybrene, and washed according to the manufacturer’s instructions. The average and combined repetitive amino acid yields determined by the instrument software were not lower than 90% for each sequenced peptide. The theoretical initial yields were not lower than 50%. Acetylation Appropriate amounts of purified native SV-IV (trimeric form, 4300 gÆmL )1 ) were acetylated in the presence of excess acetic anhydride (6 : 1, w/v) over total amino groups, and then purified by HPLC. HPLC/electrospray MS HPLC was performed using a C 18 ,5lm reverse-phase column (2.1 mm internal diameter · 250 mm; Vydac) with a flow rate of 0.5 mLÆmin )1 on a Kontron modular system. The column effluent was split 1 : 25 with a Valco tee to give a flow rate of about 20 lLÆmin )1 into the electrospray nebuliser. The bulk of the flow was run through the detector for peak collection after reading of peptide absorbance at 220 nm. Solvent A was 0.03% trifluoroacetic acid in water (v/v); solvent B was 0.02% trifluoroacetic acid in acetonitrile. The electrospray device was a Platform single-quadrupole mass spectrometer (Micromass, Manchester, UK). The source temperature was 120 °C. Mass scale calibration was carried out using myoglobin as the reference compound. Quantitative analysis of components was performed by integration of the multiple charged ions of the single species. For protein analysis, the separation was attained with a linear gradient of 20–40% solvent B over 40 min and mass spectra were acquired in the range 1800–500 m/z at a scan cycle of 5 s/scan. For peptide analysis, the separation was carried out with a linear gradient of 8–40% solvent B over 60 min, and mass spectra were acquired in the range 1600–400 m/z at a scan cycle of 5 s/scan. 264 C. Caporale et al.(Eur. J. Biochem. 271) Ó FEBS 2003 Endoproteinase Glu-C digestion Endoproteinase Glu-C (Boehringer-Mannheim Italia) hydrolytic digestion was carried out in 0.4% ammonium bicarbonate, pH 8, at 40 °C for 18 h at a substrate/enzyme ratio of 50 : 1 (w/w). The reaction was stopped by freeze- drying. MALDI-TOF MS a-Cyano-4-hydroxycinnamic acid (Fluka, Buchs, Switzer- land) was used as matrix. The protein or peptide samples (1 lLfromasolution1gÆL )1 in water) were loaded on the target and dried. Afterwards, 1 lLofa10mgÆmL )1 solution of matrix in a mixture of 0.1% trifluoroacetic acid in water and acetonitrile. The samples were analysed with a Voyager DE-Pro (PerSeptive Biosystem, Framing- ham, MA, USA) mass spectrometer operating either in linear or in reflector mode for post source decay tandem MS. Structure predictions and modeling Software and databases publicly available on the net were used for the sequence analyses and structure predictions. BLAST [29] was used to search for amino-acid sequence similarities between the SV-IV sequence and proteins collected in databases. 3D-PSSM [30], genetTHREAD [31], and TOPITS [32] were used to apply the fold recognition strategy, searching for known protein folds compatible with the SV-IV sequence. PHD [33], JPRED [34], and PSI- PRED [35] web services were used to predict the secondary structure. The 3D model of the peptide corresponding to the segment 70–90 of SV-IV was created by using the INSIGHTII package (Accelrys, San Diego, CA, USA). The Biopolymer module was used to build the chain of amino acids, folded as an a-helix in agreement with the secondary-structure prediction and CD spectra results. The initial model was geometrically optimized by energy minimization according to the standard settings of the Optimize option. Results Conformational study of SV-IV Structural modifications between the monomeric and trimeric form of the SV-IV protein are evident on CD spectra (Fig. 1). The far-UV CD spectra, as characterized by an isodichroic point located at about 208 nm indicate the presence of two-state equilibria between the monomeric and trimeric forms. The self-association process was accompan- ied by structural changes in the protein. The secondary- structure analysis program estimated that the a-helix content is 24% in the monomeric form and 45% in the trimeric form, and b-structure is absent from both forms. The CD spectra of the 1–70 and 70–90 regions of the protein are reported in the inset of Fig. 1. The secondary-structure analysis program estimated about 100% a-helix for the 70–90 fragment and 2–3% for the 1–70 segment. This suggests that the C-terminal segment of the protein is organized as a-helix in the whole protein, and the 1–70 region is poorly structured. SDS increases the a-helix content of proteins revealing the helical potential. The a-helix content of the monomeric form of SV-IV is approximately doubled by the addition of 5.4 m M SDS (Fig. 1), whereas in the 1–70 region, the a-helix content increases from 2–3% to  23% in the presence of SDS (data not shown). This suggests that the effect of SDS on the whole protein is exerted in the 1–70 region, which probably plays a fundamental role in the self-association process, with secondary-structure reorganization occurring in going from the monomeric to the trimeric form. Predictive methods have been applied to obtain a theoretical model of the structural organization of SV-IV protein. The amino-acid sequence was analyzed using the BLAST program to find similar proteins in the ÔnrÕ database (nonredundant database consisting of all protein sequences Fig. 1. Structural characterization of the monomeric (0.01 lgÆlL )1 ) and trimeric (0.1 lgÆlL )1 ) form of SV-IV protein. Far-UV CDspectraatdifferentconcentrationsofthe protein (A, 0.01 lgÆlL )1 monomeric form; B, 0.05 lgÆlL )1 monomeric/trimeric mixture; C, 0.1 lgÆlL )1 trimeric form). Monomeric form in SDS (j,5.4m M ). The CD spectra of the 1–70 and 70–90 fragment of SV-IV are reported in the inset. Each spectrum represents an average of five scans. The SV-IV samples are in 50 m M Tris/HCl, pH 7.2. Ó FEBS 2003 Structural properties of SV-IV (Eur. J. Biochem. 271) 265 present in the databases). No protein of known 3D structure was found to have sequence similarity suitable to apply the homology modeling strategy, i.e. at least 20–30% sequence identity. As an alternative, the fold-recognition approach was applied by using three independent servers on the net: 3D-PSSM, GenetTHREAD, TOPITS. None of the meth- ods identified a known fold suitable for modeling the SV-IV protein or the 1–70 region. Therefore, in conclusion, the two most reliable strategies for predicting the 3D model of a protein, i.e. homology modeling and fold-recognition strat- egies, were unable to create a model for either the whole SV-IV protein or the 1–70 region, and this suggests that this protein assumes a global structure that is not similar to any protein of known 3D structure. Secondary-structure predictions were performed by dif- ferent methods, i.e. JPRED, PHD, PSI-PRED (Fig. 2). A Ôconsensus predictionÕ based on the agreement between different methods can be considered more successful than the single method used. The consensus prediction suggests a few helical regions (48–53 and the C-terminal region) covering  20% of the protein, which is in good agreement with the secondary-structure content revealed by CD studies of 24% a-helix for the monomeric form. Protein digestion and fragment characterization SV-IV is a 90-amino-acid protein lacking disulfide bridges and possessing nine lysine and seven arginine residues, which represents a large number of potential hydrolysis sites for trypsin. For this reason, we selected this protease to investigate the different accessibility of crucial sites sup- porting molecule aggregation and characterizing the mono- meric and trimeric forms. Both forms of SV-IV were digested separately using the same enzyme/substrate ratio. Aliquots of the incubation mixtures were withdrawn at various times, and the formation of fragments was moni- tored by RP-HPLC. Figure 3 shows chromatograms of the digestion mixtures of the trimeric (Fig. 3A) and monomeric (Fig. 3B) forms after 12 h incubation. Lower amounts of all the peptides were also produced after 15 min incubation, indicating that both monomeric and trimeric forms were readily digested by trypsin (not shown). Each fragment collected was submitted to automatic sequence analysis. The corresponding start-end position in the protein sequence is indicated in the figure. Some differences in the hydrolytic pathways were found. The protein is hydrolyzed at Arg57 only in the monomeric form. In fact, whereas fragments 40–56 and 60–63 are common to both chromatograms, fragments 40–57 and 58–63 arose only from the monomeric form (Fig. 3B). The tripeptide 57–59 complementing frag- ments 40–56 and 60–63 which originated from digestion of the trimer was not identified in the chromatogram (Fig. 3A). Furthermore, fragment 80–83, complementary to fragments 64–79 and 84–90, was generated only from hydrolysis of the trimeric form (Fig. 3A), whereas fragment 82–90 arose only from the monomeric form (Fig. 3B), indicating further digestion at the level of Arg81. The dipeptide Lys80–Arg81, complementary to fragments 64–79 and 82–90, was not identified in the chromatogram (Fig. 3B). Both peptide bonds Lys80–Arg81 and Arg81– Ser82 seem to be hidden in the trimer, as the whole fragment Lys80-Arg81-Ser82-Arg83 was found (Fig. 3A), whereas no fragment ending at Lys80 or starting at Arg81 arose from digestion of the monomer (Fig. 3B). As a consequence, Arg81 should be accessible only in the monomeric form, whereas Lys80 also seems to be quite hidden in the monomeric form. SV-IV is not fully acetylated by acetic anhydride An aliquot was directly analysed by HPLC/electrospray MS to characterize the acetylated form of SV-IV protein. As shown by its transformed spectrum (Fig. 4), several com- ponents were present, differing with respect to the number of acetyl groups (mass increase of 42 for each acetyl group incorporated), and indicating that the reaction was not complete, but generated a mixture of incompletely acetyl- ated forms of the protein. These contained from three to eight acetyl groups (the maximum expected was 10, considering nine lysine residues and the N-terminal amino group), the most abundant ranging from four to six. To identify the acetylated residues, another aliquot of protein was first digested with endoproteinase Glu-C and then analysed by HPLC/electrospray MS to obtain the relevant peptide map. The peptides identified are shown in Table 1. We were therefore able to screen the whole protein sequence to identify the acetylated peptides. Peptides were identified by their molecular mass on the basis of the known protein sequence and the endoproteinase Glu-C specificity. In most cases, a mixture of the native and acetylated peptides was observed and identified by the mass increase of 42 mass units. The relative level of acetylation of a peptide was estimated on the basis of the intensity ratio of the native and acetylated species. From the data summar- ized in Table 1, it can be seen that some of the peptides were almost completely acetylated whereas some showed low or minimal acetylation. To locate acetylated Lys residues on peptides containing more than one Lys, the fractions collected from the HPLC separation (Fig. 6) were analysed by tandem MS using MALDI-TOF PSD-MS. As an example, peptide 6–12, containing Lys6, was acetylated only to 5%; peptide 72–90, containing Lys78, Lys79, and Lys80, showed partial acetylation at one of the three residues, because the signal of the triacetylated species was less intense than that of the diacetylated species. The MS/MS Fig. 2. Secondary-structure prediction. Amino-acid sequence and sec- ondary-structure predictions performed with PHD, JPRED, and PSI- PRED (see Materials and methods). a-Helix is indicated by H and b-strand conformation is indicated by E. 266 C. Caporale et al.(Eur. J. Biochem. 271) Ó FEBS 2003 analysis of the chromatographic fraction showed that the two Lys residues at positions 78 and 79 were both acetylated, whereas Lys80 was not (Fig. 5). Molecular modeling of 70–90 region CD spectra of SV-IV fragment 70–90 and the secondary- structure prediction suggested that the region 70–90 should have a high a-helix content. We created a computer model of the 70–90 peptide. The initial conformation of the backbone was imposed as a-helix, and energy minimization was performed in order to optimize the peptide structure. As a consequence of such optimization, the initial backbone conformation was approximately conserved only in the region corresponding to the 70–81 segment (Fig. 6). In the 82–90 segment, a helical conformation was conserved, but it was not consistent with a-helix features, as the Kabsch and Sander assignment of secondary structure did not define helix in this segment of the peptide. The initial conformation of side chains was also modified under energy minimization, Fig. 3. Protein digestion. Chromatograms of digestion mixtures of trimeric (A) and monomeric (B) forms of SV-IV after 12 h incubation with trypsin. Each peak is labelled with the corresponding protein fragment. Peaks present in both chromatograms refer to the protein segments 60–63, 33–39, 64–78, 64–79, 84–90, 40–56, 5–32 + 7–32, 5–39 + 7–39. Peaks present in chromatogram A but not in B refer to segment 80–83. Peaks present in chromatogram B but not in A refer to the protein segments 50–63, 82–90, 40–57. Ó FEBS 2003 Structural properties of SV-IV (Eur. J. Biochem. 271) 267 and some interesting results were obtained. In particular, the initial extended conformation of the Tyr76 and Lys79 side chains were modified and assumed an orientation suitable for hydrogen-bond formation (Fig. 6). This finding is in good agreement with other experimental results and will be discussed below. Discussion SV-IV is a protein with immunomodulatory, anti-inflam- matory, and procoagulant activity. Its physiological concentration ranges from 2 to 48 l M , i.e. from 0.019 to 0.47 lgÆlL )1 , in different conditions and organs [19]. We have recently demonstrated that, in the same concentra- tion range, the protein shows a monomer fi dimer fi trimer quaternary organization, and the equilibrium of self-association appears to control the biological properties of the protein [7]. Moreover, the immunomodulatory activity is related to the structural integrity of the whole molecule, whereas the anti-inflammatory and procoagu- lant activity is located in the unstructured 1–70 region of the molecule. In this work, structural differences between the monomeric and trimeric form of SV-IV have been confirmed from CD spectra, which revealed double the content of a-helix in the trimeric form compared with the monomeric form. As suggested by CD spectra of the 1–70 and 71–90 fragments, as well as by prediction methods, the C-terminal region has high propensity to form a-helix, so this region may be responsible for the a-helix observed by CD in the monomer. On the other hand, the increase in a-helix in the trimeric form may result from rearrange- ment of the 1–70 region, where some predictive methods assign a-helix conformation. This region is poorly struc- tured, but the addition of SDS revealed a hidden ability to form helical structure. To find functional differences related to the structural modifications occurring in the monomer–trimer transition, we investigated how proteolysis and post-translational modifications could be affected by self-association. Limited proteolysis showed that both monomeric and trimeric forms are very sensitive to trypsin hydrolysis, Lys80 being the only putative proteolytic site not hydrolyzed in both forms. It is interesting to note that Arg57 and Arg81 are hydrolyzed in the monomeric but not the trimeric form. These differences Fig. 4. Electrospray mass spectrum of the protein SV-IV acetylated with acetic anhydride. SV-IV, purified by gel filtration and ion-exchange chromatography, was incubated with acetic anhydride under the conditions described in Materials and methods, desalted, and then analysed by electrospray MS. Table 1. Analysis of the endoproteinase Glu-C digest of acetylated SV-IV by HPLC/electrospray MS. Acetylated SV-IV was digested with endo- proteinase Glu-C. The resulting peptide mixture was analyzed using a Vydac C 18 column (250 · 2.1 mm, 5 lm) on-line with a Platform mass spectrometer. The experimental details are given in Materials and methods. The measured mass is the mean ± SD molecular mass calculated by integrating the multiple peaks corresponding to each molecular species and differing only in the total number of charges measured by electrospray MS. Theoretical mass is the mass calculated on the basis of the protein amino-acid sequence. The relative abundance refers to the ratio of the acetylated/unacetylated SV-IV forms. ID, identification number of peaks. HPLC peak ID Measured mass (Da) Theoretical mass (Da) Peptide a Acetylated residues Relative abundance (% of acetylation) 1 432.5 ± 0.1 432.5 13–16 869.1 ± 0.2 869.9 6–12 Lys 6 0 2 786.5 ± 0.4 786.5 1–5 Lys 2 and 4; N-term 100 435.1 ± 0.2 435.4 49–52 3 2075.5 ± 0.4 2076.3 53–71 2117.9 ± 0.5 2118.3 53–71 Lys59 24 4 1214.7 ± 0.1 1214.1 17–29 5 2261.6 ± 0.9 2262.4 53–73 2303.5 ± 0.2 2304.4 53–73 Lys59 24 6 2022.4 ± 0.9 2022.3 30–48 7 2064.4 ± 0.9 2064.3 30–48 Lys 34 or 39 45 2106.3 ± 0.9 2106.3 30–48 Lys 34 and 39 5 8 2436.9 ± 0.5 2437.9 30–52 2479.0 ± 0.5 2479.9 30–52 Lys 34 or 39 45 2521.5 ± 0.5 2521.9 30–52 Lys 34 and 39 5 9 2080.6 ± 0.4 2081.2 74–90 Lys 78 or 79 or 80 45 10 2123. 6 ± 0.4 2123.2 74–90 Lys 78, 79 and 80 55 a Numbers refer to the N-terminus and C-terminus of each peptide. 268 C. Caporale et al.(Eur. J. Biochem. 271) Ó FEBS 2003 can be compared with structural predictions and structural features of both forms. Lysine acetylation gave us further information about the structural environment of the lysines, as acetylated lysines can be considered to be exposed to the surface of the protein, whereas non-acetylated lysines are probably not. Some of the data appear to contradict the results of limited proteolysis. In particular, Lys6 appears not to be acetylated and therefore not exposed to the surface, but proteolytic cleavage occurs at this residue. These contrasting data may be explained by the possibility that Lys6 becomes exposed only after the hydrolysis of Lys2 and Lys4. Moreover, Lys34 is partially acetylated (5%) but is not hydrolysed by trypsin in both monomeric and trimeric forms. The fact that Lys34 is followed by Pro35 and the poor efficiency of trypsin in cleaving Lys–Pro bonds may explain why Lys34 is not hydrolyzed in both monomeric and trimeric forms. Moreover, it may be possible that this Lys is exposed in the monomeric but not the trimeric form. In fact, at the protein concentration used for acetylation, the trimeric form is predominant, so the low acetylation observed may be related to the low amount of the monomeric form always present in equilibrium with the trimeric form. Most of the peptide bonds hydrolyzed by trypsin are located in regions without secondary-structure elements such as helices or b-strands, which may confer protease resistance on the backbone [36–39]. A long helix is predicted in the 75–88 region. It is interesting to note that Lys78, Lys79, and Arg81, located in such a helical region, are hydrolyzed by trypsin, while the enzyme does not hydrolyze Lys80. Secondary-structure predictions allow us to hypothesize that Lys80 could not be hydrolyzed because the amino group of its side chain might be hydrogen-bonded to the -OH group of the Tyr76 side chain. It is known that helical residues in position i and i +3/i + 4 expose their side chains on the same side of the helical surface and may interact by forming salt bridges or hydrogen bonds. Therefore, our hypothesis is supported by two experimental observations: (a) tyrosine titration does not act on all three tyrosines of SV-IV protein [7]; (b) the peptide containing Lys78, Lys79, and Lys80 is only partially acetylated. The partial titration of tyrosine may be explained by the formation of tyrosinate. The modeling of a peptide corresponding to the 70–90 region of SV-IV suggests that Tyr76 may form a hydrogen-bond with Lys79, supporting this hypothesis. As tyrosinate formation is not evident in the trimeric form, the Tyr76 side chain should be suitable to form a transient hydrogen-bond with Lys79 or Lys80 in the monomeric form, whereas, in the trimeric form, the region that includes Lys79, Lys80 and Arg81 may be involved in the oligomerization, as demonstrated by the change in sensitivity to trypsin hydrolysis, in agreement with Fig. 6. Molecular model of the peptide corresponding to the 70–90 region of SV-IV. Top, initial model, with the imposed a-helix backbone conformation. Bottom, conformation reached after energy minimiza- tion. The loss of a-helix conformation on the C-terminal side is evident. A dashed line indicates the hydrogen-bond between the Tyr76 and Lys79 side chains. It can be seen how, after minimization, the back- bone is modified in the middle, and the helix is interrupted. Fig. 5. MALDI-TOF mass spectrum in post- source decay mode of the peptide at 2081.7 m/ z. The peptide at m/z 2081.7 corresponded to the diacetylated peptide 74–90 from the endo- proteinase Glu-C digest of acetylated protein SV-IV. Signal diagnostics of peptide structure are indicated in the figure. Ó FEBS 2003 Structural properties of SV-IV (Eur. J. Biochem. 271) 269 the absence of tyrosinate. In fact, Arg81 is hydrolyzed only in the monomeric form of SV-IV. There are two different explanations for these data. In the first, the resistance of Arg81, as well as Arg57, to attack by trypsin in the trimeric form is due to subunit association and the consequent loss of exposed surface. Arg57 and Arg81 may be located in the region of interaction, and therefore would be exposed in the monomeric form and buried in the trimeric form. The second hypothesis is based on the observation of a higher a-helix content in the trimeric form of SV-IV. The long helices predicted in segments 48–60 and 75–88 may be responsible for a rigid conformation, which is resistant to proteases, thus preventing hydrolysis at the level of Arg57 and Arg81. However, the three prediction methods do not agree in predicting these two long helices. It may be possible that such differences in secondary-structure predictions are caused by regions being able to adopt different secondary structures under different quaternary structure conditions. JPRED predicted in the 75–88 region two short helices, connected by a short nonhelical segment, which includes Arg81 and Ser82. It is possible that this region is folded differently in the monomeric and trimeric forms of SV-IV: a long helix is formed in the trimeric protein, whereas two short helices are present in the monomeric form. Such conformations are compatible with the different responses to trypsin hydrolysis; Arg81 may be in a loop region when the protein is in the monomeric form, and therefore sensitive to hydrolysis, whereas it might be in a long a-helix when the protein is in the trimeric form, making it resistant to protease attack. Similarly, the 48–60 region is only predicted to be a-helix by PSI-PRED, the other two methods predicting a shorter helix, leaving Arg57 in a loop region. Finally, we note that the differences in proteolytic sensi- tivity of the monomeric and trimeric forms of SV-IV are located at residues Arg57 and Arg81, coinciding with peptide bonds proteolysed in vivo. We have previously demonstrated that the partially purified SV-IV fraction includes detectable amounts of SV-IV peptides, i.e. 1–16, 42–90, 81–88, 58–90, 1–80. The hypothesis that such peptides play a functional role is in good agreement with the opportunity to control proteolysis via the monomer– trimer equilibrium. Conclusions The aim of this work was to understand the molecular mechanisms that produce different structural and functional behavior in the monomeric and trimeric forms of SV-IV. We have previously demonstrated that SV-IV is active in different biological assays as three different functional states: monomeric, trimeric, and proteolytically cleaved. In this paper, we show that self-association induces a con- formational change mainly in the 1–70 region, which appears to be partially a-helix in the trimer but without secondary structure in the monomer. This conformational change may modulate the proteolysis of SV-IV, which in vivo generates active peptides. The different physiological levels of the protein in different conditions and organs may activate SV-IV by shifting the structure between monomeric and trimeric forms, producing two forms with different activities and different sensitivities to proteolysis, which generates active peptides. In conclusion, this study indicates that conformational changes between the monomeric and trimeric forms is an important aspect of the activity modulation. Acknowledgements This work was supported by a grant from Regione Campania. References 1. Ostrowski, M.C., Kistler, M.K. & Kistler, W.S. (1979) Purifica- tion and cell-free synthesis of a major protein from rat seminal vesicle secretion. A potential marker for androgen action. J. Biol. Chem. 254, 383–390. 2. Mansson, P.E., Sugino, A. & Harris, S.E. (1981) Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression. Nucleic Acids Res. 9, 935–946. 3. Abrescia, P., Corbo, L. & Metafora (1986) S. Maturation in dif- ferent translational systems of the protein RSV-IV secreted from the rat seminal vesicle epithelium. Bull. Mol. Biol. Med. 11, 19–33. 4. Metafora, S., Facchiano, F., Facchiano, A., Esposito, C., Peluso, G. & Porta, R. (1987) Homology between rabbit uteroglobin and the rat seminal vesicle sperm binding protein: prediction of structural features of glutamine substrates for transglutaminase. J. Protein Chem. 6, 353–359. 5. Metafora, S., Lombardi, G., De Rosa, M., Quagliozzi, L., Rav- agnan, G., Peluso, G. & Abrescia, P. (1987) A protein family immunorelated to a sperm-binding protein and its regulation in human semen. Gamete Res. 16, 229–241. 6. Abrescia, P., Lombardi, G., De Rosa, M., Quagliozzi, L., Guardiola, J. & Metafora, S. (1985) Identification and preliminary characterization of a sperm-binding protein in normal human semen. J. Reprod. Fertil. 73, 71–77. 7. Stiuso, P., Metafora, S., Facchiano, A.M., Colonna, G. & Ragone, R. (1999) The self association of protein SV-IV and its possibile functional implications. Eur. J. Biochem. 266, 1029–1035. 8. Harris, S.E., Mansson, P E., Tully, D.B. & Burkhart, B. (1983) Seminal vesicle secretion IV gene: allelic difference due to a series of 20-base-pair direct tandem repeats within an intron. Proc. Natl Acad. Sci. USA 80, 6460–6464. 9. Kandala, C., Kistler, M.K., Lawther, R.P. & Kistler, W.S. (1983) Characterization of a genomic clone for rat seminal vesicle secre- tory protein IV. Nucleic Acids Res. 11, 3169–3186. 10. McDonald, C., Williams, L., McTurck, P., Fuller, F., McIntosh, E. & Higgins, S. (1983) Isolation and characterisation of genes for androgen-responsive secretory proteins of rat seminal vesicles. Nucleic Acids Res. 11, 917–930. 11. D’Ambrosio, E., Del Grosso, N., Ravagnan, G., Peluso, G. & Metafora, S. (1993) Cloning and expression of the rat genomic DNA sequence coding for the secreted form of the protein SV-IV. Bull. Mol. Biol. Med. 18, 215–223. 12. Galdiero, F., Tufano, M.A., De Martino, L., Capasso, C., Porta, R., Ravagnan, G., Peluso, G. & Metafora, S. (1989) Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium. J. Reprod. Immunol. 16, 269–284,. 13. Metafora, S., Peluso, G., Persico, P., Ravagnan, G., Esposito, C. & Porta, R. (1989) Immunosuppressive and anti-inflammatory properties of a major protein secreted from the epithelium of the rat seminal vesicles. Biochem. Pharmacol. 38, 121–131. 14. Metafora,S.,Porta,R.,Ravagnan,G.,Peluso,G.,Tufano,M.A., De Martino, L., Ianniello, R. & Galdiero, F. (1989) Inhibitory effect of SV-IV, a major protein secreted from the rat seminal 270 C. Caporale et al.(Eur. J. Biochem. 271) Ó FEBS 2003 vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes. J. Leukoc. Biol. 46, 409–416. 15. Camussi,G.,Tetta,C.,Bussolino,F.,Metafora,S.,Peluso,G., Esposito, C. & Porta, R. (1990) An anti-inflammatory protein secreted from rat seminal vescicle epithelium inhibits the synthesis of platelet: activating factor and the release of arachidonic acid and prostacyclin. Eur. J. Biochem. 192, 481–485. 16. Vuotto, M.L., Peluso, G., Mancino, D., Colonna, G., Facchiano, A., Ielpo, M.T., Ravagnan, G. & Metafora, S. (1993) Inhibition of interleukin-1 release and activity by the rat seminal vesicle protein SV-IV. J. Leukoc. Biol. 53, 214–222. 17. Peluso, G., Porta, R., Esposito, C., Tufano, M.A., Toraldo, R., Vuotto, M.L., Ravagnan, G. & Metafora, S. (1994) Suppression of rat epididymal sperm immunogenicity by a seminal vesicle secretory protein (SV-IV) and transglutaminase both in vivo and in vitro. Biol. Reprod. 50, 593–602. 18. Romano-Carratelli, C., Galdiero, M., Nuzzo, I., Bentivoglio, C., Porta, R., Peluso, G., Ravagnan, G. & Metafora, S. (1995) In vivo inhibition of cell-mediated and humoral immune responses to cellular antigens by SV-IV, a major protein secreted from the rat seminal vesicle epithelium. J. Reprod. Immunol. 28, 15–30. 19. Tufano, M.A., Porta, R., Farzati, B., Di Pierro, P., Rossano, F., Catalanotti, P., Baroni, A. & Metafora, S. (1996) Rat seminal vesicle protein SV-IV and its transglutaminase-synthesized poly- aminated derivative Spd 2 -SV-IV induce cytokine release from human resting lymphocytes and monocytes in vitro. Cell. Immunol. 168, 148–157. 20. Di Micco, B., Colonna, G., Porta, R. & Metafora, S. (1994) Rat protein SV-IV accelerates human blood coagulation in vitro by selective inhibition of antithrombin III. Biochem. Pharmacol. 48, 345–352. 21. Di Micco, B., Stiuso, P., Colonna, C., Porta. R., Marchese, M., Schinina ` , M.E., Macalello, M.A. & Metafora, S. (1997) A peptide derivative (1–70 fragment) of protein SV-IV accelerates human blood coagulation in vitro by selective inhibition of the heparin- induced antithrombin III acivation. J. Peptide Res. 49, 179–182. 22. Di Micco, B., Caen, J., Colonna, C., Macalello, M.A., Marchese, M., Stiuso, P., Di Micco, P., Morelli, F. & Metafora, S. (2000) Inhibition of antithrombin by protein SV-IV normalizes the coa- gulation of hemophilic blood. Eur. J. Pharmacol. 391, 1–9. 23. Metafora, V., Franco, P., Massa, O., Morelli, F., Stiuso, P., Ferranti, P., Mamone, G., Malori, A., Stoppelli, M.P. & Meta- fora, S. (2001) Phosphorilation of seminal vesicle protein IV on Ser58 enhances its peroxidase-stimulating activity. Eur. J. Bio- chem. 268, 3858–3869. 24. Ferrant, P., Mamone, G., Malorni, A., Guardiola, J., Stiuso, P. & Metafora, S. (1997) Structural heterogeneity, post-traslational modification, and biological activities of SV-IV, a major protein secreted from that rat seminal vesicle epthelium. Rapid Commun. Mass Spectrom. 11, 1007–1014. 25. Porta, R., Esposito, C., Gentile, V., Mariniello, L., Peluso, G. & Metafora, S. (1990) Transglutaminase-catalyzed modifications of SV-IV, a major protein secreted from the rat seminal vesicle epi- thelium. Int. J. Peptide Protein Res. 35, 117–122. 26. Porta, R., Esposito, C., Metafora, S., Malorni, A., Pucci, P., Siciliano, R. & Mari (1991) Mass spectrometric identification of the amino donor receptor sites in a transglutaminase protein substrate secreted from rat seminal vesicles. Biochemistry 30, 3114–3120. 27. Yin,E.T.,Galanos,C.,Kinsky,S.,Bradshaw,R.A.,Wessler,S., Luderig, O. & Sarmiento, M.F. (1972) Picogram-sensitive assay for endotoxin: gelation of Limulus polyphemus blood cell lysate induced by purified lipopolysaccharides and lipid A from Gram- negative bacteria. Biochim. Biophys. Acta 261, 284–289. 28. Rubin, B.Y., Anderson, S.L., Sullivan, S.A., Williamson, B.D., Carswell, E.A. & Old, L. (1985) Purification and characterization ofahumantumornecrosisfactorfromtheLuKIIcellline.Proc. Natl Acad. Sci. USA 82, 6637–6641. 29. Altschul, S.F., Madden, T.L., Scha ¨ ffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. 30. Kelley, L.A., MacCallum, R.M. & Sternberg, M.J.E. (2000) Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299, 499–520. 31. Jones, D.T. (1999) GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J. Mol. Biol. 287, 797–815. 32. Rost, B. (1995) TOPITS: threading one-dimensional predictions into three-dimensional structures. In Third International Confer- ence on Intelligent Systems for Molecular Biology (ISMB); Cambridge, UK (July 16–19, 1995) (Rawlings, C., Clark, D., Altman, R., Hunter, L., Lengauer, T. & Wodak, S., eds), pp 314– 321. AAAI Press, Menlo Park, CA. 33. Rost, B. & Sander, C. (1993) Improved prediction of protein secondary structure by use of sequence profiles and neural net- works. Proc.NatlAcad.Sci.USA90, 7558–7562. 34. Cuff, J.A., Clamp, M.E., Siddiqui, A.S., Finlay, M. & Barton, G.J. (1998) JPred: a consensus secondary structure prediction server. Bioinformatics 14, 892–893. 35. Jones, D.T. (1999) Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202. 36. Caporale, C., Caruso Facchiano, C., Nobile, A., Leonardi, M., Bertini,L.,Colonna,L.&Buonocore,G.(1999)Probingthe modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures. Proteins 36, 192–204. 37. Hubbard, S.J., Beynon, R.J. & Thornton, J.M. (1998) Assessment of conformational parameters as predictors of limited proteolytic sites in native protein structures. Protein Eng 11, 349–359. 38. Hubbard, S.J., Eisenmenger, F. & Thornton, J.M. (1994) Mod- eling studies of the change in conformation required for cleavage of limited proteolytic sites. Protein Sci. 3, 757–768. 39. Hubbard, S.J., Campbell, S.F. & Thornton, J.M. (1991) Mole- cular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors. J. Mol. Biol. 220, 507–530. Ó FEBS 2003 Structural properties of SV-IV (Eur. J. Biochem. 271) 271 . obtain a theoretical model of the structural organization of SV-IV protein. The amino-acid sequence was analyzed using the BLAST program to find similar proteins. to control the biological properties of the protein [7]. Moreover, the immunomodulatory activity is related to the structural integrity of the whole molecule,