Chemical synthesis of the RGD-protein decorsin: Pro->Ala replacement reduces protein thermostability

Erica Frare1, Patrizia Polverino de Laureto1, Elena Scaramella1, Fiorella Tonello1, Oriano Marin1, Renzo Deana2 and Angelo Fontana1,3

1CRIBI Biotechnology Centre and 2Department of Biological Chemistry, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy

3 To whom correspondence should be addressed. E-mail: angelo.fontana{at}unipd.it


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Decorsin is a 39-residue polypeptide chain, crosslinked by three disulfide bridges, that strongly inhibits platelet aggregation. We report the chemical synthesis and characterization of analogs of decorsin with the aim of investigating the role of proline residues in protein structure, stability and biological activity. Decorsin analogs have been synthesized in which one (P23A and P24A decorsin) or two (P23,24A decorsin) proline residues have been substituted by alanine. The crude synthetic polypeptides were purified by reversed-phase HPLC in their reduced form and allowed to refold oxidatively to their disulfide-crosslinked species. The homogeneity of the synthetic mini-proteins, and also the correct pairing of the three disulfide bridges, were established by a number of analytical criteria, including fingerprinting analysis of the refolded synthetic analogs by using thermolysin and proteinase K as proteolytic enzymes. Replacement of proline by alanine results in a significant and cumulative decrease of the high thermal stability (Tm 74°C) of native decorsin. The mono-substituted analogs display a Tm of 66–67°C, while the double-substituted analog a Tm of 50°C. On the other hand, the overall secondary and tertiary structures were not affected by the Pro->Ala exchanges, as judged from circular dichroism measurements. Platelet aggregation assays established that the proline substitutions do not impair significantly the biological activity of decorsin. The results of this study clearly indicate that proline residues contribute significantly to the protein thermal stability. Our results are in line with the ‘proline rule’, previously advanced for explaining the unusual thermal stability of thermophilic enzymes, which usually show an enhanced content of proline residues with respect to their mesophilic counterparts.

Keywords: circular dichroism/decorsin/peptide synthesis/RGD-protein/thermal stability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Decorsin is a 39-residue RGD-protein isolated from the leech Macrobdella decora belonging to the family of the GP IIb–IIIa antagonists and acting as a potent inhibitor of platelet aggregation (Seymour et al., 1990Go; Salzet, 2001Go; Yang et al., 2004Go). The overall structural features of decorsin are similar to those of the small proteins, called disintegrins, isolated from snake venom (McLane et al., 1995Go) and to those obtained from leeches, such as ornatin, antistasin and hementin (Hynes, 1992Go; Lazarus and McDowell, 1993Go). All these proteins are small disulfide-rich polypeptide chains that can affect the hemostatic process (Arocha-Pinango et al., 1999Go; Salzet, 2001Go). The 3D structure of decorsin in solution has been elucidated by NMR spectroscopy (Krezel et al., 1994Go, 2000Go) and shown to be given by two ß-sheets, the first formed by three strands (S1, S2 and S3) and the second by two strands (S4 and S5) (see Figure 1). The RGD-containing loop in disintegrins is usually rather exposed and fairly flexible, whereas in decorsin it is rather well defined but still flexible (Krezel et al., 1994Go, 2000Go). Amino acid sequence analyses showed that, despite the high structural homology, the different proteins affecting blood clotting show little amino acid sequence similarities (Seymour et al., 1990Go). In particular, the overall structure of decorsin much resembles that of the N-terminal core domain 1–40 of hirudin, a potent thrombin inhibitor isolated from the leech Hirudo medicinalis not containing the RGD sequence (Folkers et al., 1989Go; Haruyama and Wüthrich, 1989Go; Rydel et al., 1991Go; Szyperski et al., 1992Go). Whereas the N-terminal core domain of hirudin does not contain Pro residues, decorsin instead contains up to six Pro residues (Pro2, Pro5, Pro23, Pro24, Pro30 and Pro36). These residues, together with the ‘cystine knot’ given by the three disulfide bridges (Cys7–Cys15, Cys17–Cys27 and Cys22–Cys38) probably contribute a great deal to the high conformational stability and rigidity of decorsin.



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Fig. 1. Amino acid sequence (top), schematic 3D structure (middle) and secondary structure (bottom) of decorsin. The disulfide bonds (Cys7–Cys15, Cys17–Cys27 and Cys22–Cys38) are indicated by solid lines in the 39-residue chain of decorsin (Seymour et al., 1990Go). The drawing of the decorsin 3D structure (Krezel et al., 1994Go) (middle) was made using a Silicon Graphics 02 workstation and the program MolScript (Kraulis, 1991Go), using the coordinates deposited in the Brookhaven Data Bank (file 1dec.pdb). The five ß-strands (S1–S5) are colored in blue, the RGD segment 33–35 in magenta and the disulfide bridges in yellow. The location of the two Pro residues replaced by Ala in this study is indicated by red spheres in the drawing of the secondary structure of decorsin (bottom). Also the location of the RGD motif is indicated here.

 
We have previously described the successful solid-phase peptide synthesis of the N-terminal core domain 1–47 of hirudin (De Filippis et al., 1995Go) and decorsin (Polverino de Laureto et al., 1998Go). However, at variance from the hirudin domain, the refolding process of the reduced synthetic decorsin was not complete, since non-native species (~30%) were present in the refolding mixture. We hypothesized that the incomplete oxidative refolding of decorsin could be related to the presence of the large number of Pro residues along its polypeptide chain (Polverino de Laureto et al., 1998Go). Actually, in native decorsin all peptide bonds are in a trans conformation (Krezel et al., 1994Go). Therefore, cis–trans transitions at X–Pro peptide bonds probably have to occur during refolding, thus slowing down the overall process and allowing the formation of misfolded molecules with non-native disulfide bonds, sufficiently stable to be trapped along the folding pathway (Brandts et al., 1975Go; Creighton, 1978Go; Levitt, 1981Go; Kelley and Richards, 1987Go; Schmid, 1992Go; Kiefhaber, 1995a,GobGo; Eyles and Gierash, 2000Go). Of interest, ~50% of the recombinant decorsin produced in Escherichia coli was a misfolded species with a non-native pairing of disulfides and showing a much lower activity (IC50 12 µM) in inhibiting platelet aggregation than the native correctly folded protein (IC50 0.27 µM) (Krezel et al., 2000Go). Analogous misfolding was evidenced also with recombinant ornatin E, for which both correctly folded and misfolded disulfide variants were obtained (Mazur et al., 1991Go).

Here, we report the chemical synthesis of Pro->Ala variants of decorsin using solid-phase methodology. Two adjacent Pro residues P23 and P24 were selected for replacement and decorsin analogs were produced with one (P23A and P24A decorsin) or two (P23,24A decorsin) Pro residues replaced by Ala. These residues are located in the loop connecting strands S3 and S4 of the molecule and between Cys22 and Cys27 (see Figure 1). The synthetic analogs were purified to homogeneity and their chemical identity and correct pairing of disulfide bridges determined by protein chemistry methods. It has been found that the Pro->Ala replacements do not impair the overall structure and biological activity of decorsin, whereas they have a significant and cumulative effect on protein thermal stability. However, the Pro->Ala exchanges at positions 23 and 24 of the polypeptide chain of decorsin did not prevent the reduced decorsin from forming misfolded species under oxidative refolding conditions. These observations are discussed in the framework of the possible role of Pro residues in protein stability and folding.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Materials

Natural leech-derived decorsin was purchased from Calbiochem (La Jolla, CA). Fmoc-L-amino acids were obtained from Novabiochem (Laufelfingen, Switzerland). The solvents and reagents used for peptide synthesis and those for peptide/protein sequencing were obtained from Applied Biosystems (Foster City, CA). Thermolysin from Bacillus thermoproteolyticus (EC 3.4.24.4) and proteinase K from Tritirachium album (EC 3.4.21.64) were supplied by Sigma (St. Louis, MO). Other chemicals were of analytical reagent grade and were obtained from Fluka (Buchs, Switzerland).

Solid-phase peptide synthesis

Decorsin and its analogs were synthesized by the solid-phase method (Atherton and Sheppard, 1989Go) on 4-hydroxymethylphenoxymethyl-co-polystyrene–1% divinylbenzene resin (0.96 mmol/g) using an automated peptide synthesizer (Applied Biosystems, Model 431A). First, the peptide corresponding to the sequence 25–39 of decorsin was synthesized on a 0.25 mmol scale, then the resin was divided into five parts and the synthesis of the rest of polypeptide chain was carried out on each fraction on a 0.05 mmol scale. Fmoc-protected amino acids (Fields and Noble, 1990Go) were used with the following side-chain protection: tert-butyl ether (tBu) for Tyr, tert-butyl ester (OtBu) for Glu and Asp, trityl (Trt) for Cys, Asn and Gln, tert-butyloxycarbonyl (Boc) for the {varepsilon}-amino group of Lys and 2,2,5,7,8-pentamethylchromane-6-sulfonyl (Pmc) for the guanidino group of Arg. Deprotection of the Fmoc group, at every cycle, was obtained by a 10 min treatment with 20% piperidine in N-methylpyrrolidone (NMP). Chain elongation was performed using a 10-fold excess (0.5 mmol) of Fmoc-amino acid, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetrametyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) (1:1:1) in the presence of a 20-fold excess of N,N-diisopropylethylamine (DIEA) (Knorr et al., 1989Go). The deprotection of the Fmoc N-protecting group was monitored by absorbance measurements at 301 nm after every coupling step in order to determine the extent of cleavage. Capping with acetic anhydride was routinely carried out after introduction of hydrophobic (Phe, Tyr, Leu) and Arg residues, as suggested by Milton et al. (1990)Go. After completion of the last cycle, the resin was washed with NMP and dichloromethane (DCM)–methanol (1:1, v/v) and then dried in vacuo. The synthetic peptide was cleaved from the resin and deprotected by treating the peptide–resin with TFA–1,2-ethanedithiol (EDT) (95:5, v/v) for 2 h at 0°C. The resin was filtered and cold diethyl ether was added to the solution in order to precipitate the crude reduced peptide. The pellet was recovered by centrifugation at 3000 r.p.m. and then resuspended in diethyl ether and centrifuged three times. The sample of crude reduced peptide was stored at –20°C.

Isolation and oxidative refolding of the reduced peptides

The crude reduced peptides were purified by reversed-phase (RP) high-performance liquid chromatography (HPLC) using a Vydac C18 column (4.6 x 150 mm; The Separations Group, Hesperia, CA), which was eluted with a gradient of 0.1% TFA in water–0.085% TFA in acetonitrile from 5 to 21% of acetonitrile in 4 min and from 21 to 46% in 15 min. The eluate from the column was monitored by absorbance measurements at 226 nm. The peptide material obtained after RP-HPLC was analyzed by N-terminal sequencing and mass spectrometry (MS). The oxidative refolding of synthetic peptides was conducted at room temperature in 0.1 M Tris–HCl buffer, pH 8.5, containing 0.2 M NaCl, 2 mM glutathione (GSH) and 1 mM oxidized glutathione (GSSG) (Chatrenet and Chang, 1993; Chang, 1995). Aliquots (15 µl, 1 mg/ml) were withdrawn at intervals from the refolding mixture and analyzed by RP-HPLC using a Vydac C18 column.

Peptide mapping

The pairing of disulfide bonds was assessed by fingerprinting analysis of the oxidatively refolded polypeptides using thermolysin and proteinase K as proteolytic enzymes. Proteolysis of the synthetic analogs with thermolysin was conducted at 40°C in 50 mM Tris–HCl, pH 7.0, containing 5 mM CaCl2, for 5 h at an E/S ratio of 1:20 (w/w). The digestion mixtures were analyzed by RP-HPLC on a Vydac C18 column, eluted as above described. The identity of the proteolytic fragments was established by N-terminal sequencing (Applied Biosystems protein sequencer, Model 477A) and matrix-assisted laser desorption/ionization (MALDI) MS (Kompact MALDI-I; Kratos-Shimadzu, Manchester, UK). The analysis of a single digest of the decorsin analogs was not sufficient for determining unequivocally the complete disulfide pairing. Therefore, some thermolytic peptides were further digested with another protease in order to prepare additional peptide fragments for the purposes of defining the disulfide topology. For example, in the case of the P23,24A analog, a peptide fragment corresponding to the sequence 16–39 was isolated by RP-HPLC from the thermolytic peptide mixture and then further digested with proteinase K at room temperature in 50 mM Tris–HCl buffer, pH 8.3, for 4 h, utilizing an E/S ratio of 1:50 (w/w). The separation of the resulting peptides was performed by RP-HPLC.

Spectroscopic characterization

The peptide concentration was determined by absorption measurements at 280 nm on a double-beam spectrophotometer (Lambda-20, Perkin-Elmer, Forster City, CA). Extinction coefficients ({varepsilon}) at 280 nm were calculated according to Gill and von Hippel (1989)Go and are 0.376 for P23A and P24A decorsin and 0.379 for P23,24A decorsin. Circular dichroism (CD) spectra were recorded with a Jasco (Tokyo, Japan) J-710 spectropolarimeter equipped with a thermostated cell holder and a Neslab RTE-110 circulating water bath. The instrument was calibrated with d-(+)-10-camphorsulfonic acid (Toumadje et al., 1992Go). Near-UV CD spectra were recorded at 25°C at a protein concentration of 0.08 mg/ml using 0.1 cm pathlength quartz cells. Each spectrum was the average of four scans and was corrected for spurious signals generated by the solvent. The results were expressed as mean residue ellipticity, [{theta}]MRW = ({theta}obs/10) (MRW/lc), where {theta}obs is the observed ellipticity at a given wavelength, MRW is the mean residue weight taken as 112.5 Da for decorsin, 111.6 for P23A and P24A decorsin and 110.9 for P23,24A decorsin, l is the cuvette pathlength in cm and c is the protein concentration in g/ml. The thermal unfolding of synthetic decorsin was monitored by recording the decrease of the CD signal at 265 nm as a function of the sample temperature (heating rate 50°C/h). Both CD signal and temperature data were recorded simultaneously by a computer program provided by Jasco. The melting temperature (Tm) was determined from the derivative curve of the CD signal at 265 nm versus temperature. The reversibility of the thermal unfolding process was determined by measuring the recovery of the CD signal upon cooling to 15°C.

Platelet aggregation assay

The biological activity of decorsin analogs was tested by assaying the inhibition of platelet aggregation (Polverino de Laureto et al., 1998Go). Fresh blood was drawn from a healthy volunteer and immediately mixed with one-tenth volume of the anticoagulant solution (85 mM sodium citrate, 70 mM citric acid, 110 mM dextrose), then incubated with prostacyclin (0.8 µg/ml) and apyrase (20 µg/ml) to prevent unspecific platelet aggregation. The blood was centrifuged at 1000 r.p.m. for 15 min at room temperature to obtain the platelet-rich plasma. Platelets were then resuspended in 20 mM HEPES buffer, pH 7.4, containing 0.15 M NaCl, 5 mM KCl, 1 mM MgSO4 and 10 mM glucose. The platelet count was adjusted to 2.5 x 108 cells/ml and the suspension was kept at room temperature and used within 2–3 h. Prior to measurements, the external calcium concentration was adjusted to 0.5 mM and the cells were equilibrated at 37°C for 3 min. Platelet aggregation, induced by thrombin (0.02 U/ml), was evaluated with an Elvi Logos 840 aggregometer at 1000 r.p.m. stirring velocity in the presence of a known concentration of decorsin or its analogs.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chemical synthesis and oxidative refolding of the reduced decorsin analogs

The solid-phase synthesis of decorsin analogs was performed in two phases. First, the peptide corresponding to the sequence 25–39 of decorsin was synthesized using a scale of 0.25 mmol, then the resin was divided into five parts and the synthesis of decorsin and its analogs P23A, P24A and P23,24A was continued on each aliquot at a 0.05 mmol scale. The synthetic procedure used here was similar to that described previously for the wild-type decorsin, in which the standard protocol was slightly modified in order to avoid cleavage of the Asp35–Pro36 peptide bond and the possible cyclization of Asp residues to succinimide moieties (Polverino de Laureto et al., 1998Go). The synthesis of decorsin analogs was successful, since the yields of synthetic crude peptides were 90% for P23A decorsin, 86% for P24A decorsin and 82% for P23,24A decorsin. The RP-HPLC traces of the crude reduced analogs were similar and these results are not shown in detail here. Figure 2 (top) shows the RP-HPLC trace of the crude reduced analog P23,24A after cleavage from the resin, and also that, as a control, of reduced wild-type leech-derived natural decorsin. The identities of the reduced analogs were confirmed by N-terminal sequencing (not shown) and MALDI-MS. The experimental molecular mass of the reduced peptides, after their RP-HPLC purification, was 4384.0 Da for wild-type decorsin, 4354.8 Da for P23A, 4356.8 Da for P24A and 4330.3 Da for P23,24A decorsin. These figures compare very favorably with those calculated (not given here) from the amino acid sequences of the decorsin analogs.



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Fig. 2. (Left) RP-HPLC of the crude synthetic P23,24A analog of decorsin after cleavage from the resin (top) and after 24 h of oxidative refolding of the reduced peptide purified by RP-HPLC (bottom). (Right) RP-HPLC of a sample of reduced natural leech-derived decorsin (top) and after its oxidative refolding (bottom). Analyses were conducted utilizing a Vydac C18 column (4.6 x 150 mm), which was eluted at a flow rate of 0.6 ml/min with a linear gradient of acetonitrile containing 0.085% TFA versus 0.1% TFA from 5 to 21% in 4 min and from 21 to 46% in 15 min. The eluate from the column was monitored by absorbance measurements at 226 nm. R and N indicate the fully reduced and the correctly folded protein, respectively, and III refers to a misfolded species (see text).

 
The oxidative refolding of the reduced decorsin analogs was performed by dissolving them at room temperature in 0.1 M Tris–HCl buffer, pH 8.5, containing 0.2 M NaCl, 2 mM GSH and 1 mM GSSG, as described previously for synthetic reduced decorsin (Polverino de Laureto et al., 1998Go). The experimental conditions for the oxidative refolding of the reduced peptides were analyzed in detail by RP-HPLC of aliquots taken from the refolding mixture at intervals. Figure 2 shows the RP-HPLC trace of P23,24A decorsin after 24 h of incubation in the refolding buffer, and also that of natural leech-derived, reduced decorsin. The peak corresponding to the reduced species of the P23,24A analog (named R) completely disappeared and two major peaks (named N and III) of oxidized products appeared in the chromatogram at lower retention time. A series of experiments were performed on natural, leech-derived reduced decorsin by varying the experimental conditions in order to find the optimal conditions for its oxidative refolding (Polverino de Laureto et al., 1998Go). Complete conversion of the reduced protein to its fully and correctly oxidized species was not achieved. The best conditions for refolding, besides the correctly folded species N, led to a ~30% of a species devoid of SH groups, but containing non-native disulfide bonds (Figure 2, bottom, peak III; see also below). Analogous behavior to that of the P23,24A analog was observed in the case of the two analogs P23A and P24A, but for simplicity these experiments are not shown here. To summarize, the oxidative refolding of the reduced decorsin species leads to two species, N and III (see Figure 2), both devoid of SH groups and thus with the three disulfide bridges formed. Indeed, the experimental molecular masses of the oxidized species (N and III), determined by MALDI-MS, were lower on average by about 6 Da with respect to the masses of the reduced species, as follows: 4377.0 Da for wild-type decorsin, 4351.8 Da for P23A, 4351.3 Da for P24A and 4326.0 Da for P23,24A decorsin.

Peptide mapping

The peptide mapping technique was used to define the disulfide pairing of the decorsin analogs after their oxidative refolding and purification by RP-HPLC (species N, Figure 2). These analyses were performed on all synthetic decorsin analogs. The correct disulfide topology in the P23A and P24A analogs was established following the procedure used for the P23,24A analog. Here, for simplicity, only the experiments conducted on the P23,24A analog are presented.

The peptide material corresponding to peak N in the RP-HPLC chromatogram of the oxidatively refolded P23,24A analog (see Figure 2) was treated with thermolysin and the proteolytic mixture thus obtained was separated by RP-HPLC (Figure 3A). The identification of the peptide material contained in each chromatographic peak was obtained by MALDI-MS and N-terminal sequencing and the results are summarized in Table I. In particular, fragments corresponding to sequences 1–15, 3–15 and 4–15 (Th3, Th2 and Th1, respectively) were clearly identified, thus providing evidence for the formation of the Cys7–Cys15 disulfide (see Figure 3C). The chromatographic peak named Th4 in Figure 3A was identified as fragment 16–39 and it is expected to contain the disulfides Cys17–Cys27 and Cys22–Cys38 of the native protein. Since Th4 was particularly resistant to proteolytic attack by thermolysin, it was subjected to further proteolysis using the more aggressive proteinase K, leading to several peptide fragments (Figure 3B). The multiple chain fragments 16–19/20–27/38–39 and 16–24/25–27/34–39 were identified in the proteolytic mixture of Th4 by N-terminal sequencing and MS data (see Table I and Figure 3C). The identity of these disulfide-crosslinked peptide species was taken as a clear indication of the existence in the P23,24A analog of the correct disulfides Cys17–Cys27 and Cys22–Cys38 of natural decorsin.



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Fig. 3. RP-HPLC of (A) P23,24A decorsin digested with thermolysin and (B) the thermolytic fragment Th4 (chain segment 16–39) digested with proteinase K. Proteolysis of the decorsin analog was conducted with thermolysin at 40°C in 50 mM Tris–HCl, pH 7.0, containing 5 mM CaCl2, at an E/S ratio of 1:20 (w/w). The peptide fragment Th4 was isolated by RP-HPLC and then further digested with proteinase K in 50 mM Tris–HCl, pH 8.3, at room temperature, at an E/S ratio of 1:50 (w/w) (see Materials and methods). (C) Scheme of the proteolytic digestion of the 39-residue chain of the P23,24A analog of decorsin by thermolysin and proteinase K. The sites of proteolysis along the chain by thermolysin and proteinase K are indicated by open and filled arrows, respectively.

 

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Table I. Analytical characterization of proteolytic fragments of the P23,24A analog of decorsin obtained by proteolysis with thermolysin (Th1–4) and of its thermolytic fragment Th4 (peptide 16–39) reacted with proteinase K (K1–7)

 
A similar fingerprinting analysis was conducted also on the peptide material of peak III obtained in the RP-HPLC of the oxidatively refolded decorsin analogs. Briefly, following the procedures outlined above for the fingerprinting analysis of species N, evidence was provided that species III contains mostly the non-native disulfides Cys7–Cys15, Cys17–Cys22 and Cys27–Cys38 (not shown). Therefore, peak III contains a decorsin analog characterized by a ‘beads’ topology (Zhang and Snyder, 1991Go). The formation of this isomer is favored for symmetry and proximity reasons and results from the statistical encounter of –SH groups along the polypeptide chain (Benham and Jafri, 1993Go). However, since these analyses revealed for species III also the presence of some peptide fragments with the correct disulfide topology, the results were not considered to be conclusive. It is possible that the oxidative refolding of reduced decorsin analogs produces, in addition to the correctly folded species N, also misfolded species III containing both correct and wrong pairing of disulfides. Therefore, we can tentatively propose that decorsin analogs with correct disulfide connectivities can be eluted in two chromatographic peaks (N and III) from an RP-HPLC column (Figure 2). Indeed, peak splitting in RP-HPLC has been observed previously with some Pro-containing peptides and proteins (see Discussion).

Spectroscopic characterization

In Figure 4, the far-UV (A) and near-UV (B) CD spectra of refolded oxidized decorsin and its analogs are shown. The far-UV CD spectrum of decorsin in the region between 200 and 230 nm is unusual and mostly given by the positive contributions of the aromatic residues and the three disulfide bonds of the protein (see also Polverino de Laureto et al., 1998Go). This strong positive CD signal overwhelms the weak negative CD contribution of peptide bonds in a ß-structure at 215–220 nm. The shape of the far-UV CD spectra of the analogs is not significantly changed, whereas the ellipticity ratio between the bands centered at 204 and 218 nm is slightly different. Because these two CD bands are due to the contributions of Tyr, Phe and Cys–Cys (Strickland, 1974Go; Khan, 1979Go; Manning and Woody, 1989Go; Vuilleumier et al., 1993Go), it is reasonable to attribute the change in intensity of the maxima to some variation of the micro-environment of these residues induced by the substitution of Pro residues. It is important to note that in the region of the far-UV CD spectra (230–245 nm) in which interference by aromatic residues and disulfide bonds does not occur and only the contribution of the ß-structure is expected, decorsin analogs show the same dichroic signal, implying that the Pro->Ala substitutions do not influence the content and type of secondary structure.



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Fig. 4. Spectroscopic characterization of the synthetic analogs of decorsin. Far-UV (A) and near-UV (B) CD spectra of decorsin (solid line) and its synthetic analogs P23A (dashed line), P24A (dotted line) and P23,24A (dashed and dotted line). CD spectra were taken at 25°C in 10 mM Na2HPO4, pH 7.0, using a 1 or 5 mm pathlength quartz cuvette in the far- and near-UV region, respectively. (C) Thermal unfolding of decorsin and its synthetic analogs P23A, P24A and P23,24A, followed by monitoring the CD signal at 265 nm. Measurements were conducted in 10 mM Na2HPO4, pH 7.0. Data are given as the [{theta}]/[{theta}]0 ratio, where [{theta}]0 is the mean residue ellipticity value measured at 15°C.

 
The near-UV CD spectrum of refolded decorsin (Figure 4B) shows a broad positive band in the 250–290 nm region, due to the optical activity of the three disulfide bonds (Strickland, 1974Go; Kahn, 1979Go), as expected from the fact that they are in a right-handed conformation (Krezel et al., 1994Go; see also Polverino de Laureto et al., 1998Go). The shape and intensity of the near-UV CD spectra of the decorsin analogs are similar to those of natural decorsin, providing evidence that the three disulfides have the same conformation in all species. Considering that the near-UV CD spectra of proteins is a very sensitive fingerprint of tertiary structure (Strickland, 1974Go; Kahn, 1979Go), the almost identical spectra shown in Figure 4A allow us to conclude that the overall structure of the decorsin analogs is very similar (see also Polverino de Laureto et al., 1998Go, for additional comments).

The thermal stability of decorsin and its analogs was compared by monitoring the decrease of the CD signal at 265 nm as a function of temperature between 15 and 90°C (Figure 4C). The decrease in CD signal at 265 nm is diagnostic of conformational variations mainly around the disulfide bridges of the protein (Kahn, 1979Go; De Filippis et al., 1995Go). The denaturation profile of decorsin is characterized by a broad conformational transition in the temperature range 60–90°C (Polverino de Laureto et al., 1998Go). By differentiation of the melting curve, it was possible to calculate a melting temperature (Tm) of 74°C for native decorsin. The two analogs with a single Pro exchanged show about the same Tm of 66–67°C, while the Tm of the double substituted analog P23,24A is ~50°C. The thermal denaturation process of decorsin and its analogs was highly reversible, as given by the 95–97% recovery of the ellipticity value at 265 nm upon cooling. Therefore, the figures for Tm observed with the decorsin analogs indicate that the destabilization by the Pro->Ala exchanges is roughly cumulative.

Biological activity

The dose-dependent inhibition of human platelet aggregation induced by decorsin and its Pro->Ala analogs is reported in Figure 5. The complete inhibition of the thrombin-induced aggregation is obtained by incubating the platelet suspension in the presence of 1 µM synthetic oxidized decorsin at 37°C for 3 min before adding the agonist (Polverino de Laureto et al., 1998Go) (Figure 5), in agreement with the results obtained with natural leech-derived decorsin (Seymour et al., 1990Go). Decorsin analogs P24A, P23A and P23,24A were able to inhibit completely aggregation of platelet approximately to the same extent. In fact, whereas the IC50 of synthetic oxidized decorsin was calculated to be ~0.1 µM, IC50 values of ~0.1, 0.12 and 0.14 µM were calculated for the decorsin analogs P24A, P23A and P23,24A, respectively. Therefore, the results shown in Figure 5 indicate that the replacement of some Pro residues in the decorsin chain does not influence significantly the biological acitivity of the protein.



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Fig. 5. Inhibition of human platelet aggregation by decorsin (closed circles), P24A decorsin (open circles), P23A decorsin (open triangles) and P23,24A decorsin (closed triangles) (see Materials and Methods).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Decorsin is one of the most potent inhibitors of platelet aggregation and, being a powerful antagonist of the platelet receptor for fibrinogen, decorsin-like peptides may eventually find an application in the treatment of thrombotic disorders (Lazarus and McDowell, 1993Go). The successful chemical synthesis of decorsin and analogs suggests the possibility of obtaining by chemical methods variants of this protein with the aim of providing useful information on the structure–activity relationships and to engineer new biologically active molecules. The main advantage of chemical synthesis with respect to recombinant methods resides in the unique possibility of introducing non-native amino acid residues in the polypeptide chain, in order to modulate the structural features of the protein in terms of steric hindrance, hydrophobicity and binding properties (Kent, 1988Go). The chemical synthesis of decorsin analogs was not expected to be an easy task, if one considers that this is a mini-protein of 39 residues containing three disulfides. Therefore, the reduced species containing six SH groups, as obtained by cleaving the peptide–resin in acid, had to be correctly refolded to its oxidized species. This means that, in order to reach the correct disulfide topology of native decorsin, only one among the 15 different disulfide-crosslinked species had to form (Benham and Jafri, 1993Go). Nevertheless, the main component in the refolding mixture was the correctly folded species N, accompanied however by misfolded variant(s) III (Figure 2; see also below). Determination of the correct disulfide topology in the synthetic decorsin analogs by using the fingerprinting approach was elaborate (see Figure 3 and Table I), but the identification of the various peptides produced by cleaving the synthetic mini-protein with thermolysin and proteinase K allowed us to conclude that the disulfide pairing was correct. Of note, in previous studies the disulfide connectivities in decorsin have been deduced only from the NMR structure of the protein (Krezel et al., 1994Go, 2000Go).

The replacement of Pro residues in position 23 and 24 of the 39-residue chain of decorsin does not cause substantial variations in the global secondary and tertiary structure of the protein, as judged from far- and near-UV CD measurements. However, a major effect of Pro->Ala replacements was observed on the thermal stability of decorsin. Indeed, the analogs with a single mutation show a decrease in Tm of ~10°C with respect to that of decorsin (Tm {approx} 74°C), while the replacement of two Pro residues leads to an analog with a Tm lower by ~24°C. The thermal unfolding process of decorsin analogs is characterized by a fairly high Tm for a very small 39-residue protein and a low cooperativity of unfolding (see Figure 4C). Therefore, the characteristics of the heat denaturation of decorsin analogs parallel those of other small proteins, such as BPTI, neurotoxin II, parvalbumin and the IgG binding domain of streptococcal protein G. Indeed, with these small proteins, the stability curves are broader and shallower than the curves for larger proteins (see Alexander et al., 1992Go, for a discussion).

The unusual thermal stability of decorsin can be correlated with its large number of Pro residues, in agreement with the ‘proline rule’ previously advanced for explaining the high thermal stability of thermophilic enzymes, which usually show an enhanced content of Pro residues with respect to their mesophilic thermolabile counterparts (Suzuki et al., 1987Go; Watanabe et al., 1994Go). The relative abundance of Pro residues (15.3%) in decorsin is much higher than that normally observed in natural proteins (5.1%) (McCaldon and Argos, 1988Go). The molecular mechanism of Pro stabilization appears to be related to the fact that the pyrrolidine ring of Pro restricts the number of conformations that it can adopt, compared with other residues. It has been proposed that mutant proteins with Pro->X exchanges display a reduced stability due to the entropy gain in the denatured state (Matthews et al., 1987Go). Indeed, a Pro->Ala replacement was calculated to contribute 4 cal/mol.K to the entropy of denaturation (Nemethy et al., 1966Go). In particular, it has been estimated that the native form of a protein is stabilized by 0.5–1 kcal/mol when a Pro residue is introduced in a protein chain at a location that does not alter the protein secondary structure (Matthews et al., 1987Go; Nicholson et al., 1992Go; Allen et al., 1998Go). Therefore, the results of this study parallel those that have shown that Pro has a remarkable influence on the intrinsic stability of protein structure, as for example in the case of tryptophan synthase {alpha}-subunit (Yutani et al., 1991Go), human lysozyme (Ueda et al., 1993Go), T4 lysozyme (Matthews et al., 1987Go) and others. In some cases, however, the decrease in protein stability upon Pro replacements was only marginal (Chen et al., 1992Go) and, in rare cases, even an enhanced stability was observed (Green et al., 1992Go; Maki et al., 1999Go). Therefore, the role of Pro on protein stability may depend on the location of the Pro residues in the protein fold (Gray et al., 1996Go) and, consequently, the stabilizing role of Pro cannot be taken as a rule. In fact, it has been shown recently that Pro->Ala replacements in ubiquitin can produce context-dependent effects, since they had opposite effects in reducing or enhancing protein stability depending on the site of mutation along the protein chain (Crespo et al., 2004Go).

Proline residues have a major effect also on the process of protein folding (Brandts et al., 1975Go). This is caused by the fact that peptide bonds involving Pro residues have a high probability of existing in a cis conformation (between 0.1 and 0.3, in comparison with 10–3 for non-Pro residues) (Brandts et al., 1975Go; Ramachandran and Mitra, 1976Go; MacArthur and Thornton, 1991Go). Given that the rate of the cistrans conversion is rather slow under physiological conditions, it can be anticipated that the cistrans isomerization would slow the protein folding process and, in particular, eventually lead to the formation of parallel folding or misfolding processes (Brandts et al., 1975Go; Nall, 1994Go; Kiefhaber, 1995aGo,bGo; Wu and Matthews, 2003Go). Indeed, the substitution of Pro residues in a protein can simplify the folding process, owing to the elimination of slow phase(s) given by the cistrans isomerization of Pro–X bonds (Creighton, 1990Go; Ogasahara and Yutani, 1997Go; Wu and Matthews, 2003Go). In this study, it is seen that decorsin analogs with Pro replacements do not fold better than the wild-type, leech-derived decorsin, since with both natural and synthetic decorsin analogs the formation of misfolded forms is observed (species III in Figure 2). This indicates that misfolding is not connected with Pro23 or Pro24, but perhaps with other Pro residues or even other non-Pro peptide bonds (Odefey et al., 1995Go). The results obtained here with decorsin contrast with the clean and high yield oxidative refolding process of the reduced hirudin domain 1–47 (De Filippis et al., 1995Go). We have found that misfolded species III (Figure 2) contains a decorsin isomer with a ‘beads’ topology of disulfides (Cys7–Cys15, Cys17–Cys22 and Cys27–Cys38), i.e. a protein chain crosslinked by disulfides whose formation is favored by the proximity of SH groups along the chain and not by the overall structure of the protein (Zhang and Snyder, 1991Go; Benham and Jafri, 1993Go). However, evidence has been obtained that species III contains also decorsin species with the correct pairing of disulfides as species N (see Results and Figure 2). This would imply that the same protein molecule adopts two conformational states which are stable enough to be separated by RP-HPLC. This interpretation is supported by previous observations that have indicated that some Pro-containing peptides are eluted from an RP-HPLC column in two chromatographic peaks, as a result of the slow cistrans isomerization of Pro-containing peptide bonds (Gray et al., 1996). In particular, the 42-residue protein myotoxin from Crotalus viridis viridis, containing three disulfide bridges as in the case of decorsin, was shown to exist in two conformational states that can be separated by RP-HPLC, as a result of a cistrans isomerization at Pro20 (Nedelkov et al., 1997Go). Therefore, we may conclude that species III (Figure 2) is a misfolded form(s) due to either different disulfide pairing or different Pro conformers resulting from the cistrans isomerization. Similar explanations have been advanced also for the formation of misfolded form(s) of decorsin obtained by recombinant methods (Krezel et al., 2000Go).

The effects of the substitution of Pro on the biological activity of decorsin have been found to be marginal. The IC50 values, i.e. the concentrations of peptides that lead to a 50% inhibition of platelet aggregation, are about the same for the analogs and natural decorsin (see Figure 5). This indicates that the Pro->Ala exchanges at positions 23 and 24 do not modify significantly the RGD active site of decorsin. Actually, the biological activity of decorsin is similar to that observed with other disintegrins, all containing a flexible RGD site and little secondary structure. Krezel et al. (2000)Go have shown that the RGD loop in decorsin is the most dynamic part of the molecule, even if somewhat more restricted than in other proteins with similar activities. Since many different scaffolds, such as kistrin, echistatin, albolabrin and mambin, can present the flexible RGD binding site for the integrin receptors, it may well be that the Pro->Ala exchanges described here are tolerated by the decorsin scaffold without affecting both the protein overall structure and the capability of the dynamic RGD loop to interact with integrin receptors (Krezel et al., 1994Go). Therefore, the RGD epitope can be presented by a variety of protein scaffolds (Vita, 1997Go), including proteins (Lee et al., 1993Go; Zanetti et al., 1993Go; Yamada et al., 1995Go; Nie and Tang, 1998Go; Kellenberger et al., 1999Go) and small peptides (see Lazarus and McDowell, 1993Go, for a review).

Overall, this study demonstrates that the Pro residues in decorsin play a major role in the extraordinary thermal stability of the protein. Such high thermostability is likely to have an important physiological role, conferring on decorsin resistance to proteolytic degradation and structural rigidity.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by the Italian Ministry of Universities and Research (FIRB Project on Protein Folding and PRIN-2003). We thank Mr Marcello Zambonin for his expert technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received July 27, 2005; accepted July 28, 2005.

Edited by Lynne Regan and Andreas Plückthun





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