Primary Structure and Post-Translational Modifications of Silicatein Beta from the Marine Sponge Petrosia ficiformis (Poiret, 1789) Andrea Armirotti,†,| Gianluca Damonte,‡,| Marina Pozzolini,‡ Francesca Mussino,| Carlo Cerrano,§ Annalisa Salis,‡,| Umberto Benatti,‡,| and Marco Giovine*,†,‡,⊥ Centro Biotecnologie Avanzate, Largo Rosanna Benzi, 10, 16132 Genova, Italy, Center of Excellence for Biomedical Research, Viale Benedetto XV, 7 16132 Genova, Italy, Dipartimento per lo Studio del Territorio e delle sue Risorse, Corso Europa 26, 16132 Genova, Italy, Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Viale Benedetto XV, 1 16132 Genova, Italy, and Dipartimento di Biologia, Universita` degli Studi di Genova, Via Pastore 3, 16132 Genova, Italy Received April 15, 2009
Biosilica is an amazing example of natural order and complexity. Siliceous sponge spicules, in particular, are characterized by a large variety of dimensions and shapes, with an ultrastructure based on silica nanoparticles strictly packaged around an axial filament constituted by a family of proteins called silicateins. These peculiar proteins have a high sequence homology with cathepsins and they play a double role of enzyme and template in the control of biosilica precipitation. However, their natural structural organization inside the spicules is far from being understood in details. In this work, axial filaments extracted from spicules of Petrosia ficiformis have been extensively analyzed by mass spectrometry, exploiting MALDI and ESI analysis of both the intact protein and the peptides coming from digestion of the axial filament with different proteases. Results demonstrate that P. ficiformis spicules contain almost only silicatein beta. Several post-translational modifications, like methylations at the N-terminal region, three phosphorylation sites, and the oxidation of a histidine and of a cysteine to cysteic acid, are described. Keywords: silicatein • sponge • biosilica • MALDI-MS • ESI-MS • tandem mass spectrometry • posttranslational modifications
Introduction Many marine organisms build their skeleton catalyzing the precipitation of a variety of distinct biominerals.1 Sponge and diatoms, in particular, have the fascinating capability of building very complex biosilica structures showing a high degree of order up to the nanometric level.2,3 Sponges (phylum Porifera) are simple animals arranged into three classes according to their anatomy/morphology and their skeletons, composed by discrete elements called spicules. These elements can be made of calcium carbonate, as in the Calcarea class, or made of silica. The class Hexactinellida (which is the phylogenetically oldest) and many species of the Demospongiae class have a siliceous skeleton.4 Spicules may show very different and complex shapes and the molecular mechanism leading to their formation is only partially understood and some aspects are not completely disclosed.5 Despite their high diversity in shapes and dimensions (from some micrometers to 2-3 m length), their nanostructural features are very similar: they are formed by concentric arrays of silica nanoparticles organized around * To whom correspondence should be addressed. E-mail: mgiovine@ unige.it. † Centro Biotecnologie Avanzate. | Dipartimento di Medicina Sperimentale. ‡ Center of Excellence for Biomedical Research. § Dipartimento per lo Studio del Territorio e delle sue Risorse. ⊥ Dipartimento di Biologia, Universita` degli Studi di Genova. 10.1021/pr900342y CCC: $40.75
2009 American Chemical Society
a proteic axial filament.3,6 Spicules are initially produced within specialized cells (sclerocytes) and successively extruded in the extracellular environment where their growth is completed. It is generally assumed that their growth is a bidirectional process: the increase in length is affected by the elongation of the filament, while the increase in width is determined by the apposition of silica. Researches into the mechanisms controlling these processes highlighted a new family of proteins called silicateins as possible control molecules.5 Silicateins seem to have a peculiar dual role in the Demospongiae biosilicification process: they act as both structural proteins and enzymes able to catalyze and structurally direct the deposition of biosilica.5 Several silicateins from different sponges have been nowadays described7-14 and all these proteins exhibit a high sequence homology with the cystein proteases family of cathepsins, suggesting a common evolutionary origin, even if some peculiar differences justify the grouping of these proteins in a specific new family.15 The comparison between the primary sequences of silicateins and cathepsin L shows the presence of a “catalytic triad” in both proteins15 and this evidence suggested a hydrolytic activity in the mechanism of action of silicateins.8 The active site residues are in equivalent positions in the silicateins and in cathepsin L, even though the nucleophilic site in the active site of silicatein is a serine, rather than a cysteine as in cathepsin L.16 Another relevant homology is the persistence Journal of Proteome Research 2009, 8, 3995–4004 3995 Published on Web 06/13/2009
research articles in both protein families of six cysteine residues. In several cathepsins, these residues are linked in intramolecular disulfide bridges, while the structural role of these cysteines in silicateins is still unknown. Despite the high sequence homology with the extremely soluble cathepsins, it has been published that, after their extraction from the silica envelope with NH4F/HF, the silicatein monomers aggregate into oligomers. These oligomeric units assemble into an insoluble fractal network that subsequently condenses and organizes into a filamentous structure, that was originally considered structurally similar to the spicules axial filament.8,17 These filamentous structures have been demonstrated to catalyze the in vitro precipitation of silica when incubated with the organosilicon compound tetraetoxysilane (TEOS), thus, indicating that silicateins act as templates on which biosilica is precipitated in the Demospongiae sclerocytes.18 Several questions on the biosilicification process in sponges are nevertheless still open and the present in vitro model of silicatein is not completely satisfying. In particular, the structural organization inside the spicules and the natural silica substrata used by these proteins to build up the highly ordered and complex structure of spicules are far from being understood in detail. To achieve a more complete and satisfying description of silicatein structural organization, a proteomic approach could be useful. In this work, axial filaments extracted from spicules of Petrosia ficiformis have been extensively analyzed by mass spectrometry. The experimental results here described were obtained exploiting different techniques with the aim to verify the protein composition of axial filament and to characterize the mature isoform of silicatein beta, whose cDNA sequence was described in a previous work of this research group.14 Both MALDI and Nanospray mass spectrometry experiments on the protein content extracted by HF dissolution of spicules have been performed, in order to estimate the molecular mass of the proteins contained inside the axial filament. Furthermore, a “bottom-up” analysis, where the sample protein is digested with proteases and the resulting peptides are analyzed by tandem MS, was performed on the total axial filament protein content, in order to identify the N-terminus of mature silicatein and to put in evidence possible post-translational modifications (PTMs). An extensive de novo analysis of tandem mass spectra of tryptic and chimotryptic peptides yielded a complete coverage of the predicted cDNA sequence and allowed the identification of several PTMs.
Materials and Methods Sponges Collection. Specimens of the Demospongiae P. ficiformis were collected on the rocky cliff of the Marine Protected Area of the Portofino Promontory (Ligurian Sea, Italy) between 15-20 m depth. P. ficiformis is a common Mediterranean sponge living in different levels of symbiosis with cyanobacteria.19 This sponge is rich in monoaxonic spicules and it was already used for previous studies by this research group.14,20 Preparation of Spicules and Axial Filament (Silicatein). The axial filament was extracted from spicules as described by Shimizu in 1998.16 Briefly, samples of dry tissues of P. ficiformis were treated with H2SO4/HNO3 (4:1) overnight. The silica spicules obtained were rinsed extensively with Milli-Q purified water until the pH was above 6. The samples were dried using a vacuum drier and 5 g of this material was dissolved in 500 mL of 2 M HF/8 M NH4F, pH 5 at room temperature for 3 h, then dialyzed against Milli-Q water, and finally collected by centrifugation. For the determination of protein concentration 3996
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Armirotti et al. in insoluble axial filament extract, aliquots of 100 µL of this suspension were centrifuged for 5 min at maximum speed and the protein precipitate was dissolved in 100 µL of 8 M urea. The protein content in the samples was determined by Bradford assay, using BSA as standard. Electron Microscopy Analysis. Spicules were air-dried and filaments were critical point dried using a CO2 Pabish CPD apparatus and then coated with gold-palladium in a Balzers Union evaporator. Samples were examined with a Philips EM 515 SEM. Silicatein Activity Assay. Aliquots of 150 µg of silicatein extract were incubated in 300 µL of 25 mM Tris buffer, pH 6.8, with 500 µL (2.2 mmol) of tetraethoxysilane (TEOS, Sigma), for 18 h at room temperature under rotator agitation. The enzymatic reaction was terminated by centrifugation (16000g, 15 min). The precipitate was washed three times with ethanol, airdried, redissolved in 300 µL of 1 M NaOH, and incubated for 15 min at 90 °C. After neutralization with 1 M HCl, the concentration of dissolved silica was determined as described by Cha in 1999,18 using the Silicon Test colorimetric assay kit (Merck). Incubations with tetraethoxysilane and silicatein extract alone were performed as negative controls. For a semiquantitative analysis of silicatein activity, the silica precipitate, after ethanol wash, was air-dried and directly analyzed with electron microscopy. SDS PAGE of Silicatein. Ten milligrams of purified spicules and 10 µg of silicatein extract were dissolved in 1X gel loading buffer (62.5 mM tris-HCl, pH 6.8, 25% glycerol, 2% SDS, and 0.01% bromo-phenol blue), boiled for 10 min, and subjected to SDS-Page (12% polyacrylamide, 0.1% SDS). The gel was stained for 20 min with Coomassie blue R250 solution (BioRad). Bottom-Up Analysis of Silicatein. Protein samples coming from dissolution of spicules were resuspended in 50 mM ammonium bicarbonate, reduced with 10 mM dithiothreitol (DTT) (60 °C for 1 h), and subjected to cysteine alkylation with 50 mM iodoacetamide (room temperature (RT) for 30 min). Protease digestion was performed with trypsin or chimotrypsin (peptide sequencing grade, purchased from Sigma-Aldrich) in 50 mM ammonium bicarbonate at 37 °C overnight. Resulting peptides were dried in vacuo and resuspended in a 90/10 water/acetonitrile solution. Initial sample cloudiness, due to extremely low solubility of silicatein, was reduced during the process and the sample was completely dissolved after digestion. Peptides were injected on a LC-Packings Ultimate NanoLC system (Dionex Corporation Sunnyvale, CA) coupled with an Applied Biosystems QSTAR XL qTOF mass spectrometer (Foster City, CA). Eluents were water and acetonitrile added with 0.1% formic acid. Gradient was 5% acetonitrile for 5 min, then to 90% in 120 min. Flow rate was set to 200 nL/min. Ionization was achieved with a Nanospray ion source (Applied Biosystems). Mass spectra were acquired in positive ion mode in the 300-1800 m/z mass range. Tandem mass spectra of doubly and triply charged precursors were automatically acquired by the control software. Collision gas was nitrogen and the applied collision energy was set in the 20-80 eV range, accordingly to the precursor ion mass to charge ratio. Source parameters were set as follows: spray voltage, 2400 V; declustering potential, 60 V; focusing potential, 220 V; curtain gas, 25; detector, 2400 V. Bioinformatics. Both Web-based MASCOT (MatrixScience, London, U.K.) and in-house ProteinPilot Demo Version (by Applied Biosystems) sequence search algorithms were used for sequence coverage analysis and PTMs search. Manual de novo
Primary Structure and PTMs of Silicatein Beta from P. ficiformis sequencing, though assisted by data analysis software (Analyst QS and Protein Pilot, Applied Biosystems), was extensively exploited to analyze ambiguous results, to confirm borderline peptide identifications and unusual PTMs. Silicatein sequence homology search was performed with the ScanPS tool hosted in the Expasy Web server,21 using the UniProt KB/SwissProt database. Hydropathy calculations were performed using the ProtScale tool hosted in Expasy, with the Kite-Doolittle scale22 and a window size of 9. Prediction on silicatein aggregation in aqueous solutions was performed with PASTA Web-based algorithm.23 Details on Database Search. Tandem mass spectra were automatically converted in .mgf format peaklist by the Analyst software and submitted to the search algorithm. Parameters used for MASCOT search: MSDB database; other Metazoa taxonomy; trypsin or chimotrypsin as enzymes; one missed cleavage allowed; carboxamidomethylation as fixed modification; variable modifications, oxidation (H, W, M); peptide tolerance 0.3 Da; MS/MS tolerance 0.2 Da; peptide charge +2 and +3; instrument qTOF. Parameters used for Protein Pilot search (Paragon Algorithm): Swiss-Prot database (uniprot_sprot_2007 0123); other species taxonomy; trypsin or chimotrypsin digestion; carboxamidomethyl cysteine alkylation; ID focus, biological modifications (according to Applied Biosystems PTMs database); thorough ID search effort. MALDI Analysis of Intact Silicatein. Since silicatein is insoluble in MS-compatible solvents, the sample was resuspended in a water/acetonitrile 50/50 solution and 1 µL of such resuspension was quickly dropped on the sample plate, followed, after dryness, by 1 µL of matrix. MALDI analysis was performed on the same qTOF instrument described above, using an oMALDI 2 ion source (Applied Biosystems). Ionization was achieved using a nitrogen UV laser (337 nm) with power ranging from 8.8 to 13.2 µJ with a pulse rate of 20 Hz. Spectra were acquired in positive ion mode using sinapic acid as matrix (30 mg/mL in water/acetonitrile 50/50 with 0.1% TFA). NanoLC-MS Analysis of Intact Silicatein. The instrumental setup was the same as that described above for tandem mass analysis of peptides, with acetonitrile gradient from 30% to 95%. Acquisition range was set to 500-1800 m/z and declustering potential was raised to 80 V. After centrifugation at 14000 rpm for 3 min of the protein suspension described above for MALDI analysis, 10 µL of the supernatant was loaded onto the column.
Results Preparation of Spicules and Axial Filament (Silicatein). Acid dissolution of 62 g of dry tissue from different specimens of P. ficiformis yielded 14 g of silica spicules (Figure 1, panel A). After this treatment, all detectable proteins were removed from the outside of the spicules. The axial filament contained inside the silica spicules (Figure 1, panel B) was obtained after treatment with HF/NH4F. Typically, 2 g of silica material yielded 2 mg of protein extract. The silicate component present in the sample was 0.06 nmol/µg of protein. Silicatein Activity Assay. The activity present in the silicatein extract incubated for 18 h with TEOS and expressed in terms of polymerized silica was 143.2 ( 16.3 nmol/µg of protein. In the negative control sample, without protein, the TEOS spontaneous silica precipitate was 90.9 ( 2.3 nmol, whereas the silicon background present in silicatein sample without TEOS was 10.0 ( 0.4 nmol/µg of protein (Table 1). The SEM analysis of silica precipitate reveals that no detectable silica particles were present in silicatein sample without TEOS (Figure 2A); 2
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nm silica particles were evident around all the axial filament surfaces in sample with TEOS (Figure 2B). Amorphous silica aggregate was obtained in sample without silicatein (Figure 2C). Electrophoresis of Axial Filament. When 10 mg of purified spicules was run in a 12% polyacrylamide gel, no protein bands were observed, whereas a predominant band around 50 kDa, corresponding to a dimeric form of silicatein, was observed when 10 µg of axial filament extract was analyzed in the same conditions (Figure 3). N-Terminal Analysis. The genomic sequence of silicatein beta from P. ficiformis was originally characterized by this group in 200414 and its full-length cDNA codifies for an immature protein with expected mass of 37645 Da. This protein is subjected to post-translational processes leading to the removal of a pre-propeptide and of a propeptide, with the generation of a shorter mature protein. Several tandem mass experiments on the total protein content of spicules were performed in order to identify the N-terminus of the effectively expressed protein sequence. With the use of trypsin as protease, with C-terminal side of K and R as expected cleavage sites, peptide LPETVDWR was identified as the N-terminus, because leucine follows a serine residue in the genomic sequence and no cutting was expected here (see Supporting file 1). Moreover, no peptides from the genomic sequence above this N-terminus were detected in tandem mass experiments. Mature silicatein sequence is therefore: LPETVDWRTGGAVTHVKDQLRCGCSYAFAAVGALEGAAALARGRTASLSEQNVLDCSVPYGNHGCSCEDVNNAFMYVIDNGGLDTTSSYPYVSRQYYCKFKSSGVGATATGIVTISSGDESSLESALATAGPVAVYIDASHSSFQFYKYGVLNVPNCSRSKLSHAMILIGYGTTSSKKYWLLKNSWGPNWGISGYIKMSRGMSNQCGIATYASFPTL. The expected molecular mass for this protein is 23094 Da with a calculated isoelectric point of 8.12. Solubilization of Silicatein and Analysis of the Intact Protein. To confirm the molecular mass of the intact protein, several experiments on silicatein dissolution were carried out. With the use of both aqueous and organic solvents, the protein remains poorly soluble. MALDI mass spectrometry was then performed on a suspension of silicatein in water and acetonitrile, exploiting the sensitivity of this technique and the possibility to ionize proteins from the solid phase. After several attempts to achieve the best experimental conditions, a complex pattern of poorly resolved signals was detected in the 23-24 kDa range, with an apex at 23,5 kDa (see Figure 4, panel A). Concentrated (30 g/L) sinapic acid was used as matrix. Lower matrix concentrations (1-10 g/L) did not produce mass spectra. Reported results show an effective protein sequence significantly shorter than the putative one based upon the cDNA sequence (37645 Da), but very similar to the expected mature silicatein, deprived of its propeptide. It should be noted here that signal complexity might be significantly increased due to adducted species with sinapic acid molecules (a common occurrence in MALDI-MS with concentrated matrices, as showed, for example, by Jensen and colleagues24). Despite the above-mentioned difficulties with silicatein solubilization, effective nanoLC/MS analysis of the intact protein was achieved. Silicatein was resuspended in 8 M urea (detergents help silicatein solubilization but were avoided because of their complete incompatibility with MS), passed through a desalting column, and then loaded onto the nanobore column. Silicatein is eluted as a sharp peak at 27.5 min (see Figure 4, panel B) and appears in the mass spectrum as a multiply charged pattern of three molecules, very close in mass, at 46865, 47010, Journal of Proteome Research • Vol. 8, No. 8, 2009 3997
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Figure 1. SEM pictures of purified spicules from P. ficiformis (panel A) and axial filaments obtained from spicules dissolution (panel B). A single axial filament is indicated by an arrow. Table 1. Silicatein Enzymatic Activitya axial filament (silicatein)
TEOS
polymerized silica (ng silicon/µg protein)
+ + -
+ +
90.9 ( 2.3 143.2 ( 16.3 10.0 ( 0.4
a The silicatein assay was performed in presence (+) or in absence (-) of 2.2 mmol of tetraethoxysilane (TEOS) as the substrate. The amount of polymerized silica was quantified after hydrolyzation with NaOH and subsequent determination of released silicic acid. The values are given in nanograms of silicon, and if silicatein was added, normalized to 1 µg of protein.
and 47125 Da, respectively. These masses correspond to dimeric forms of three molecules of 23432, 23505, and 23562 Da, respectively (see Figure 4, panels C and D). This inclination of silicatein to form dimeric aggregates is in good agreement with literature data and with the results of gel electrophoresis. Since no direct evidence for covalent peptide linking was collected in bottom-up experiments with trypsin and chimo3998
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trypsin, these silicatein dimers should form in a noncovalent way. This conclusion is supported by the aggregation profile of silicatein calculated with the PASTA algorithm.23 Silicatein has two highly aggregating sequence parts (Supporting file 10): residues 70-78 (VNNAFMYVI) and residues 133-154 (VAVYIDASHSSFQFYKYGVLNV). The calculated PASTA energy values for these sequences are -7.2 and -6.8, respectively. As a benchmark, the highest calculated PASTA energy for the aggregation of human amyloid protein, residues 12-20, is -6.1.25 That means that silicatein monomers are estimated to be highly aggregating in aqueous solutions. Moreover, silicatein remained in dimeric form even after overnight incubation with 50 mM DTT, therefore, excluding the formation of intermolecular disulfide bridges. Furthermore, after DTT reduction, no mass shifts in multicharged peaks of silicatein were collected in LC-MS experiments on the intact protein. Peak at 27.5 min represents the large majority of the protein content in the axial filament. Traces of two shorter proteins, with a molecular mass
Primary Structure and PTMs of Silicatein Beta from P. ficiformis
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Figure 2. SEM analysis of silica precipitate obtained after incubation of silicatein extract whitout or with TEOS as substrate. Silicatein extract incubated without TEOS (A), silicatein extract incubated with 2.2 mmol of TEOS (B), spontaneous silica precipitate of TEOS (C).
and a calculated primary sequence mass of 23.1 kDa, leaving approximately 400 Da to be assigned as PTMs.
Figure 3. Electrophoretic analysis of purified spicules and axial filament extract. Standard of molecular weight (lane a); 10 mg of purified and boiled spicules (lane b); 10 µg of axial filament extract (lane c).
of 17195 and 16327 Da, respectively, were recorded in dimeric forms, at 25.7 and 26.3 min (see Figure 4, panel D). These two proteins should correspond to shorter forms of silicatein, because no other protein except silicatein was identified by MASCOT and ProteinPilot in bottom-up experiments. Protein solubilization was enhanced with SDS 1% in 8 M urea and a small amount of silicatein was purified on a conventional RP chromatography, analogous to that performed for the nanoLCMS analysis, but monitoring 280 nm absorbance with an UV detector (data not shown). This separation also yielded a single peak, that was collected, reduced, alkylated and digested. Subsequent tandem mass experiments proved that peak at 27.5 min of the LC-MS run is indeed silicatein in dimeric form. Those minor peaks present in nanoLC/MS analysis were not detected in conventional HPLC analysis at 280 nm, possibly because even in these “strong” conditions the amount of protein in solution is low and UV detection is not adequately sensitive. It should be reported here that the molecular mass recorded in MALDI experiments (23.5 kDa) might correspond to both a single molecule and to a doubly charged dimer, and neither the resolution of our instrument nor the maximum mass range (40 kDa) can help address this issue. Data collected on the intact sample depict therefore the axial filament content as an almost unique protein with a recorded mass of 23.5 kDa
Results of MASCOT Search and Sequence Coverage of Silicatein. Silicatein beta from P. ficiformis (Q6YD92) was the only protein confidentially identified in the sample, with high MASCOT scores, typically above 400 (MudPit score). No other protein was identified in the data set. The focus was then kept on achieving the complete sequence coverage of silicatein beta. A consistent number of tandem mass experiments was required in order to confirm silicatein primary structure. With conventional tryptic digestion, large part of the sequence remains uncovered by tandem mass analysis, due to the absence of lysine and arginine residues in the central part of the sequence. Resulting peptides (3-5 kDa) are therefore too large and retain too few charges in ESI conditions for effective tandem mass analysis. Chimotrypsin, cleaving proteins at C-terminus of F, L, M, W and Y residues, usually produces a large number of smaller peptides in the 500-1000 Da mass range. Unfortunately, the tandem mass behavior of these peptides is often affected by the absence of a positively charged residue at the C-terminus (K or R). In chimotryptic peptides, basic residues are therefore present inside the sequence, instead of the Nand C-terminals. These basic residues usually “trap” the mobile proton (the migration of a “mobile proton” is at present the most widely accepted model for peptide fragmentation26), producing tandem mass spectra abnormally rich in multiply charged species and whose automated identification is generally less good than that of tryptic peptides. An accurate overlap of the parts of the sequence covered by trypsin with those covered by chimotrypsin and an accurate spectrum-byspectrum analysis yielded a complete sequence coverage. In particular, an extensive use of manual interpretation of MS/ MS spectra, in combination with de novo results proposed by ProteinPilot, was a crucial breakthrough in silicatein analysis, allowing the complete coverage of those part of the sequence still to be verified. The genomic sequence previously reported in literature for silicatein beta was completely confirmed. No evidence of covalent peptide linking was collected in tandem mass experiments. Analysis of Post-Translational Modifications. Search for PTMs was performed in a semiautomated way: tandem mass spectra were submitted to ProteinPilot, looking for conventional and unusual PTMs reported in the Applied Biosystems PTMs database included in the software. Results proposed by the alghorithm were then confirmed, rejected or modified closely analyzing the corresponding tandem mass spectrum, often combining data from experiments at slightly different experimental conditions, such as different collision energies. With this approach, several PTMs were detected. Three phosphorylation sites, Ser 66, Tyr 97, and Ser 213, were found in Journal of Proteome Research • Vol. 8, No. 8, 2009 3999
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Figure 4. MS analysis of intact silicatein. Panel A shows the MALDI mass spectrum of silicatein extracted from spicules by HF dissolution. Panel B shows the Total Ion Chromatogram (arbitrary units) of the nanoLC-MS analysis of intact silicatein. Panel C shows the recorded mass spectrum of peak at 27.5 min. Panel D summarizes the calculated molecular masses of the monomeric forms of silicatein (detected in the LC run as dimers). Table 2. Results of Tandem Mass Analysis of the Effectively Expressed Form of Silicateina meLPETVDWRTGGAVToxH15VKDQLRCGCSYAFAAVGALEGAAALARGRTASLSEQNVVDCSVPYGNHGCpS66CEDVNNAFMYVIDNGGLDTTSSYPYVSRQYpY97CKFKSSGVGATATGIVTISSGDESSLESALATAGPVAVYIDASHSSFQFYKYGVLNVPNcaC157SRSKLSHAMILIGYGTTSSKKYWLLKNSWGPNWGISGYIKMSRGMSNQCGIATYApS213FPTL a Detected PTMs are underlined. The position of the modification is indicated as superscript. Abbreviations used: me, methylation; ox, oxidation; p, phosphorylation; ca, cisteic acid.
functional silicatein from P. ficiformis (Supporting files 2-4). It should nevertheless be noted that the reported phosporylations are those surviving the HF extraction procedure and might not correctly represent the quantitative level of phosphorylation of silicatein. For this reason, experiments to assess the correct grade of phosphorylation cannot be taken into account on silicatein extracted with HF. Along with LPETVDWR N-terminus peptide of effectively expressed silicatein, less abundant mono and bimethylated (Me)LPETVDWR and (Me)2LPETVDWR peptides were found in the samples (Supporting files 5 and 6). Two other PTMs were pointed out by ProteinPilot and manually confirmed. Histidine 15 is extensively oxidized (Supporting file 7) and in Cys 157, the thiol group is modified to cisteic acid (Supporting file 8). It should be noted here that these PTMs are believed to be important indicators of redox phenomena occurring in vivo.27 The primary structure of the silicatein effectively expressed in P. ficiformis, resulting from proteolytic digestion and tandem mass analysis of peptides, is summarized in Table 2.
Discussion The great interest in biomaterials sciences and in biomimesis processes have determined a remarkable increase in the number of studies in the field of biosilicification. In particular, diatoms and sponges are the two models principally studied and the highly ordered biosilica structures synthesized by these organisms represent one of the most intriguing example of natural complexity. Different approaches are necessary for a complete disclosure of the fine structure of these materials originated by the reactions of organic molecules, prevalently 4000
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proteins, and soluble silicon-containing molecules. In marine sponges, the classical approaches used for the material science studies have recently given several information on the ultrastructure and on the chemical-physical properties of biosilica,28-30 and some research groups have begun to describe the organic content of these structure. Ten years of literature on this specific topic suggest that the high level of order, up to nanoscale level, is specifically governed by a complex network of proteins, and some of them have been recently characterized in their cDNA and cloned, but very few information is still present on the native structure of them. In particular, their low solubility after HF extraction is the main cause of uneasy handling of these proteins into aqueous solution with consequent difficulties for their biochemical purification. Notwithstanding these well-known difficulties, a primary structure analysis of silicatein, hence performed on a very uneasy material, is reported in this paper, in order to give a contribution to the structural characterization of this protein. Tandem mass analysis proves that the primary sequence of this protein corresponds to the gene-predicted one, with a few PTMs. Results presented here show that the mature form of P. ficiformis from silicatein beta has a molecular weight of 23.1 kDa. This shorter molecule is generated upon the cleavage of a large propeptide, yielding the N-terminus shown in Supporting file 1. A similar behavior has already been predicted for silicatein alpha by Morse16 and Krasko.7 Furthermore, in P. ficiformis, only one protein has been observed: one single band is present in SDS-PAGE; LC-MS experiment yielded only one sharp peak; in bottom-up analysis of the axial filament, no protein other than silicatein beta was identified by MASCOT;
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Primary Structure and PTMs of Silicatein Beta from P. ficiformis
Table 3. Sequence Alignment of Silicatein from P. ficiformis (Upper) and Cathepsin L from S. peregrine (Lower)a
a
Cysteine residues positions and the four residue propeptide cleaved in cathepsin are underlined.
and, finally, molecular biology experiments from cDNA obtained from adult sponge tissues led to the amplification of silicatein beta gene only (data not shown). According to these data, only one protein is present in the axial filament. In recent literature, nevertheless, it has been reported9,32 that in the genome of other sponges different genes of silicatein are present and expressed. In some of the cited examples, different typologies of spicules are present in the sponge and it has also been shown that the gene expression levels change during sponge development. Since only one kind of spicules is present in P. ficiformis, it should be assumed that in the adult sponge only one gene is expressed. According to the data reported in Results, silicatein beta is present in three unresolved forms very close in mass and shows mono and bimethylation at the N-terminus. Taking into account three phosphorylation sites and the other PTMs, the difference with the mass calculated on the basis of the genomic sequence is under 100 Da and might correspond to an undetected PTM or to a noncovalently bound small molecule yet to be detected. Since 17.2 and a 16.3 kDa proteins share with silicatein the same primary sequence, they should be considered as products of a proteolytic activity of silicatein itself. Such behavior has already been shown by Mu ¨ller and colleagues.8 As mentioned above, the biological role of those oxidation-related PTMs detected by tandem mass analysis deserves further investigation, because they might indicate that spicules represent an environment relatively transparent to oxidative phenomena. The analysis of PTMs can be introduced in the more vast issue about silicatein extremely low solubility. It is well-known that silicateins have a high degree of homology with cathepsins. However, they are soluble in aqueous solutions. To address this issue, a sequence homology search performed using the effective silicatein sequence reported in Results indicates that cathepsin L from Sarcophaga peregrine (CATL_SARPE) has the highest sequence homology with silicatein beta from P. ficiformis (E-value 10 residues). In addition, the H-value of the hydrophilic regions is always above -2. Furthermore, no evidence of silicatein glycosilation was collected so far and three PTMs that might help solubilization, such as two phosporylations (pS66 and pY97) and the cisteic acid residue (C157), occur in regions of silicatein that are already hydrophilic, thus not significantly helping protein dissolution. Finally, the extensive N-terminal methylation, that increases the hydrophobicity, is proved for silicatein and not reported for cathepsin. These simple considerations on the hydropathy of these two proteins and the role of silicatein PTMs might perhaps help understanding the reason of such dramatic differences in the behavior of these two proteins toward aqueous buffers. Hydrophobicity of silicatein can also be involved in dimers formation through noncovalent forces. Discussion on silicatein structure, if not supported by computer-aided calculations, cannot be definitely addressed until crystallographic data will be collected. A few very simple considerations can nevertheless be made. According to the Expasy database (Web page: http://www.uniprot.org/ uniprot/Q26636,31), cathepsins carry three disulfide bridges, with the third (Cys 157) holding together a heavy and a light chain produced by the cleavage of a four residues propeptide (DESG, residues 295-298 for CATL_SARPE). The corresponding Journal of Proteome Research • Vol. 8, No. 8, 2009 4001
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Table 4. Sequence Alignment of the Primary Sequence of Effective Silicatein Beta from P. ficiformis (Upper), Compared with Silicatein Beta from S. domuncula (Middle) and from T. aurantium (Bottom)a
a
PTMs reported in the paper for P. ficiformis are underlined.
part of silicatein sequence was nevertheless identified in the tandem mass experiments on tryptic peptides (see Supporting file 9). This fact shows another significant difference with cathepsins: silicatein does not cleave this propeptide, maybe because the corresponding fourth residue in the sequence is missing (TSS-, see Table 3). Furthermore, as described above, silicatein remains in dimeric form after treatment with DTT, meaning that dimer formation does not occur through SS linking. Furthermore, no mass shifts on the multiply charged peaks of intact silicatein were observed. If silicatein should carry three SS bridges, as cathepsin does, then the total mass of the dimer should decrease by 12 Da, corresponding to roughly 0.4 m/z on a +39 multiply charged silicatein molecule (1206.05 m/z vs 1206.44 m/z). This difference can be confidently detected by the MS instrument but was not recorded. That means that silicatein does not form SS bridges, despite its perfect homology on the number and position of cysteine residues involved in the SS bridging in cathepsin (see Table 2). This might explain why Cys 157 is oxidized to cisteic acid in silicatein: this protein, unlike cathepsin, does not cleave the propeptide and does not need SS bridges. Since such crosslinking reflects a particular molecular folding that brings the two cysteine residues close enough to form a SS link, then significant differences between three-dimensional structures of cathepsin and silicatein should perhaps be taken into account. It should be noted that these findings do not openly contradict with previous studies that postulate mechanisms of silica deposition on the basis of the predicted position of catalytic residues in cathepsins,18 because the active site of the protein might be conserved despite the structural differences suggested in the present paper. The protein folding provided by the third disulfide bridge in cathepsin L (the link holding the heavy and light chains together) might be conserved by the presence of the TSS tripeptide that, unlike cathepsin DESG tetrapeptide, is not cleaved in silicatein (see Table 3). Furthermore, the experimental results previously reported by another research group,8 demonstrating the proteolytic activity of silicatein, support the hypothesis of a significant conservation of the active site. The sequence alignment of silicatein beta of P. ficiformis, Suberites domuncula and Tethya aurantium has been performed (Table 4), in order to assess whether the modified residues are conserved in the three sponges or not: two posphorylations occur on Ser66 and Tyr97 that are present in silicatein beta of Petrosia only, the third phosphorylation occurs on Ser213 that is conserved in P. ficiformis, S. domuncula and T. aurantium. His15, oxidized in P. ficiformis, is 4002
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replaced by a lysine in Suberites and by a glycine in Tethya; Cis157, on the contrary, is conserved in all the three species. The presence of phosporylations in silicatein from S. domuncula has been previously reported in literature,5 estimating a decrease in protein pI of 0.2 units per phosphorylation. According to this evaluation, the calculated isoelectric point of silicatein from P. ficiformis should decrease to approximately 7.1, taking also into account the cisteic acid residue at position 157. Unfortunately, two papers reporting molecular evidence of phosphorylations of silicatein (ref 5 and the present one) may report an underestimated phosphorylation grade due to the HF extraction procedure, as mentioned above. Despite the identification of the precise phosphorylation sites supported by tandem mass spectra and reported in the present paper, the biological role of these modifications is still unclear. According to Muller and colleagues,5 the phosphorylation occurs in the Golgi apparatus and silicatein is subsequently transported into vesicles where it forms the axial filaments.
Conclusions Some conclusive remarks should be given after the discussion of the structural data here reported and in particular the authors consider of high relevance to underline the still open questions on the protein behavior in aqueous solution after its extraction with HF. The gel electrophoresis shows silicatein beta as a predominant band near 50 kDa which corresponds to the protein dimer. This result should be discussed in relation with the findings of other groups describing similar structural organization of silicateins from T. aurantium17 and S. domuncola.8 In the first case, a self-assembly following a fractal scheme of silicatein is described, and in particular, it was suggested a covalent organization of silicatein oligomers by means of disulfide bonds.17 In this specific case, treatment with strongly reducing agent disaggregate the oligomers. In the experimental conditions used for the present work, on the contrary, noncovalent interactions were observed as principal cause of silicatein dimerization. Dimers, indeed, still remain as such even after strong reducing agent treatment and this noncovalent dimeric form is also confirmed by the interpretation of ESI-MS spectra. Data here reported better fit the results of another research group, which described the reassembly of silicatein alpha/beta of S. domuncola.8 In this case, noncovalent dimers, tetramers and hexamers were observed after HF extraction, and this reassembly was not affected by strong reducing agent. In the same paper, Mu ¨ller and colleagues report
Primary Structure and PTMs of Silicatein Beta from P. ficiformis different electrophoretic behaviors of silicateins alpha and beta depending on the extraction procedures and on the presence/ absence of strong reducing agent and/or chaotropic substances (urea in particular). The formation of monomers of silicateins from S. domuncula was thus demonstrated. These findings suggest that, after their extraction from silica, silicateins are highly susceptible to structural modifications depending on the solution environment and these structural modifications strongly affect the oligomeric organization of the protein. In this perspective, it should be noted that T. aurantium produces three different silicateins isoforms,17 while two different silicatein are produced by S. domuncola.5 In the present study, nevertheless, the only isoform clearly detected with a proteomic approach is silicatein beta, and no cDNA of silicatein alpha was detected with various PCR approaches. Moreover, in the present work, only dimeric forms of silicatein beta were observed in gel electrophoresis and MS, even after urea treatment of the sample. These three different conditions might be the cause of the different behaviors toward aqueous solutions. The interest for these proteins in the scientific community is relevant, because of their high potential in biotechnological applications such as electronics and lithography. The handling of these molecules is nevertheless very difficult. These difficulties are indeed significantly slowing down the worldwide research activity on these proteins and the present work will hopefully represent an important step forward in the understanding of the interactions between biomolecules and silicon. Abbreviations: MS, mass spectrometry; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; PAGE, polyacrilamide gel electrophoresis; SEM, scanning electron microscope; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization; qTOF, quadrupole-time-of-flight.
Acknowledgment. This work was partially supported by EC fund (NMP4-CT-2006-031541) to M.G., by Liguria Region, area marine biotechnology funds to M.G., by Italian research ministry fund (PRIN 2007) to M.G.. We thank Mauro Michetti for technical support in the SEM analysis. Supporting Information Available: File 1, silicatein N-terminal tryptic peptide LPETVDWR (precursor 508.3 m/z, charge state +2). File 2, phosphoserine 66 in tandem mass spectrum of chimotryptic peptide GNHGCpSCEDVNNAF (precursor m/z 831.27, charge state +2). Cysteine residues are carboxamidomethylated. File 3, phosphotyrosine 97 in tandem mass spectrum of chimotryptic peptide VSRQYpY (precursor 448.2 m/z, charge state +2). File 4, phosphoserine 213 in tandem mass spectrum of chimotryptic peptide ATYApSFPTL (precursor 525.7 m/z, charge state +2). File 5, silicatein N-terminal methylated tryptic peptide (Me)LPETVDWR (precursor 515.3 m/z, charge state +2). File 6, silicatein N-terminal dimethylated tryptic peptide (Me)2LPETVDWR (precursor 522.3 m/z, charge state +2). File 7, oxydation of His 15: tandem mass spectrum of tryptic peptide TGGAVTHoxVK (precursor 443.3 m/z, charge state +2). File 8, oxydation of Cys 157 to cisteic acid: tandem mass spectrum of chimotryptic peptide NVPNCcaSRSKL (precursor 583.3 m/z, charge state +2). File 9, silicatein, unlike cathepsin L, does not cleave the propeptide: tandem mass spectrum of tryptic peptide LSHAMILIGYGTTSSK (precursor 839.9m/z, charge state +2). File 10, silicatein beta aggregation profile in water, calculated by PASTA Web-based software. This material is available free of charge via the Internet at http://pubs.acs.org.
research articles
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PR900342Y
