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Phosphorylated serine clusters of phosvitin plays a crucial role in the regulatory mineralization
International Journal of Biological Macromolecules 115 (2018) 1109–1115
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Phosphorylated serine clusters of phosvitin plays a crucial role in the regulatory mineralization Xiaowei Zhang a,b, Xi Huang b, Meihu Ma b,⁎ a State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, College of Food Engineering and Biotechnology, Tianjin University of Science &Technology, Tianjin 300457, China b National R&D Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China
a r t i c l e
i n f o
Article history: Received 27 December 2017 Received in revised form 23 April 2018 Accepted 24 April 2018 Available online 25 April 2018 Keywords: Phosvitin Phosphopeptides Mineralization
a b s t r a c t Phosphorylation of phosvitin has been proved to play an important role in the mineralization, but the active region of phosvitin is still not known yet. Four phosvitin phosphopeptides (PPPs) were obtained after separating from ion exchange column and desalting from gel filtration, and named as PPP0, PPP1, PPP3 and PPP4, respectively. The effect of PPP on the mineralization was investigated by pH-stat system, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscope. SDS-PAGE and circular dichroism were applied to study the composition and the structure of PPP. Results showed that the order of promoting mineralization was as follows: PPP3 N phosvitin N PPP4 N PPP1 N control N PPP0. PPP0 and PPP1 was not involved in the mineralization, while the structure of PPP4 was too compact to promote mineralization because of its high β-sheet conformation. PPP3, which contained a 10 kDa peptide, is the most effective promoter for mineralization. LTQ-MS/ MS results indicated that the phosphorylated serine clusters of phosvitin was the active region for promoting mineralization. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Egg provides all the material needed for the development of chicken embryos, such as phosphorus and calcium required for the bone development in chicken embryo. Phosvitin is a highly phosphorylated protein derived from egg yolk that contains about 10% phosphorus, and represents about 80% of the protein-bound phosphorus in egg yolk [1]. Phosvitin has an exceptional chelating power for cations, and thus plays an important role in the bone development in chicken embryo. However, the biological function of phosvitin is not yet well understood. Many studies have clarified the significant regulation roles of phosphorylated proteins in biomineralization such as dentin phosphoprotein (DPP) and osteopontin, and the phosphorylated serine and threonine are critical as mediators of mineral crystallization. Phosvitin has a similar structure and properties to other phosphorylated proteins: acidic proteins with flexible random coil structures, rich in glutamic
⁎ Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. E-mail address: [email protected] (M. Ma).
https://doi.org/10.1016/j.ijbiomac.2018.04.130 0141-8130/© 2018 Elsevier B.V. All rights reserved.
acid, aspartic acid, and phosphorylated serine/threonine (P-Ser/P-Thr) residues. Additionally, they possess calcium-binding capacity and hydroxyapatite affinity [2,3]. Phosvitin has the closest evolutionary relationship with DPP through bioinformatics analysis. This suggests that phosvitin, as a representative of phosphorylated protein, should have biological properties similar to DPP and may play a vital role in biomineralization. Recently, phosvitin has been shown to be involved in bone formation in chicken embryos. In cultured calvarial osteoblasts, phosvitin is capable of stimulating the differentiation of osteoblasts, collagen synthesis, hydroxyproline formation, and biomineralization [4,5]. During the embryonic development, yolk phosvitin is degraded to provide phosphorus to enhance the absorption and accumulation of Ca in bones [6]. Our early study demonstrated that there has a decrease in phosphorus content in phosvitin and phosvitin is gradually decreased during the incubation period, especially on the 12th day [7]. This indicates that phosvitin is degraded into phosvitin phosphopeptide to play its regulatory role in mineralization. Our previous study proved that phosvitin significantly promoted the mineral formation and growth, and phosvitin-Ca2+ interaction played a key role in the regulation of mineralization [2,8]. Previous study also showed that phosvitin had a dose-dependent effect on the regulation of mineralization and the phosphorylation of phosvitin were closely
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related to this regulation. Phosvitin is not only dephosphorylated but also hydrolyzed in the presence of high concentration of alkaline solution, and the alkaline-hydrolyzed phosvitin significantly improved the acceleration of mineralization [9]. However, this alkaline-hydrolyzed phosvitin is too complex to analyze its active area, due to the non-specificity of alkali hydrolysis. Thus, to investigate the active area of phosvitin involved in mineralization further, and to clarify the regulation mechanism of phosvitin on mineralization, it's necessary to prepare phosvitin phosphopeptides through the specified enzyme-hydrolysis. In this study, phosvitin phosphopeptides (PPP) was prepared by hydrolyzing phosvitin with trypsin. The effect of PPP on mineralization was investigated using the pH-stat system and the active area of phosvitin that played the role in mineralization regulation was determined using tandem mass spectrometry (MS/MS).
0.22 μm membrane filter, then the precipitate (mineral calcium phosphate) was collected and lyophilized for later examination after rinsing three times with Milli-Q water. Milli-Q water and phosvitin were used as control instead of PPP. 2.4. Characterization and analysis of mineral Characterization of mineral phosphate generated with PPP in the mineralization reaction was evaluated according to the previous description [2]. In short, the structural properties of mineral phosphate were identified by X-ray diffraction (XRD, Bruker D8 Advance X-ray diffractometer with Cu Kα radiation) and Fourier transform infrared spectroscopy (FTIR, Nicolet Nexus 470 FTIR Spectrometer). The surface morphology was measured by scanning electron microscopy (SEM) with a JEOL JSM-6390LV SEM equipped with an Oxford INCA EDS detector.
2. Materials and methods 2.5. Circular dichroism (CD) analysis of phosvitin phosphopeptides 2.1. Preparation of phosvitin phosphopeptides Phosvitin was prepared from hen egg yolk as previously described [10] with the purity of 93.14% examined by HPLC. PPP was prepared according to the method of Jiang and Mine [11] with some modifications. Before the enzyme hydrolysis, phosvitin was partially dephosphorylated with 0.1 M NaOH solution at 37 °C for 3 h. Then, the sample solution was adjusted to pH 8.0 with 1 M HCl, added trypsin (E/S = 1/50, w/w) and incubated at 37 °C for 12 h. At the end of the reaction, the solution was heated at 100 °C for 5 min to inactivate the enzyme, followed by centrifugation at 5900 g for 15 min. Finally, the clear supernatant was lyophilized to obtain PPP. 2.2. Separation and desalination of phosvitin phosphopeptides PPP was separated into several fractions by anion-exchange chromatography (IEC) with Toyopeal DEAE-650 M column (TOSOH). PPP sample was dissolved (10 mg/mL) with 0.05 M TrisHCl buffer (pH 7.5) and filtered through a 0.22 μm membrane filter. Then the sample solution was injected to the column that was previously equilibrated with 0.05 M Tris-HCl buffer (pH 7.5). The elution was performed with an increasing gradient of NaCl (0–0.5 M) in the 0.05 M Tris-HCl buffer (pH 7.5) at a flow rate of 3 mL/min. The eluate was monitored at 280 nm and the IEC fractions were collected. Based on the elution time with a gradient of NaCl (0–0.5 M) solution, the IEC fractions were named as PPP0, PPP1, PPP3, PPP4 and PPP5, respectively. Eluted IEC fractions were further desalted by gel filtration with Sepharose G-25 (GE Healthcare). The elution was performed with MilliQ water at a flow rate of 1 mL/min, and the eluate was monitored with an UV–Vis detector at 280 nm and a conductivity meter. Desalted PPP was collected and lyophilized. 2.3. Dynamic monitor of the phase transformation of calcium phosphate The phase transformation of calcium phosphate was monitored and assessed by pH-stat titration assay at 25 °C. The mineralization reaction was performed as previously described [9]. The reaction solution was prepared by mixing 0.2 mg/mL desalted PPP solution first with 20 mM CaCl2 solution and then with 12 mM NaH2PO4 solution. All the concentration of the reaction solution mentioned in this study was final concentrations, unless otherwise specified. The initial Ca/P molar ratio of the solution was 1.67. The reaction was initiated and monitored by continuous titration with a 0.15 M NaOH solution using an automated titration system (Titrando 907, Metrohm), and monitored to pH 7.0 ± 0.1, which was set as a fixed end point. After reaction, the mineralization solution was filtered through a
CD spectra were recorded according to the method of Zhang et al. [8], with a Jasco J-1500 spectro-polarimeter (Jasco, Tokyo, Japan) using a quartz cell of 0.1 cm optical path length at room temperature. CD spectra were scanned at the far-UV range (190–250 nm) with three replicates at 100 nm/min. The band width was 1.0 nm. CD experiments were conducted using 0.20 mg/mL of PPP. CD data was displayed as observed ellipticity θ (mdeg) vs. wavelength (nm). 2.6. Tris-Tricine SDS-PAGE Tris-Tricine SDS-PAGE is commonly used to separate peptides and to measure the molecular mass of peptides in the electrophoretic system [12]. The molecular mass marker with the range of 10– 170 kDa was used as marker. PPP sample (10 μg) was loaded onto the gel and subjected to electrophoresis at a constant current of 120 V. After migration, gels were stained as described previously [10]. The gel was analyzed by Gel-Pro Analyzer 4.0 (Media Cybernetics, USA). The selected band (PPP3-S2) was cut out from the gel for the identification of bioactive peptides through the subsequent gel digestion and LC-MS/MS. 2.7. Identification of PPP3-S2 by LC-MS/MS The selected protein bands in SDS-PAGE were destained with 30% acetonitrile (ACN)/100 mM ammonium bicarbonate (NH4HCO3) and dried in a vacuum centrifuge. The in-gel proteins were reduced with dithiothreitol (10 mM dithiothreitol/100 mM NH4HCO3) for 30 min at 56 °C, then alkylated with iodoacetamide (200 mM indoleacetic acid/100 mM NH4HCO3) at room temperature for 30 min in darkness. Gel pieces were briefly rinsed with 100 mM NH4HCO3 and ACN, respectively. Then, the gel pieces were digested overnight in 12.5 ng/μL trypsin in 25 mM NH4HCO3. The peptides were extracted three times with 60% ACN/ 0.1% formic acid (TFA). The extracts were pooled and dried completely by a vacuum centrifuge. Ettan™ MDLC system (GE Healthcare, USA) was used for desalting and separating tryptic peptides mixtures. In this system, samples were desalted on the RP trap columns (Zorbax 300 SB C18, 0.3 × 5 mm, Agilent Technologies, USA), and then separated on a RP column (Zorbax 300 SB C18, 0.15 × 100 mm, Column Technology Inc., Fremont, CA). Mobile phase A (0.1% TFA in water) and mobile phase B (0.1% TFA in ACN) were selected. Tryptic peptide mixture (20 μg) was loaded onto the columns, and the separation was done at a flow rate of 2 μL/min by using a linear gradient of 4–50% buffer B for 50 min, 50–100% buffer B for 4 min and 100% buffer B for 6 min. LTQ Velos (Thermo Scientific, USA) equipped with a micro-spray interface was connected to the LC setup for eluted peptides detection.
X. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 1109–1115
Data-dependent MS/MS spectra were obtained simultaneously. Each scan cycle consisted of one full scan mass spectrum (m/z 300–1800) followed by 20 MS/MS events of the most intense ions with the following dynamic exclusion settings: repeat count 2, repeat duration 30 s, and exclusion duration 90 s. The Thermo LTQ Velos Pro dual-pressure linear ion trap mass spectrometer was used for MS/MS experiment with an ion transfer capillary at 160 °C with pray voltage of 3 kV. Normalized collision energy was 35.0%. MS/MS spectra were automatically searched on Gallus gallus database (P02845, vitellogenin sequences, phosvitin precursor) using the Bioworks-Browser rev. 3.1 (Thermo Electron, San Jose, CA.). Protein identification results were extracted from SEQUEST out files with Build Summary. The peptides were constrained to be tryptic and up to two missed cleavages were allowed. Carbamidomethylation of cysteines were treated as a fixed modification, whereas oxidation of methionine residues, phosphorylation of serine/threonine, and phosphorylation of tyrosine were considered as variable modifications. Mass tolerance allowed for the precursor ions was 2.0 Da and fragment ions was 0.8 Da, respectively. The protein identification criteria were based on Delta CN (≥0.1) and cross-correlation score (Xcorr, one charge ≥ 1.9, two charges ≥ 2.2, three charges ≥ 3.75).
3. Results and discussion 3.1. Effect of PPP on regulation of mineralization The IEC profile of PPP in Fig. 1 presented five elution peaks, designated as PPP1, PPP2, PPP3, PPP4 and PPP5, respectively. However, PPP5 was discarded because the trace amount of PPP5 could not meet the experimental requirements. Thus, four fractions (PPP1, PPP2, PPP3 and PPP4) were selected for following studies. The titration curves for all samples were divided into two regions: a faster titration rate followed by a slower one, as indicated in Fig. 2. Greater amount of NaOH consumed per unit time meant more rapid phase transformation. With added PPP, the trend of NaOH consumption during mineralization reaction was as follows: PPP3 N phosvitin N PPP4 N PPP1 N Control N PPP0, indicating that PPP3, PPP4 and PPP1 could accelerate the mineralization reaction while PPP0 slowed down the reaction. PPP3 accelerated the mineralization reaction faster than phosvitin.
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Fig. 2. The titration curve of the phase transformation of calcium phosphate during the mineralization reaction, with added 0.20 mg/mL of PPP, phosvitin and control. Control: Milli-Q water.
FTIR, XRD and SEM were employed to characterize and analyze the phase identification and the microstructures of the mineral phosphate generated by the PPP in the mineralization reaction. FTIR can be used to reliably identify dicalcium phosphate dihydrate (DCPD) and hydroxyapatite (HAP) based on their characteristic bands [13], and the assignments of the FTIR spectra were given in Table S1. As shown in Fig. 3 and Fig. S1, the characteristic XRD peaks and FTIR bands of DCPD and HAP, indicated that the mineral generated with added PPP0, PPP1 and PPP4 was DCPD at the end of the reaction (1 h), while the one with added PPP3 was HAP, and that with phosvitin was the mixture of DCPD and HAP. The dominant XRD peak of the mineral with added PPP0, PPP1 and PPP4 appeared at 11.63°, which was assigned to the (020) plane of DCPD crystal. However, the most dominant XRD peaks of mineral with added PPP3 appeared at 11.63° and 31.65°, which corresponded to the (020) plane of DCPD and that (211) of HAP crystal, respectively [14]. Moreover, an increase in preferred orientation in the c-plane was
Fig. 1. Isolation of PPP by anion exchange chromatography. Five fractions were obtained with a gradient NaCl elution: PPP0, PPP1, PPP3, PPP4, PPP5, respectively.
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Fig. 3. FTIR spectra of the calcium phosphate generated with 0.20 mg/mL PPP, phosvitin and control. Control: Milli-Q water.
observed in mineral with added PPP3 as the intensity ratios of the crystal plane (020) and (211) was 0.058 [15], suggesting that HAP become the main component of mineral. Reference intensity ratio (RIR) method is commonly used to quantitative phase analysis [16]. As the results showed in Table 1, the weight fraction of HAP with added PPP3 was highest with 97.93%. This indicated that the acceleration effect of PPP3 on mineralization was stronger than phosvitin, while the other PPPs (PPP0, PPP1 and PPP4) were weaker than phosvitin. The morphology changes of calcium phosphate generated with PPP were examined with the SEM micrographs. As shown in Fig. 4, minerals with PPP0 and PPP1 were in the shape of a plate with a smooth surface, which corresponded to the DCPD signatures observed in FTIR and XRD. With PPP4, the mineral was gradually converted from plate particles to amorphous particle. With PPP3, the crystal became irregular aggregates, which corresponded to HAP. Previous reports have demonstrated that the phase transformation from DCPD to HAP was accomplished via dissolution-recrystallization pathway [2,17]. This result suggested that PPP3 could accelerate the phase transformation from DCPD to HAP and promoted HAP crystal growth. 3.2. Characteristic of PPP Tris-Tricine SDS-PAGE patterns of PPP were shown in Fig. 5. After enzyme hydrolysis, there were no detectable bands in PPP0 and
Table 1 Weight fractions of the mineral calcium phosphate generated with PPP. Mineral
PPP0 PPP1 PPP3 PPP4 Phosvitin Control
Weight fraction (%) DCPD
HAP
100.00 100.00 2.07 100.00 16.96 100.00
0.00 0.00 97.93 0.00 83.04 0.00
PPP1, probably because they were oligopeptides with low molecular weight or single amino acid residues containing a benzene ring. PPP4 contained three bands between the molecular weight of 15 kDa–35 kDa (S1). However, PPP3 contained more protein bands in the gel than PPP4, blurry band around 10 kDa (S2) was appeared in PPP3. In combination with the results of the acceleration effect of PPP on mineralization, it revealed that the active region of the PPP3 was small peptides (PPP3-S2) that responded to the acceleration of mineralization. Thus, PPP3-S2 was subsequently identified through LCMS/MS. The MS/MS results (Table 2 and Fig. S2) indicated that the highly phosphorylated serine cluster region with the sequence of D1165R1258 was identified as the active region of PPP that accelerated the mineralization. Aspartate-serine-serine (DSS) is the fundamental repeat unit within DPP and believed to play a critical role in promoting the formation of HAP [18]. In PPP, Lysine/arginine/histidine-serine-serine (K/R/H-SS) was the fundamental repeat unit, which are consisted of nonaromatic, hydroxylated, and charged amino acid residues, showed beneficial effects in the binding and nucleation of hydroxyapatite [19]. The CD spectra of PPP (Fig. 6) indicated that PPP0 and PPP1 exhibited no change of mean residue ellipticity. The combined results of SDS-PAGE indicated that PPP0 and PPP1 might be small oligopeptides. PPP3 and PPP4 had a very strong negative peak around 200 nm. Yang's fitting calculation points out that the main conformation of PPP3 and PPP4 was β-sheet. Compared to native phosvitin, both PPP3 and PPP4 contained higher β-sheet and lower β-turn and γ-random, because negative charge and the electrostatic repulsion, which were reduced after enzyme hydrolysis. The result in the conversion of random structure into β-sheet structure. This indicated that the acceleration of mineralization was not only related to amino acid composition of phosvitin, but also related to the structure of phosvitin. Report demonstrated that the β-sheet structure of phosphorylated protein can serve as the template to orient growth of crystal in mineralization [20,21]. Phosphopeptides hydrolyzed from phosvitin can enhance the acceleration effect, consistent with the result of George and Veis [22], who demonstrated that phosphorylation of dentin phosphophoryn peptides played a key role on the
X. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 1109–1115
Fig. 4. The morphologies of the calcium phosphate generated with 0.20 mg/mL PPP, phosvitin and control. Control: Milli-Q water. Left: ×4000; Right: ×10,000.
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more nucleation sites, and increased mineralization template. Thus, PPP3 significantly enhanced the mineralization. 4. Conclusions In summary, PPP was obtained through IEC method, and the order of mineralization effect was: PPP3 N phosvitin N PPP4 N PPP1 N Control N PPP0. The active region for promoting mineralization, identified through LTQ-MS/MS, was phosphorylated serine clusters of phosvitin. Acknowledgments This work was supported by the Earmarked Fund for Modern Agroindustry Technology Research System (Project No. CARS-41-K23) and the National Natural Science Foundation of China (Grant No. 31471602). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2018.04.130. References
Fig. 5. Protein patterns of PPP by Tris-Tricine-SDS-PAGE.
Table 2 The identified active region of PPP3-S2 through LTQ-MS/MS. Peptide position 1165–1180 1218–1234 1235–1252 1238–1258
Sequence #
K.DASSSSRSSKSSNSS K.R K.SSSSSSKS#SSSSSRSR.S R.SSSKSS#S#SSS#SSS#S#SSSSK.S K.S#S#SSSSSSSSSSSSKSSSS#R.S
MH+
Diff (MH+)
1682.567 1671.544 2155.519 2110.678
0.0346 −1.8641 0.6997 −1.3562
oriented growth of HAP. β-sheet and highly phosphorylated serine clusters were necessary for the acceleration of mineralization, because they exposed more active binding area for calcium ions and
[1] F.J. Joubert, W.H. Cook, Preparation and characterization of phosvitin from hen egg yolk, Can. J. Biochem. Physiol. 36 (1958) 399–408. [2] X. Zhang, F. Geng, X. Huang, M. Ma, Acceleration of the initial phase transformation of mineralization by phosvitin, J. Cryst. Growth 409 (2015) 44–50. [3] A.L. Boskey, Matrix proteins and mineralization: an overview, Connect. Tissue Res. 35 (1996) 357–363. [4] J. Liu, D. Czernick, S.-C. Lin, A. Alasmari, D. Serge, E. Salih, Novel bioactivity of phosvitin in connective tissue and bone organogenesis revealed by live calvarial bone organ culture models, Dev. Biol. 381 (2013) 256–275. [5] Q. Liu, C. Li, F. Geng, X. Huang, M. Ma, Hen egg yolk phosvitin stimulates osteoblast differentiation in the absence of ascorbic acid, J. Sci. Food Agric. 97 (2017) 4532–4538. [6] H. Samaraweera, W.G. Zhang, E.J. Lee, D.U. Ahn, Egg yolk phosvitin and functional phosphopeptides-review, J. Food Sci. 76 (2011) R143–R150. [7] C. Li, F. Geng, X. Huang, M. Ma, X. Zhang, Phosvitin phosphorus is involved in chicken embryo bone formation through dephosphorylation, Poult. Sci. 93 (2014) 3065–3072. [8] X. Zhang, F. Geng, X. Huang, M. Ma, Calcium binding characteristics and structural changes of phosvitin, J. Inorg. Biochem. 159 (2016) 76–81. [9] X. Zhang, X. Huang, M. Ma, Role of phosphorylation of phosvitin in the phase transformation of mineralization, Int. J. Biol. Macromol. 101 (2017) 712–718. [10] X. Zhang, N. Qiu, F. Geng, M. Ma, Simply and effectively preparing high-purity phosvitin using polyethylene glycol and anion-exchange chromatography, J. Sep. Sci. 34 (2011) 3295–3301. [11] B. Jiang, Y. Mine, Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin, J. Agric. Food Chem. 48 (2000) 990–994.
Fig. 6. CD spectra and secondary structure analysis of PPP.
X. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 1109–1115 [12] H. Schägger, Tricine-sds-page, Nat. Protoc. 1 (2006) 16–22. [13] I.A. Karampas, C.G. Kontoyannis, Characterization of calcium phosphates mixtures, Vib. Spectrosc. 64 (2013) 126–133. [14] J.S. Lim, J.H. Kim, New application of poly (butylene succinate) (PBS) based ionomer as biopolymer: a role of ion group for hydroxyapatite (HAP) crystal formation, J. Mater. Sci. 44 (2009) 6398–6403. [15] O. Kalinkevich, S. Danilchenko, A. Kalinkevich, V. Kuznetsov, J. Lü, J. Shang, S. Yang, Formation of nanocrystalline hydroxyapatite in presence of some aminoacids, J. Nano-Electron. Phys. 6 (2014) 4014-1. [16] R.L. Snyder, The use of reference intensity ratios in X-ray quantitative analysis, Powder Diffract. 7 (1992) 186–193. [17] A. Shkilnyy, S. Schöne, C. Rumplasch, A. Uhlmann, A. Hedderich, C. Günter, A. Taubert, Calcium phosphate mineralization with linear poly (ethylene imine): a time-resolved study, Colloid Polym. Sci. 289 (2011) 881–888.
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[18] D.K. Yarbrough, E. Hagerman, R. Eckert, J. He, H. Choi, N. Cao, K. Le, J. Hedger, F. Qi, M. Anderson, Specific binding and mineralization of calcified surfaces by small peptides, Calcif. Tissue Int. 86 (2010) 58–66. [19] M. Melcher, S.J. Facey, T.M. Henkes, T. Subkowski, B. Hauer, Accelerated nucleation of hydroxyapatite using an engineered hydrophobin fusion protein, Biomacromolecules 17 (2016) 1716–1726. [20] S. Dahlin, J. Angström, A. Linde, Dentin phosphoprotein sequence motifs and molecular modeling: conformational adaptations to mineral crystals, Eur. J. Oral Sci. 106 (1998) 239–248. [21] E. Villarreal-Ramirez, D. Eliezer, R. Garduño-Juarez, A. Gericke, J.M. Perez-Aguilar, A. Boskey, Phosphorylation regulates the secondary structure and function of dentin phosphoprotein peptides, Bone 95 (2017) 65–75. [22] A. George, A. Veis, Phosphorylated proteins and control over apatite nucleation, crystal growth, and inhibition, Chem. Rev. 108 (2008) 4670–4693.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Phosphorylated serine clusters of phosvitin plays a crucial role in the regulatory mineralization Xiaowei Zhang a,b, Xi Huang b, Meihu Ma b,⁎ a State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, College of Food Engineering and Biotechnology, Tianjin University of Science &Technology, Tianjin 300457, China b National R&D Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China
a r t i c l e
i n f o
Article history: Received 27 December 2017 Received in revised form 23 April 2018 Accepted 24 April 2018 Available online 25 April 2018 Keywords: Phosvitin Phosphopeptides Mineralization
a b s t r a c t Phosphorylation of phosvitin has been proved to play an important role in the mineralization, but the active region of phosvitin is still not known yet. Four phosvitin phosphopeptides (PPPs) were obtained after separating from ion exchange column and desalting from gel filtration, and named as PPP0, PPP1, PPP3 and PPP4, respectively. The effect of PPP on the mineralization was investigated by pH-stat system, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscope. SDS-PAGE and circular dichroism were applied to study the composition and the structure of PPP. Results showed that the order of promoting mineralization was as follows: PPP3 N phosvitin N PPP4 N PPP1 N control N PPP0. PPP0 and PPP1 was not involved in the mineralization, while the structure of PPP4 was too compact to promote mineralization because of its high β-sheet conformation. PPP3, which contained a 10 kDa peptide, is the most effective promoter for mineralization. LTQ-MS/ MS results indicated that the phosphorylated serine clusters of phosvitin was the active region for promoting mineralization. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Egg provides all the material needed for the development of chicken embryos, such as phosphorus and calcium required for the bone development in chicken embryo. Phosvitin is a highly phosphorylated protein derived from egg yolk that contains about 10% phosphorus, and represents about 80% of the protein-bound phosphorus in egg yolk [1]. Phosvitin has an exceptional chelating power for cations, and thus plays an important role in the bone development in chicken embryo. However, the biological function of phosvitin is not yet well understood. Many studies have clarified the significant regulation roles of phosphorylated proteins in biomineralization such as dentin phosphoprotein (DPP) and osteopontin, and the phosphorylated serine and threonine are critical as mediators of mineral crystallization. Phosvitin has a similar structure and properties to other phosphorylated proteins: acidic proteins with flexible random coil structures, rich in glutamic
⁎ Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. E-mail address: [email protected] (M. Ma).
https://doi.org/10.1016/j.ijbiomac.2018.04.130 0141-8130/© 2018 Elsevier B.V. All rights reserved.
acid, aspartic acid, and phosphorylated serine/threonine (P-Ser/P-Thr) residues. Additionally, they possess calcium-binding capacity and hydroxyapatite affinity [2,3]. Phosvitin has the closest evolutionary relationship with DPP through bioinformatics analysis. This suggests that phosvitin, as a representative of phosphorylated protein, should have biological properties similar to DPP and may play a vital role in biomineralization. Recently, phosvitin has been shown to be involved in bone formation in chicken embryos. In cultured calvarial osteoblasts, phosvitin is capable of stimulating the differentiation of osteoblasts, collagen synthesis, hydroxyproline formation, and biomineralization [4,5]. During the embryonic development, yolk phosvitin is degraded to provide phosphorus to enhance the absorption and accumulation of Ca in bones [6]. Our early study demonstrated that there has a decrease in phosphorus content in phosvitin and phosvitin is gradually decreased during the incubation period, especially on the 12th day [7]. This indicates that phosvitin is degraded into phosvitin phosphopeptide to play its regulatory role in mineralization. Our previous study proved that phosvitin significantly promoted the mineral formation and growth, and phosvitin-Ca2+ interaction played a key role in the regulation of mineralization [2,8]. Previous study also showed that phosvitin had a dose-dependent effect on the regulation of mineralization and the phosphorylation of phosvitin were closely
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related to this regulation. Phosvitin is not only dephosphorylated but also hydrolyzed in the presence of high concentration of alkaline solution, and the alkaline-hydrolyzed phosvitin significantly improved the acceleration of mineralization [9]. However, this alkaline-hydrolyzed phosvitin is too complex to analyze its active area, due to the non-specificity of alkali hydrolysis. Thus, to investigate the active area of phosvitin involved in mineralization further, and to clarify the regulation mechanism of phosvitin on mineralization, it's necessary to prepare phosvitin phosphopeptides through the specified enzyme-hydrolysis. In this study, phosvitin phosphopeptides (PPP) was prepared by hydrolyzing phosvitin with trypsin. The effect of PPP on mineralization was investigated using the pH-stat system and the active area of phosvitin that played the role in mineralization regulation was determined using tandem mass spectrometry (MS/MS).
0.22 μm membrane filter, then the precipitate (mineral calcium phosphate) was collected and lyophilized for later examination after rinsing three times with Milli-Q water. Milli-Q water and phosvitin were used as control instead of PPP. 2.4. Characterization and analysis of mineral Characterization of mineral phosphate generated with PPP in the mineralization reaction was evaluated according to the previous description [2]. In short, the structural properties of mineral phosphate were identified by X-ray diffraction (XRD, Bruker D8 Advance X-ray diffractometer with Cu Kα radiation) and Fourier transform infrared spectroscopy (FTIR, Nicolet Nexus 470 FTIR Spectrometer). The surface morphology was measured by scanning electron microscopy (SEM) with a JEOL JSM-6390LV SEM equipped with an Oxford INCA EDS detector.
2. Materials and methods 2.5. Circular dichroism (CD) analysis of phosvitin phosphopeptides 2.1. Preparation of phosvitin phosphopeptides Phosvitin was prepared from hen egg yolk as previously described [10] with the purity of 93.14% examined by HPLC. PPP was prepared according to the method of Jiang and Mine [11] with some modifications. Before the enzyme hydrolysis, phosvitin was partially dephosphorylated with 0.1 M NaOH solution at 37 °C for 3 h. Then, the sample solution was adjusted to pH 8.0 with 1 M HCl, added trypsin (E/S = 1/50, w/w) and incubated at 37 °C for 12 h. At the end of the reaction, the solution was heated at 100 °C for 5 min to inactivate the enzyme, followed by centrifugation at 5900 g for 15 min. Finally, the clear supernatant was lyophilized to obtain PPP. 2.2. Separation and desalination of phosvitin phosphopeptides PPP was separated into several fractions by anion-exchange chromatography (IEC) with Toyopeal DEAE-650 M column (TOSOH). PPP sample was dissolved (10 mg/mL) with 0.05 M TrisHCl buffer (pH 7.5) and filtered through a 0.22 μm membrane filter. Then the sample solution was injected to the column that was previously equilibrated with 0.05 M Tris-HCl buffer (pH 7.5). The elution was performed with an increasing gradient of NaCl (0–0.5 M) in the 0.05 M Tris-HCl buffer (pH 7.5) at a flow rate of 3 mL/min. The eluate was monitored at 280 nm and the IEC fractions were collected. Based on the elution time with a gradient of NaCl (0–0.5 M) solution, the IEC fractions were named as PPP0, PPP1, PPP3, PPP4 and PPP5, respectively. Eluted IEC fractions were further desalted by gel filtration with Sepharose G-25 (GE Healthcare). The elution was performed with MilliQ water at a flow rate of 1 mL/min, and the eluate was monitored with an UV–Vis detector at 280 nm and a conductivity meter. Desalted PPP was collected and lyophilized. 2.3. Dynamic monitor of the phase transformation of calcium phosphate The phase transformation of calcium phosphate was monitored and assessed by pH-stat titration assay at 25 °C. The mineralization reaction was performed as previously described [9]. The reaction solution was prepared by mixing 0.2 mg/mL desalted PPP solution first with 20 mM CaCl2 solution and then with 12 mM NaH2PO4 solution. All the concentration of the reaction solution mentioned in this study was final concentrations, unless otherwise specified. The initial Ca/P molar ratio of the solution was 1.67. The reaction was initiated and monitored by continuous titration with a 0.15 M NaOH solution using an automated titration system (Titrando 907, Metrohm), and monitored to pH 7.0 ± 0.1, which was set as a fixed end point. After reaction, the mineralization solution was filtered through a
CD spectra were recorded according to the method of Zhang et al. [8], with a Jasco J-1500 spectro-polarimeter (Jasco, Tokyo, Japan) using a quartz cell of 0.1 cm optical path length at room temperature. CD spectra were scanned at the far-UV range (190–250 nm) with three replicates at 100 nm/min. The band width was 1.0 nm. CD experiments were conducted using 0.20 mg/mL of PPP. CD data was displayed as observed ellipticity θ (mdeg) vs. wavelength (nm). 2.6. Tris-Tricine SDS-PAGE Tris-Tricine SDS-PAGE is commonly used to separate peptides and to measure the molecular mass of peptides in the electrophoretic system [12]. The molecular mass marker with the range of 10– 170 kDa was used as marker. PPP sample (10 μg) was loaded onto the gel and subjected to electrophoresis at a constant current of 120 V. After migration, gels were stained as described previously [10]. The gel was analyzed by Gel-Pro Analyzer 4.0 (Media Cybernetics, USA). The selected band (PPP3-S2) was cut out from the gel for the identification of bioactive peptides through the subsequent gel digestion and LC-MS/MS. 2.7. Identification of PPP3-S2 by LC-MS/MS The selected protein bands in SDS-PAGE were destained with 30% acetonitrile (ACN)/100 mM ammonium bicarbonate (NH4HCO3) and dried in a vacuum centrifuge. The in-gel proteins were reduced with dithiothreitol (10 mM dithiothreitol/100 mM NH4HCO3) for 30 min at 56 °C, then alkylated with iodoacetamide (200 mM indoleacetic acid/100 mM NH4HCO3) at room temperature for 30 min in darkness. Gel pieces were briefly rinsed with 100 mM NH4HCO3 and ACN, respectively. Then, the gel pieces were digested overnight in 12.5 ng/μL trypsin in 25 mM NH4HCO3. The peptides were extracted three times with 60% ACN/ 0.1% formic acid (TFA). The extracts were pooled and dried completely by a vacuum centrifuge. Ettan™ MDLC system (GE Healthcare, USA) was used for desalting and separating tryptic peptides mixtures. In this system, samples were desalted on the RP trap columns (Zorbax 300 SB C18, 0.3 × 5 mm, Agilent Technologies, USA), and then separated on a RP column (Zorbax 300 SB C18, 0.15 × 100 mm, Column Technology Inc., Fremont, CA). Mobile phase A (0.1% TFA in water) and mobile phase B (0.1% TFA in ACN) were selected. Tryptic peptide mixture (20 μg) was loaded onto the columns, and the separation was done at a flow rate of 2 μL/min by using a linear gradient of 4–50% buffer B for 50 min, 50–100% buffer B for 4 min and 100% buffer B for 6 min. LTQ Velos (Thermo Scientific, USA) equipped with a micro-spray interface was connected to the LC setup for eluted peptides detection.
X. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 1109–1115
Data-dependent MS/MS spectra were obtained simultaneously. Each scan cycle consisted of one full scan mass spectrum (m/z 300–1800) followed by 20 MS/MS events of the most intense ions with the following dynamic exclusion settings: repeat count 2, repeat duration 30 s, and exclusion duration 90 s. The Thermo LTQ Velos Pro dual-pressure linear ion trap mass spectrometer was used for MS/MS experiment with an ion transfer capillary at 160 °C with pray voltage of 3 kV. Normalized collision energy was 35.0%. MS/MS spectra were automatically searched on Gallus gallus database (P02845, vitellogenin sequences, phosvitin precursor) using the Bioworks-Browser rev. 3.1 (Thermo Electron, San Jose, CA.). Protein identification results were extracted from SEQUEST out files with Build Summary. The peptides were constrained to be tryptic and up to two missed cleavages were allowed. Carbamidomethylation of cysteines were treated as a fixed modification, whereas oxidation of methionine residues, phosphorylation of serine/threonine, and phosphorylation of tyrosine were considered as variable modifications. Mass tolerance allowed for the precursor ions was 2.0 Da and fragment ions was 0.8 Da, respectively. The protein identification criteria were based on Delta CN (≥0.1) and cross-correlation score (Xcorr, one charge ≥ 1.9, two charges ≥ 2.2, three charges ≥ 3.75).
3. Results and discussion 3.1. Effect of PPP on regulation of mineralization The IEC profile of PPP in Fig. 1 presented five elution peaks, designated as PPP1, PPP2, PPP3, PPP4 and PPP5, respectively. However, PPP5 was discarded because the trace amount of PPP5 could not meet the experimental requirements. Thus, four fractions (PPP1, PPP2, PPP3 and PPP4) were selected for following studies. The titration curves for all samples were divided into two regions: a faster titration rate followed by a slower one, as indicated in Fig. 2. Greater amount of NaOH consumed per unit time meant more rapid phase transformation. With added PPP, the trend of NaOH consumption during mineralization reaction was as follows: PPP3 N phosvitin N PPP4 N PPP1 N Control N PPP0, indicating that PPP3, PPP4 and PPP1 could accelerate the mineralization reaction while PPP0 slowed down the reaction. PPP3 accelerated the mineralization reaction faster than phosvitin.
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Fig. 2. The titration curve of the phase transformation of calcium phosphate during the mineralization reaction, with added 0.20 mg/mL of PPP, phosvitin and control. Control: Milli-Q water.
FTIR, XRD and SEM were employed to characterize and analyze the phase identification and the microstructures of the mineral phosphate generated by the PPP in the mineralization reaction. FTIR can be used to reliably identify dicalcium phosphate dihydrate (DCPD) and hydroxyapatite (HAP) based on their characteristic bands [13], and the assignments of the FTIR spectra were given in Table S1. As shown in Fig. 3 and Fig. S1, the characteristic XRD peaks and FTIR bands of DCPD and HAP, indicated that the mineral generated with added PPP0, PPP1 and PPP4 was DCPD at the end of the reaction (1 h), while the one with added PPP3 was HAP, and that with phosvitin was the mixture of DCPD and HAP. The dominant XRD peak of the mineral with added PPP0, PPP1 and PPP4 appeared at 11.63°, which was assigned to the (020) plane of DCPD crystal. However, the most dominant XRD peaks of mineral with added PPP3 appeared at 11.63° and 31.65°, which corresponded to the (020) plane of DCPD and that (211) of HAP crystal, respectively [14]. Moreover, an increase in preferred orientation in the c-plane was
Fig. 1. Isolation of PPP by anion exchange chromatography. Five fractions were obtained with a gradient NaCl elution: PPP0, PPP1, PPP3, PPP4, PPP5, respectively.
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Fig. 3. FTIR spectra of the calcium phosphate generated with 0.20 mg/mL PPP, phosvitin and control. Control: Milli-Q water.
observed in mineral with added PPP3 as the intensity ratios of the crystal plane (020) and (211) was 0.058 [15], suggesting that HAP become the main component of mineral. Reference intensity ratio (RIR) method is commonly used to quantitative phase analysis [16]. As the results showed in Table 1, the weight fraction of HAP with added PPP3 was highest with 97.93%. This indicated that the acceleration effect of PPP3 on mineralization was stronger than phosvitin, while the other PPPs (PPP0, PPP1 and PPP4) were weaker than phosvitin. The morphology changes of calcium phosphate generated with PPP were examined with the SEM micrographs. As shown in Fig. 4, minerals with PPP0 and PPP1 were in the shape of a plate with a smooth surface, which corresponded to the DCPD signatures observed in FTIR and XRD. With PPP4, the mineral was gradually converted from plate particles to amorphous particle. With PPP3, the crystal became irregular aggregates, which corresponded to HAP. Previous reports have demonstrated that the phase transformation from DCPD to HAP was accomplished via dissolution-recrystallization pathway [2,17]. This result suggested that PPP3 could accelerate the phase transformation from DCPD to HAP and promoted HAP crystal growth. 3.2. Characteristic of PPP Tris-Tricine SDS-PAGE patterns of PPP were shown in Fig. 5. After enzyme hydrolysis, there were no detectable bands in PPP0 and
Table 1 Weight fractions of the mineral calcium phosphate generated with PPP. Mineral
PPP0 PPP1 PPP3 PPP4 Phosvitin Control
Weight fraction (%) DCPD
HAP
100.00 100.00 2.07 100.00 16.96 100.00
0.00 0.00 97.93 0.00 83.04 0.00
PPP1, probably because they were oligopeptides with low molecular weight or single amino acid residues containing a benzene ring. PPP4 contained three bands between the molecular weight of 15 kDa–35 kDa (S1). However, PPP3 contained more protein bands in the gel than PPP4, blurry band around 10 kDa (S2) was appeared in PPP3. In combination with the results of the acceleration effect of PPP on mineralization, it revealed that the active region of the PPP3 was small peptides (PPP3-S2) that responded to the acceleration of mineralization. Thus, PPP3-S2 was subsequently identified through LCMS/MS. The MS/MS results (Table 2 and Fig. S2) indicated that the highly phosphorylated serine cluster region with the sequence of D1165R1258 was identified as the active region of PPP that accelerated the mineralization. Aspartate-serine-serine (DSS) is the fundamental repeat unit within DPP and believed to play a critical role in promoting the formation of HAP [18]. In PPP, Lysine/arginine/histidine-serine-serine (K/R/H-SS) was the fundamental repeat unit, which are consisted of nonaromatic, hydroxylated, and charged amino acid residues, showed beneficial effects in the binding and nucleation of hydroxyapatite [19]. The CD spectra of PPP (Fig. 6) indicated that PPP0 and PPP1 exhibited no change of mean residue ellipticity. The combined results of SDS-PAGE indicated that PPP0 and PPP1 might be small oligopeptides. PPP3 and PPP4 had a very strong negative peak around 200 nm. Yang's fitting calculation points out that the main conformation of PPP3 and PPP4 was β-sheet. Compared to native phosvitin, both PPP3 and PPP4 contained higher β-sheet and lower β-turn and γ-random, because negative charge and the electrostatic repulsion, which were reduced after enzyme hydrolysis. The result in the conversion of random structure into β-sheet structure. This indicated that the acceleration of mineralization was not only related to amino acid composition of phosvitin, but also related to the structure of phosvitin. Report demonstrated that the β-sheet structure of phosphorylated protein can serve as the template to orient growth of crystal in mineralization [20,21]. Phosphopeptides hydrolyzed from phosvitin can enhance the acceleration effect, consistent with the result of George and Veis [22], who demonstrated that phosphorylation of dentin phosphophoryn peptides played a key role on the
X. Zhang et al. / International Journal of Biological Macromolecules 115 (2018) 1109–1115
Fig. 4. The morphologies of the calcium phosphate generated with 0.20 mg/mL PPP, phosvitin and control. Control: Milli-Q water. Left: ×4000; Right: ×10,000.
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more nucleation sites, and increased mineralization template. Thus, PPP3 significantly enhanced the mineralization. 4. Conclusions In summary, PPP was obtained through IEC method, and the order of mineralization effect was: PPP3 N phosvitin N PPP4 N PPP1 N Control N PPP0. The active region for promoting mineralization, identified through LTQ-MS/MS, was phosphorylated serine clusters of phosvitin. Acknowledgments This work was supported by the Earmarked Fund for Modern Agroindustry Technology Research System (Project No. CARS-41-K23) and the National Natural Science Foundation of China (Grant No. 31471602). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2018.04.130. References
Fig. 5. Protein patterns of PPP by Tris-Tricine-SDS-PAGE.
Table 2 The identified active region of PPP3-S2 through LTQ-MS/MS. Peptide position 1165–1180 1218–1234 1235–1252 1238–1258
Sequence #
K.DASSSSRSSKSSNSS K.R K.SSSSSSKS#SSSSSRSR.S R.SSSKSS#S#SSS#SSS#S#SSSSK.S K.S#S#SSSSSSSSSSSSKSSSS#R.S
MH+
Diff (MH+)
1682.567 1671.544 2155.519 2110.678
0.0346 −1.8641 0.6997 −1.3562
oriented growth of HAP. β-sheet and highly phosphorylated serine clusters were necessary for the acceleration of mineralization, because they exposed more active binding area for calcium ions and
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Fig. 6. CD spectra and secondary structure analysis of PPP.
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