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Role of phosphorylation of phosvitin in the phase transformation of mineralization
Accepted Manuscript Title: Role of phosphorylation of phosvitin in the phase transformation of mineralization Authors: Xiaowei Zhang, Xi Huang, Meihu Ma PII: DOI: Reference:
S0141-8130(16)32915-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.158 BIOMAC 7324
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
15-12-2016 21-3-2017 27-3-2017
Please cite this article as: Xiaowei Zhang, Xi Huang, Meihu Ma, Role of phosphorylation of phosvitin in the phase transformation of mineralization, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.158 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Role of phosphorylation of phosvitin in the phase transformation of mineralization Xiaowei Zhang, Xi Huang, Meihu Ma* National R&D Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. * Corresponding author: Meihu Ma College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China Tel: +86 27 87283177 Fax: +86 27 87283177 E-mail: [email protected]
Highlights
Phosvitin in the acceleration effect of phase transformation was dose-concentration effect.
With lower dephosphorylation degree of phosvitin (<20%), the acceleration effect was weaker than native phosvitin.
With higher dephosphorylation degree of phosvitin (>40%), the acceleration effect was enhanced compared to native phosvitin.
Phosphorylation of phosvitin played an important role in regulating mineralization.
Abstract Phosvitin is a unique highly phosphorylated protein that plays a role in the regulation of mineralization. This study investigated the role of phosphorylation of phosvitin in the phase transformation of calcium phosphate in the mineralization solution. Partially dephosphorylated phosvitins (T1, T2, T3 and T4) were prepared with 2.98, 19.46, 43.39 and 71.07% of phosphate released from native phosvitin, respectively. And their effect on regulating the phase transformation was investigated, the characterization and composition analysis was performed by circular dichroism, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. Results showed that phosvitin in the acceleration effect of phase transformation was dose-concentration effect by pH-stat titration. With lower dephosphorylation degree of phosvitin (< 20%), the acceleration effect was weaker than native phosvitin, since phosphorylation and random structure of phosvitin were reduced. However, with higher dephosphorylation degree of phosvitin (> 40%), the acceleration effect was enhanced compared to native phosvitin, in which the β-sheet structure was increased and phosvitin was partially hydrolyzed to phosphopeptides. The acceleration effect of phase transformation was as follows: T4 > T3 > phosvitin > T1 >
T2 > Control. This study clearly demonstrated that phosphorylation of phosvitin played an important role in the regulation of mineralization. Keywords: phosphorylation; phosvitin; phase transformation
1. Introduction Phosvitin is a highly phosphorylated protein derived from egg yolks and prevalent in vertebrates [1]. So far, the biological function of phosvitin is not well understood. It’s generally accepted that the phosphorylated amino acid residues of phosvitin may participate in enzymatic phosphoryl transfer or energy storage [2], and ion transport as well [1]. Phosvitin shares similar properties to phosphorylated proteins such dentin phosphophoryn. They are both acidic proteins with flexible random coil structures and are rich in Glu, Asp, and phosphorylated serine/threonine residues [3-5]. Additionally, they possess calcium-binding capacity and hydroxyapatite affinity [6, 7]. Furthermore, they have a bias codon usage pattern of AGC and AGT for serine, this correlation between codon usage and codon propensities in repeat formation in multiphosphorylated proteins is involved in the biomineralization of hard tissues [8]. These peculiar structures and properties suggest that phosvitin should have similar biological properties to those of phosphorylated proteins that are crucial mediators of biomineralization [9], and may be crucial in tissue calcification during embryonic development. Early hypothesis proposed that phosvitin was correlated with bone formation [1] and might provide continued skeletal mineralization during embryonic development [10]. Several research have shown that phosvitin could promote calcium phosphate crystal nucleation in vitro [11-14]. The phosphate esters of phosphorylated proteins were important for their functions as mediators of biomineralization [15, 16]. Our previous research showed that phosvitin phosphorus was involved in chicken embryo
bone formation through dephosphorylation [17], and we have certified that dicalcium phosphate dihydrate (DCPD) formed in the precursor phase of mineralization, then converted into hydroxyapatite (HAP) in the subsequent stepwise phase via a dissolution-recrystallization pathway. And there was no evidence of other calcium phosphate phases being present. Phosvitin significantly promotes the initiation of phase transformation,
accelerated
the
transformation
process
and
shortened
the
transformation time from 6.5 to 1 h [11]. However, the role of phosphorylation of phosvitin in the phase transformation of calcium phosphate has not been fully elucidated. Thus, in this study, we evaluated the effect of the dephosphorylation degree of phosvitin on the phase transformation of calcium phosphate, in order to investigate the role of phosphorylation of phosvitin in the regulation of mineralization. 2. Experimental methods 2.1 Alkaline dephosphorylation of phosvitin Phosvitin was prepared from hen egg yolk as previously described [18] with the purity of 93.14% examined by HPLC. Partial dephosphorylation of phosvitin was performed by the method of Jiang and Mine [19] with some modifications. Four equal phosvitin (100 mg) was dissolved in 4 mL of 0.1, 0.2, 0.3 and 0.4 M NaOH, respectively, and incubated at 37 °C for 3 h, after which the pH was reduced to 7.0 by the addition of 1.0 M HCl. The reaction solution was centrifuged at 5000 × g for 15 min to separate partially dephosphorylated phosvitin from free phosphoric acid, using centricon-3 micro-concentrators (Amicon, USA) with a cutoff range of 3 kDa, and
washed three times with Milli-Q water (Millipore corporation, USA). The permeates of sample treated by different concentrations of NaOH were collected and adjusted to 100 mL with Milli-Q water, referenced to as “P1, P2, P3 and P4”, respectively. While the retentates were also collected and adjusted to 100 mL, referenced to as “T1, T2, T3 and T4”, respectively. 2.2 Determination of phosphorus content and dephosphorylation degree of phosvitin Phosphorus content of the permeate and retentate was determined as previously described [18]. The dephosphorylation degree of phosvitin was measured based on the content of phosphate released, calculated as follows: Ri = MPi / (MPi + MTi) × 100% where i is the sample treated by different concentrations of NaOH, MPi is the amount of phosphorus content of the permeate, and MTi is the amount of phosphorus content of the retentate. 2.3 Native-PAGE Native gel electrophoresis was performed in 1.0 M Tris-HCl buffer (pH 6.8) for stacking gel (5% acrylamide) and 1.5 M Tris-HCl buffer (pH 8.8) for separating gel (12% acrylamide). Migration was performed at 80 V in the stacking gel and at 120 V in the separating gel, in 25 mM Tris and 192 mM Glycine, pH 8.3. About 50 μg sample was loaded on the gel. Prestained protein molecular weight marker was used as a marker (10 - 170 kDa, Fermentas China Co., Ltd. Shenzhen, China). After migration, gels stain
was carried out as previous described [18]. The gel was analyzed by Gel-Pro Analyzer 4.0 (Media Cybernetics, USA). 2.4 Circular dichroism (CD) analysis of phosvitin CD spectra were recorded according to the method of Zhang et al. [6], 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 (190250 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 partially dephosphorylated phosvitin (T1, T2, T3 and T4). CD data was displayed as observed ellipticity θ (mdeg) vs. wavelength (nm). 2.5 Dynamic monitor of the phase transformation of calcium phosphate The phase transformation of calcium phosphate was dynamic monitored and assessed by pH-stat titration assay at 25 °C. The mineralization reaction was performed as previous description [11]. The reaction mixtures comprised 20 mM CaCl2, 12 mM NaH2PO4 and phosvitin (0.02, 0.04, 0.10 and 0.20 mg/mL, respectively). BSA (0.20 mg/mL) was used to compare with phosvitin. 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 monitored by continuous titrating with a 0.15 M NaOH solution using an automated titration system (Titrando 907, Metrohm), and maintaining to pH 7.0 ± 0.1, which was set as a fixed end point. After reaction, the precipitate (mineral) was rinsed three times with Milli-Q
water and then freeze-dried for later examination. Milli-Q water was used as a control instead of phosvitin. In addition, the phase transformation with different dephosphorylation degrees of phosvitin (0.20 mg/mL of T1, T2, T3 and T4) was also assessed. 2.6 Characterization and analysis of mineral Characterization of mineral was evaluated according to previous description [11]. In short, the structural properties of generated calcium phosphate were identified by Xray 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 morphologies were measured by scanning electron microscopy (SEM) with a JEOL JSM-6390LV SEM equipped with an Oxford INCA EDS detector. 3. Results and discussion 3.1 Preparation of partially dephosphorylated phosvitin Partially dephosphorylated phosvitins (T1, T2, T3 and T4) were prepared by treated with NaOH solutions, with 2.98, 19.46, 43.39, and 71.07% of phosphate released from native phosvitin, respectively (Table S1). This suggested that dephosphorylation was NaOH concentration dependent. Native-PAGE patterns of partially phosphorylated phosvitin were showed in Fig. 1. Native phosvitin composed two main bands (α- phosvitin and β-phosvitin), there have insignificantly change when phosvitin was treated with 0.1 M NaOH. The
intensities of α and β bands was gradually weakened and even disappeared when increased NaOH concentration. Especially, when phosvitin was treated with 0.4 M NaOH, both of α and β bands were disappeared and lower molecular weight blurred bands (10-20 kDa) were emerged, indicating that phosvitin was not only released phosphate but also hydrolyzed to lower molecular weight peptides. Some reports have demonstrated that phosphate groups of phosphoserine were sensitive to alkaline and easier to release phosphate [19-21], meanwhile the other amino acids could be hydrolysis due to occur racemization under alkaline conditions [22]. Effect of dephosphorylation on the structure of phosvitin was evaluated by CD analysis and showed in Fig. 2. The main secondary structure of native phosvitin was γrandom and β-sheet, which was consistent with the literature [23]. With the increase of dephosphorylation degree, β-sheet was gradually increased whereas β-turn and γrandom were gradually reduced. This could be explain that the net negative charge of phosvitin was reduced, and electrostatic repulsion between phosvitin molecular was weakened due to the reduction of phosphorylation, thus resulted in molecular folding and became more compact, this phenomenon was also observed by Kato et al. [24]. 3.2 Role of phosphorylation of phosvitin in the phase transformation of calcium phosphate 3.2.1 Dynamic monitor of the phase transformation The phase transformation of calcium phosphate was monitored by pH-stat titration, where H+ generated by the mineralization reaction was titrated by NaOH solution to
maintain constant pH at 7.0 ± 0.1. The transformation reaction was accomplished when the titration curve reached a plateau and the mineral was hydroxyapatite (HAP) identified by FTIR and XRD. The titration curves for all samples can be divided into two regions as indicated in Fig. 3, a faster titration rate followed by a slower one. More amount of NaOH consumed per unit time meant more rapidly transformation. The calcium phosphate with added phosvitin was rapidly transformed into HAP within 1 h, while the control group without added protein needed more than 3 h. Furthermore, the calcium phosphate with added BSA was retarded this transformation. The unique effect of phosvitin on the phase transformation of calcium phosphate could be due to the different structures of phosvitin and BSA. Phosvitin was a small protein with a dimension of 4-6 nm while BSA was 14 nm [25, 26]. Moreover, phosvitin contained much higher net negative charge (with -179e) than BSA (with -18e) in the neutral condition [27, 28], due to the abundant phosphorylated serine/threonine residue clusters. Therefore, phosvitin could accumulate more calcium ions and provided more high-density nucleation sites for mineralization than BSA. This indicated that phosvitin significantly accelerated the phase transformation of calcium phosphate due to its highly phosphorylation. The titration curve of the phase transformation with added different concentrations of phosvitin and partially dephosphorylated phosvitin were showed in Fig. 4. There was a linear relation between the amount of NaOH consumed and concentration of phosvitin (R2 = 0.996, Fig. 4A). These results were indicated that there was a dose-dependent effect of phosvitin on the rate of phase transformation.
With added partially dephosphorylated phosvitin, the trend of amount of NaOH consumed during mineralization reaction was as follows: T4 > T3 > phosvitin > T1 > T2 > Control (Fig. 4B), demonstrating that the lower dephosphorylation degree of phosvitin (< 20%) weakened the acceleration effect of the phase transformation while the higher dephosphorylation degree of phosvitin (> 40%) enhanced the acceleration effect. 3.3.2 Characterization and composition analysis of the calcium phosphate The technologies of FTIR, XRD and SEM were employed to characterization and composition analysis of the mineral calcium phosphate generated with various phosvitins in the mineralization reaction. It should be noted that there may be formed other phases such as OCP in the present system, however, FTIR could be used to reliably identify DCPD, HAP and other phases, based on their characteristic IR bands [29]. There only existed DCPD and HAP characteristic IR bands and no other characteristic IR bands could be observed in this study, the assignments of the FTIR spectra were given in Table S2. As shown in Fig. 5A and 6A, the mineral generated without phosvitin was DCPD. When higher concentration of phosvitin was added, the characteristic FTIR bands and XRD peaks of DCPD gradually decreased while that of HAP occurred and gradually increased. Thus, the mineral was a mixture of DCPD and HAP at the end of the reaction (1 h), and the proportion of HAP was higher with higher phosvitin concentration. With added 0.02, 0.04 and 0.10 mg/mL of phosvitin, the dominant peak of mineral appeared at 11.63 °, which assigned to the (020) plane of DCPD crystal, similar to the result of mineral with control group. However, the most
dominant peaks of mineral with added 0.20 mg/mL of phosvitin appeared at 11.63 ° and 31.65 °, which corresponded to the (020) plane of DCPD and the (211) plane of HAP crystal, respectively [30]. Moreover, an increase in preferred orientation in the cplane was observed in mineral with added 0.20 mg/mL of phosvitin as the intensity ratios of the crystal plane (020) and (211) was 0.278 [31], suggested that HAP became the main component of mineral. As shown in Fig. 5B and 6B, the mineral generated with added T4 was HAP due to there were only presented the characteristic FTIR bands and XRD peaks of HAP, indicating that the acceleration effect of T4 on the phase transformation was stronger than that of phosvitin. With added T3, the mineral was mainly HAP since there only existed a weak FTIR band of DCPD (530 cm-1). The mineral generated with added T1 or T2 was a mixture of DCPD and HAP, and the FTIR band intensities of DCPD in mineral with added T1 were slightly weaker than that of mineral with added T2. Reference intensity ratio (RIR) method was commonly used to quantitative phase analysis [32]. Results showed in Table 1, on one hand, when phosphate released from phosvitin was lower than 20%, weight fraction of HAP was lower with higher dephosphorylation degree of phosvitin, suggesting that dephosphorylated phosvitin had weaker acceleration effect than that of native phosvitin. On the other hand, when phosphate released from phosvitin was higher than 40%, in which phosvitin was partially hydrolyzed to phosphopeptides, weight fraction of HAP was higher with higher dephosphorylation degree, indicated that stronger acceleration effect of the phase transformation than that of native phosvitin. These results demonstrated that
phosphorylation played an important role in the phase transformation, which is consistent with the finding of other researchers [7, 33]. The morphology changes of calcium phosphate generated with different concentrations of phosvitin were identified from the SEM micrographs. As shown in Fig. 7, the mineral in the absence of phosvitin was in the shape of a plate with a smooth surface, which corresponded to the DCPD signatures observed in FTIR and XRD. With the increase of phosvitin concentration, the smooth plate-like particles was gradually dissolved and amorphous shape was appeared. In the presence of 0.20 mg/mL of phosvitin, the crystal mainly grew into amorphous particles, which corresponded to HAP. Previous reports have demonstrated that the phase transformation from DCPD into HAP was accomplished via dissolution-recrystallization pathway [11, 34]. With higher dephosphorylation degree of phosvitin, the crystal was gradually converted from plate particles into amorphous particles, and the particles formed irregular aggregates. In addition, the mineral generated with added T4 was dense agglomerate. This result suggested
that
partially dephosphorylated
phosvitin accelerated
the phase
transformation from DCPD to HAP and promoted HAP crystal growth. Lower concentration of phosvitin (< 0.20 mg/mL) could induce formation of DCPD but not enough to promote HAP crystal growth per unit time. The acceleration effect of lower dephosphorylation degree of phosvitin (< 20%) on the phase transformation was weaker than that of native phosvitin. This could be explained that phosphorylation and random structure of phosvitin were reduced, resulted in the limited capacities to accumulate calcium ions and provide nucleation sites. The
phosphorylation and flexible random structure of phosphorylated protein was important to regulate the mineralization [35]. However, with higher dephosphorylation degree of phosvitin (> 40%), in which phosvitin was partially hydrolyzed to phosphopeptides, exposed more active binding area of calcium ions and could provide more nucleation sites. Furthermore, mineralization template was increased with increased the β-sheet structure of phosvitin. Previous study have showed that phosvitin sequestered large amounts of calcium ions since the β-sheet structure increased [6]. Reports have demonstrated that the β-sheet structure of phosphorylated protein could serve as the template to orient growth of crystal in mineralization [33, 36, 37]. This study suggested that the regulation of phase transformation of calcium phosphate was closely associated with phosphorylation of phosvitin, and phosphopeptides hydrolyzed from phosvitin could enhanced the acceleration effect, consistent with the result of George and Veis [7], who demonstrated that phosphorylation of dentin phosphophoryn peptides played a key role on the oriented growth of HAP. To elucidate the regulation mechanism of mineralization with phosvitin, further research is required on the identification of the critical mineralization-inducing active domain sequence of phosvitin phosphopeptides. 4. Conclusions The regulation of phosvitin on the phase transformation of calcium phosphate was dose-dependent effect, and the role in regulation was closely associated with the dephosphorylation degree of phosvitin. The acceleration effect was as follows: T4 > T3 > phosvitin > T1 > T2 > Control. Phosphopeptides hydrolyzed from phosvitin could enhance the regulation of mineralization.
Acknowledgments This work was supported by the Earmarked Fund for Modern Agro-industry Technology Research System (Project No. CARS-41-K23) and the National Natural Science Foundation of China (Grant No. 31471602). References [1] R.N. Finn, Vertebrate yolk complexes and the functional implications of phosvitins and other subdomains in vitellogenins, Biol. Reprod. 76 (2007) 926-935. [2] G.A. Morrill, T.L. Dowd, A.B. Kostellow, R.K. Gupta, Progesterone-induced changes in the phosphoryl potential during the meiotic divisions in amphibian oocytes: Role of Na/K-ATPase, BMC Dev. Biol. 11 (2011) 67. [3] M. Anton, O. Castellani, C. Guérin-Dubiard, Phosvitin, in: R. Huopalahti, R. LópezFandiño, M. Anton, R. Schade (Eds.), Bioactive Egg Compounds, Springer Berlin Heidelberg 2007, pp. 17-24. [4] G. Hunter, P. Hauschka, A. Poole, L. Rosenberg, H. Goldberg, Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins, Biochem. J. 317 (1996) 59-64. [5] A.L. Boskey, Matrix proteins and mineralization: an overview, Connect. Tissue Res. 35 (1996) 357-363. [6] X. Zhang, F. Geng, X. Huang, M. Ma, Calcium binding characteristics and structural changes of phosvitin, J. Inorg. Biochem. 159 (2016) 76-81. [7] A. George, A. Veis, Phosphorylated proteins and control over apatite nucleation,
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George, Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization, J. Biol. Chem. 280 (2005) 33109-33114. [17] 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. [18] 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. [19] B. Jiang, Y. Mine, Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin, J. Agric. Food Chem. 48 (2000) 990-994. [20] J. Ren, Phosvitin extraction and phosphopeptides characterization from chicken egg yolk, Edmonton: University of Alberta Libraries, 2012. [21] V.K. Pasupuleti, S. Braun, State of the art manufacturing of protein hydrolysates, Protein Hydrolysates in Biotechnology, Springer Netherlands 2010, pp. 11-32. [22] L.C. Sen, E. Gonzalez-Flores, R.E. Feeney, J.R. Whitaker, Reactions of phosphoproteins in alkaline solutions, J. Agric. Food Chem. 25 (1977) 632-638. [23] V. Renugopalakrishnan, P.M. Horowitz, M.J. Glimcher, Structural studies of phosvitin in solution and in the solid state, J. Biol. Chem. 260 (1985) 11406-11413. [24] A. Kato, S. Miyazaki, A. Kawamoto, K. Kobayashi, Effects of phosphate residues on the excellent emulsifying properties of phosphoglycoprotein phosvitin (food & nutrition), Agric. Biol. Chem. 51 (1987) 2989-2994. [25] R. Bellairs, M. Backhouse, R.J. Evans, A correlated chemical and morphological
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Figure captions Fig. 1 Native-PAGE patterns of phosvitin. Lane 1-4, T1, T2, T3 and T4 obtained from phosvitin by treated with 0.1, 0.2, 0.3 and 0.4 M NaOH solution, respectively. Lane 5, Marker; Lane 6, phosvitin. Fig. 2 CD patterns and secondary structure analysis of partially dephosphorylated phosvitin. Fig. 3 The titration curve of the phase transformation of calcium phosphate during the mineralization reaction, with added 0.20 mg/mL of phosvitin, BSA and control. Fig. 4 The titration curve of the phase transformation of calcium phosphate during the mineralization reaction. (A), with 0.02, 0.04, 0.10 and 0.20 mg/mL of phosvitin; (B), with 0.20 mg/mL of T1, T2, T3 and T4; Control, without phosvitin replaced by MilliQ water. Fig. 5 FTIR spectra of the calcium phosphate generated. (A), with 0.02, 0.04, 0.10 and 0.20 mg/mL of phosvitin; (B), with 0.20 mg/mL of T1, T2, T3 and T4; Control, without phosvitin replaced by Milli-Q water. Fig. 6 XRD patterns of the calcium phosphate generated. (A), with 0.02, 0.04, 0.10 and 0.20 mg/mL of phosvitin; (B), with 0.20 mg/mL of T1, T2, T3 and T4; Control, without phosvitin replaced by Milli-Q water.
Fig. 7 The surface morphologies of the calcium phosphate generated with native phosvitin and partially dephosphorylated phosvitin. Control, without phosvitin replaced by Milli-Q water.
Figr-1
Figr-2
Figr-3
Figr-4
Figr-5
Figr-6
Figr-7
Table 1 Weight fractions of the mineral calcium phosphate generated with partially dephosphorylated phosvitins Weight fraction (%) Mineral DCPD
HAP
T1
17.74
82.26
T2
19.97
80.03
T3
5.09
94.91
T4
0.00
100.00
phosvitin
16.96
83.04
Control
100.00
0.00
S0141-8130(16)32915-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.158 BIOMAC 7324
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
15-12-2016 21-3-2017 27-3-2017
Please cite this article as: Xiaowei Zhang, Xi Huang, Meihu Ma, Role of phosphorylation of phosvitin in the phase transformation of mineralization, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.158 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Role of phosphorylation of phosvitin in the phase transformation of mineralization Xiaowei Zhang, Xi Huang, Meihu Ma* National R&D Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. * Corresponding author: Meihu Ma College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China Tel: +86 27 87283177 Fax: +86 27 87283177 E-mail: [email protected]
Highlights
Phosvitin in the acceleration effect of phase transformation was dose-concentration effect.
With lower dephosphorylation degree of phosvitin (<20%), the acceleration effect was weaker than native phosvitin.
With higher dephosphorylation degree of phosvitin (>40%), the acceleration effect was enhanced compared to native phosvitin.
Phosphorylation of phosvitin played an important role in regulating mineralization.
Abstract Phosvitin is a unique highly phosphorylated protein that plays a role in the regulation of mineralization. This study investigated the role of phosphorylation of phosvitin in the phase transformation of calcium phosphate in the mineralization solution. Partially dephosphorylated phosvitins (T1, T2, T3 and T4) were prepared with 2.98, 19.46, 43.39 and 71.07% of phosphate released from native phosvitin, respectively. And their effect on regulating the phase transformation was investigated, the characterization and composition analysis was performed by circular dichroism, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. Results showed that phosvitin in the acceleration effect of phase transformation was dose-concentration effect by pH-stat titration. With lower dephosphorylation degree of phosvitin (< 20%), the acceleration effect was weaker than native phosvitin, since phosphorylation and random structure of phosvitin were reduced. However, with higher dephosphorylation degree of phosvitin (> 40%), the acceleration effect was enhanced compared to native phosvitin, in which the β-sheet structure was increased and phosvitin was partially hydrolyzed to phosphopeptides. The acceleration effect of phase transformation was as follows: T4 > T3 > phosvitin > T1 >
T2 > Control. This study clearly demonstrated that phosphorylation of phosvitin played an important role in the regulation of mineralization. Keywords: phosphorylation; phosvitin; phase transformation
1. Introduction Phosvitin is a highly phosphorylated protein derived from egg yolks and prevalent in vertebrates [1]. So far, the biological function of phosvitin is not well understood. It’s generally accepted that the phosphorylated amino acid residues of phosvitin may participate in enzymatic phosphoryl transfer or energy storage [2], and ion transport as well [1]. Phosvitin shares similar properties to phosphorylated proteins such dentin phosphophoryn. They are both acidic proteins with flexible random coil structures and are rich in Glu, Asp, and phosphorylated serine/threonine residues [3-5]. Additionally, they possess calcium-binding capacity and hydroxyapatite affinity [6, 7]. Furthermore, they have a bias codon usage pattern of AGC and AGT for serine, this correlation between codon usage and codon propensities in repeat formation in multiphosphorylated proteins is involved in the biomineralization of hard tissues [8]. These peculiar structures and properties suggest that phosvitin should have similar biological properties to those of phosphorylated proteins that are crucial mediators of biomineralization [9], and may be crucial in tissue calcification during embryonic development. Early hypothesis proposed that phosvitin was correlated with bone formation [1] and might provide continued skeletal mineralization during embryonic development [10]. Several research have shown that phosvitin could promote calcium phosphate crystal nucleation in vitro [11-14]. The phosphate esters of phosphorylated proteins were important for their functions as mediators of biomineralization [15, 16]. Our previous research showed that phosvitin phosphorus was involved in chicken embryo
bone formation through dephosphorylation [17], and we have certified that dicalcium phosphate dihydrate (DCPD) formed in the precursor phase of mineralization, then converted into hydroxyapatite (HAP) in the subsequent stepwise phase via a dissolution-recrystallization pathway. And there was no evidence of other calcium phosphate phases being present. Phosvitin significantly promotes the initiation of phase transformation,
accelerated
the
transformation
process
and
shortened
the
transformation time from 6.5 to 1 h [11]. However, the role of phosphorylation of phosvitin in the phase transformation of calcium phosphate has not been fully elucidated. Thus, in this study, we evaluated the effect of the dephosphorylation degree of phosvitin on the phase transformation of calcium phosphate, in order to investigate the role of phosphorylation of phosvitin in the regulation of mineralization. 2. Experimental methods 2.1 Alkaline dephosphorylation of phosvitin Phosvitin was prepared from hen egg yolk as previously described [18] with the purity of 93.14% examined by HPLC. Partial dephosphorylation of phosvitin was performed by the method of Jiang and Mine [19] with some modifications. Four equal phosvitin (100 mg) was dissolved in 4 mL of 0.1, 0.2, 0.3 and 0.4 M NaOH, respectively, and incubated at 37 °C for 3 h, after which the pH was reduced to 7.0 by the addition of 1.0 M HCl. The reaction solution was centrifuged at 5000 × g for 15 min to separate partially dephosphorylated phosvitin from free phosphoric acid, using centricon-3 micro-concentrators (Amicon, USA) with a cutoff range of 3 kDa, and
washed three times with Milli-Q water (Millipore corporation, USA). The permeates of sample treated by different concentrations of NaOH were collected and adjusted to 100 mL with Milli-Q water, referenced to as “P1, P2, P3 and P4”, respectively. While the retentates were also collected and adjusted to 100 mL, referenced to as “T1, T2, T3 and T4”, respectively. 2.2 Determination of phosphorus content and dephosphorylation degree of phosvitin Phosphorus content of the permeate and retentate was determined as previously described [18]. The dephosphorylation degree of phosvitin was measured based on the content of phosphate released, calculated as follows: Ri = MPi / (MPi + MTi) × 100% where i is the sample treated by different concentrations of NaOH, MPi is the amount of phosphorus content of the permeate, and MTi is the amount of phosphorus content of the retentate. 2.3 Native-PAGE Native gel electrophoresis was performed in 1.0 M Tris-HCl buffer (pH 6.8) for stacking gel (5% acrylamide) and 1.5 M Tris-HCl buffer (pH 8.8) for separating gel (12% acrylamide). Migration was performed at 80 V in the stacking gel and at 120 V in the separating gel, in 25 mM Tris and 192 mM Glycine, pH 8.3. About 50 μg sample was loaded on the gel. Prestained protein molecular weight marker was used as a marker (10 - 170 kDa, Fermentas China Co., Ltd. Shenzhen, China). After migration, gels stain
was carried out as previous described [18]. The gel was analyzed by Gel-Pro Analyzer 4.0 (Media Cybernetics, USA). 2.4 Circular dichroism (CD) analysis of phosvitin CD spectra were recorded according to the method of Zhang et al. [6], 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 (190250 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 partially dephosphorylated phosvitin (T1, T2, T3 and T4). CD data was displayed as observed ellipticity θ (mdeg) vs. wavelength (nm). 2.5 Dynamic monitor of the phase transformation of calcium phosphate The phase transformation of calcium phosphate was dynamic monitored and assessed by pH-stat titration assay at 25 °C. The mineralization reaction was performed as previous description [11]. The reaction mixtures comprised 20 mM CaCl2, 12 mM NaH2PO4 and phosvitin (0.02, 0.04, 0.10 and 0.20 mg/mL, respectively). BSA (0.20 mg/mL) was used to compare with phosvitin. 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 monitored by continuous titrating with a 0.15 M NaOH solution using an automated titration system (Titrando 907, Metrohm), and maintaining to pH 7.0 ± 0.1, which was set as a fixed end point. After reaction, the precipitate (mineral) was rinsed three times with Milli-Q
water and then freeze-dried for later examination. Milli-Q water was used as a control instead of phosvitin. In addition, the phase transformation with different dephosphorylation degrees of phosvitin (0.20 mg/mL of T1, T2, T3 and T4) was also assessed. 2.6 Characterization and analysis of mineral Characterization of mineral was evaluated according to previous description [11]. In short, the structural properties of generated calcium phosphate were identified by Xray 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 morphologies were measured by scanning electron microscopy (SEM) with a JEOL JSM-6390LV SEM equipped with an Oxford INCA EDS detector. 3. Results and discussion 3.1 Preparation of partially dephosphorylated phosvitin Partially dephosphorylated phosvitins (T1, T2, T3 and T4) were prepared by treated with NaOH solutions, with 2.98, 19.46, 43.39, and 71.07% of phosphate released from native phosvitin, respectively (Table S1). This suggested that dephosphorylation was NaOH concentration dependent. Native-PAGE patterns of partially phosphorylated phosvitin were showed in Fig. 1. Native phosvitin composed two main bands (α- phosvitin and β-phosvitin), there have insignificantly change when phosvitin was treated with 0.1 M NaOH. The
intensities of α and β bands was gradually weakened and even disappeared when increased NaOH concentration. Especially, when phosvitin was treated with 0.4 M NaOH, both of α and β bands were disappeared and lower molecular weight blurred bands (10-20 kDa) were emerged, indicating that phosvitin was not only released phosphate but also hydrolyzed to lower molecular weight peptides. Some reports have demonstrated that phosphate groups of phosphoserine were sensitive to alkaline and easier to release phosphate [19-21], meanwhile the other amino acids could be hydrolysis due to occur racemization under alkaline conditions [22]. Effect of dephosphorylation on the structure of phosvitin was evaluated by CD analysis and showed in Fig. 2. The main secondary structure of native phosvitin was γrandom and β-sheet, which was consistent with the literature [23]. With the increase of dephosphorylation degree, β-sheet was gradually increased whereas β-turn and γrandom were gradually reduced. This could be explain that the net negative charge of phosvitin was reduced, and electrostatic repulsion between phosvitin molecular was weakened due to the reduction of phosphorylation, thus resulted in molecular folding and became more compact, this phenomenon was also observed by Kato et al. [24]. 3.2 Role of phosphorylation of phosvitin in the phase transformation of calcium phosphate 3.2.1 Dynamic monitor of the phase transformation The phase transformation of calcium phosphate was monitored by pH-stat titration, where H+ generated by the mineralization reaction was titrated by NaOH solution to
maintain constant pH at 7.0 ± 0.1. The transformation reaction was accomplished when the titration curve reached a plateau and the mineral was hydroxyapatite (HAP) identified by FTIR and XRD. The titration curves for all samples can be divided into two regions as indicated in Fig. 3, a faster titration rate followed by a slower one. More amount of NaOH consumed per unit time meant more rapidly transformation. The calcium phosphate with added phosvitin was rapidly transformed into HAP within 1 h, while the control group without added protein needed more than 3 h. Furthermore, the calcium phosphate with added BSA was retarded this transformation. The unique effect of phosvitin on the phase transformation of calcium phosphate could be due to the different structures of phosvitin and BSA. Phosvitin was a small protein with a dimension of 4-6 nm while BSA was 14 nm [25, 26]. Moreover, phosvitin contained much higher net negative charge (with -179e) than BSA (with -18e) in the neutral condition [27, 28], due to the abundant phosphorylated serine/threonine residue clusters. Therefore, phosvitin could accumulate more calcium ions and provided more high-density nucleation sites for mineralization than BSA. This indicated that phosvitin significantly accelerated the phase transformation of calcium phosphate due to its highly phosphorylation. The titration curve of the phase transformation with added different concentrations of phosvitin and partially dephosphorylated phosvitin were showed in Fig. 4. There was a linear relation between the amount of NaOH consumed and concentration of phosvitin (R2 = 0.996, Fig. 4A). These results were indicated that there was a dose-dependent effect of phosvitin on the rate of phase transformation.
With added partially dephosphorylated phosvitin, the trend of amount of NaOH consumed during mineralization reaction was as follows: T4 > T3 > phosvitin > T1 > T2 > Control (Fig. 4B), demonstrating that the lower dephosphorylation degree of phosvitin (< 20%) weakened the acceleration effect of the phase transformation while the higher dephosphorylation degree of phosvitin (> 40%) enhanced the acceleration effect. 3.3.2 Characterization and composition analysis of the calcium phosphate The technologies of FTIR, XRD and SEM were employed to characterization and composition analysis of the mineral calcium phosphate generated with various phosvitins in the mineralization reaction. It should be noted that there may be formed other phases such as OCP in the present system, however, FTIR could be used to reliably identify DCPD, HAP and other phases, based on their characteristic IR bands [29]. There only existed DCPD and HAP characteristic IR bands and no other characteristic IR bands could be observed in this study, the assignments of the FTIR spectra were given in Table S2. As shown in Fig. 5A and 6A, the mineral generated without phosvitin was DCPD. When higher concentration of phosvitin was added, the characteristic FTIR bands and XRD peaks of DCPD gradually decreased while that of HAP occurred and gradually increased. Thus, the mineral was a mixture of DCPD and HAP at the end of the reaction (1 h), and the proportion of HAP was higher with higher phosvitin concentration. With added 0.02, 0.04 and 0.10 mg/mL of phosvitin, the dominant peak of mineral appeared at 11.63 °, which assigned to the (020) plane of DCPD crystal, similar to the result of mineral with control group. However, the most
dominant peaks of mineral with added 0.20 mg/mL of phosvitin appeared at 11.63 ° and 31.65 °, which corresponded to the (020) plane of DCPD and the (211) plane of HAP crystal, respectively [30]. Moreover, an increase in preferred orientation in the cplane was observed in mineral with added 0.20 mg/mL of phosvitin as the intensity ratios of the crystal plane (020) and (211) was 0.278 [31], suggested that HAP became the main component of mineral. As shown in Fig. 5B and 6B, the mineral generated with added T4 was HAP due to there were only presented the characteristic FTIR bands and XRD peaks of HAP, indicating that the acceleration effect of T4 on the phase transformation was stronger than that of phosvitin. With added T3, the mineral was mainly HAP since there only existed a weak FTIR band of DCPD (530 cm-1). The mineral generated with added T1 or T2 was a mixture of DCPD and HAP, and the FTIR band intensities of DCPD in mineral with added T1 were slightly weaker than that of mineral with added T2. Reference intensity ratio (RIR) method was commonly used to quantitative phase analysis [32]. Results showed in Table 1, on one hand, when phosphate released from phosvitin was lower than 20%, weight fraction of HAP was lower with higher dephosphorylation degree of phosvitin, suggesting that dephosphorylated phosvitin had weaker acceleration effect than that of native phosvitin. On the other hand, when phosphate released from phosvitin was higher than 40%, in which phosvitin was partially hydrolyzed to phosphopeptides, weight fraction of HAP was higher with higher dephosphorylation degree, indicated that stronger acceleration effect of the phase transformation than that of native phosvitin. These results demonstrated that
phosphorylation played an important role in the phase transformation, which is consistent with the finding of other researchers [7, 33]. The morphology changes of calcium phosphate generated with different concentrations of phosvitin were identified from the SEM micrographs. As shown in Fig. 7, the mineral in the absence of phosvitin was in the shape of a plate with a smooth surface, which corresponded to the DCPD signatures observed in FTIR and XRD. With the increase of phosvitin concentration, the smooth plate-like particles was gradually dissolved and amorphous shape was appeared. In the presence of 0.20 mg/mL of phosvitin, the crystal mainly grew into amorphous particles, which corresponded to HAP. Previous reports have demonstrated that the phase transformation from DCPD into HAP was accomplished via dissolution-recrystallization pathway [11, 34]. With higher dephosphorylation degree of phosvitin, the crystal was gradually converted from plate particles into amorphous particles, and the particles formed irregular aggregates. In addition, the mineral generated with added T4 was dense agglomerate. This result suggested
that
partially dephosphorylated
phosvitin accelerated
the phase
transformation from DCPD to HAP and promoted HAP crystal growth. Lower concentration of phosvitin (< 0.20 mg/mL) could induce formation of DCPD but not enough to promote HAP crystal growth per unit time. The acceleration effect of lower dephosphorylation degree of phosvitin (< 20%) on the phase transformation was weaker than that of native phosvitin. This could be explained that phosphorylation and random structure of phosvitin were reduced, resulted in the limited capacities to accumulate calcium ions and provide nucleation sites. The
phosphorylation and flexible random structure of phosphorylated protein was important to regulate the mineralization [35]. However, with higher dephosphorylation degree of phosvitin (> 40%), in which phosvitin was partially hydrolyzed to phosphopeptides, exposed more active binding area of calcium ions and could provide more nucleation sites. Furthermore, mineralization template was increased with increased the β-sheet structure of phosvitin. Previous study have showed that phosvitin sequestered large amounts of calcium ions since the β-sheet structure increased [6]. Reports have demonstrated that the β-sheet structure of phosphorylated protein could serve as the template to orient growth of crystal in mineralization [33, 36, 37]. This study suggested that the regulation of phase transformation of calcium phosphate was closely associated with phosphorylation of phosvitin, and phosphopeptides hydrolyzed from phosvitin could enhanced the acceleration effect, consistent with the result of George and Veis [7], who demonstrated that phosphorylation of dentin phosphophoryn peptides played a key role on the oriented growth of HAP. To elucidate the regulation mechanism of mineralization with phosvitin, further research is required on the identification of the critical mineralization-inducing active domain sequence of phosvitin phosphopeptides. 4. Conclusions The regulation of phosvitin on the phase transformation of calcium phosphate was dose-dependent effect, and the role in regulation was closely associated with the dephosphorylation degree of phosvitin. The acceleration effect was as follows: T4 > T3 > phosvitin > T1 > T2 > Control. Phosphopeptides hydrolyzed from phosvitin could enhance the regulation of mineralization.
Acknowledgments This work was supported by the Earmarked Fund for Modern Agro-industry Technology Research System (Project No. CARS-41-K23) and the National Natural Science Foundation of China (Grant No. 31471602). References [1] R.N. Finn, Vertebrate yolk complexes and the functional implications of phosvitins and other subdomains in vitellogenins, Biol. Reprod. 76 (2007) 926-935. [2] G.A. Morrill, T.L. Dowd, A.B. Kostellow, R.K. Gupta, Progesterone-induced changes in the phosphoryl potential during the meiotic divisions in amphibian oocytes: Role of Na/K-ATPase, BMC Dev. Biol. 11 (2011) 67. [3] M. Anton, O. Castellani, C. Guérin-Dubiard, Phosvitin, in: R. Huopalahti, R. LópezFandiño, M. Anton, R. Schade (Eds.), Bioactive Egg Compounds, Springer Berlin Heidelberg 2007, pp. 17-24. [4] G. Hunter, P. Hauschka, A. Poole, L. Rosenberg, H. Goldberg, Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins, Biochem. J. 317 (1996) 59-64. [5] A.L. Boskey, Matrix proteins and mineralization: an overview, Connect. Tissue Res. 35 (1996) 357-363. [6] X. Zhang, F. Geng, X. Huang, M. Ma, Calcium binding characteristics and structural changes of phosvitin, J. Inorg. Biochem. 159 (2016) 76-81. [7] A. George, A. Veis, Phosphorylated proteins and control over apatite nucleation,
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Figure captions Fig. 1 Native-PAGE patterns of phosvitin. Lane 1-4, T1, T2, T3 and T4 obtained from phosvitin by treated with 0.1, 0.2, 0.3 and 0.4 M NaOH solution, respectively. Lane 5, Marker; Lane 6, phosvitin. Fig. 2 CD patterns and secondary structure analysis of partially dephosphorylated phosvitin. Fig. 3 The titration curve of the phase transformation of calcium phosphate during the mineralization reaction, with added 0.20 mg/mL of phosvitin, BSA and control. Fig. 4 The titration curve of the phase transformation of calcium phosphate during the mineralization reaction. (A), with 0.02, 0.04, 0.10 and 0.20 mg/mL of phosvitin; (B), with 0.20 mg/mL of T1, T2, T3 and T4; Control, without phosvitin replaced by MilliQ water. Fig. 5 FTIR spectra of the calcium phosphate generated. (A), with 0.02, 0.04, 0.10 and 0.20 mg/mL of phosvitin; (B), with 0.20 mg/mL of T1, T2, T3 and T4; Control, without phosvitin replaced by Milli-Q water. Fig. 6 XRD patterns of the calcium phosphate generated. (A), with 0.02, 0.04, 0.10 and 0.20 mg/mL of phosvitin; (B), with 0.20 mg/mL of T1, T2, T3 and T4; Control, without phosvitin replaced by Milli-Q water.
Fig. 7 The surface morphologies of the calcium phosphate generated with native phosvitin and partially dephosphorylated phosvitin. Control, without phosvitin replaced by Milli-Q water.
Figr-1
Figr-2
Figr-3
Figr-4
Figr-5
Figr-6
Figr-7
Table 1 Weight fractions of the mineral calcium phosphate generated with partially dephosphorylated phosvitins Weight fraction (%) Mineral DCPD
HAP
T1
17.74
82.26
T2
19.97
80.03
T3
5.09
94.91
T4
0.00
100.00
phosvitin
16.96
83.04
Control
100.00
0.00