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Prion protein (PrPc) interacts with histone H3 confirmed by affinity chromatography
Journal of Chromatography B, 929 (2013) 40–44
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Prion protein (PrPc ) interacts with histone H3 confirmed by affinity chromatography Hanning Cai 1 , Ying Xie 1 , Lingyin Hu, Jingjing Fan, Renqiang Li ∗ Department of Biotechnology, Jinan University, Shipai, Guangzhou 510632, China
a r t i c l e
i n f o
Article history: Received 14 February 2013 Accepted 6 April 2013 Available online 12 April 2013 Keywords: Prion protein Histone H3 Interaction Affinity chromatography
a b s t r a c t The histones including H2a, H2b, H3 and H4 purified from pig liver tissue were immobilized onto Sepharose 4B to create a histone-Sepharose column. During chromatography of cow milk casein by histone-Sepharose column, two isoforms of prion protein (PrPc ) with 34 and 30 kDa molecular mass corresponding to diglycosylated and monoglycosylated PrPc respectively were found to be captured by histone ligands. To further verify the interaction between histones and PrPc , the PrPc -Sepharose column was prepared and used to separate the histones. Two chromatography processes and SDS-PAGE demonstrated that only H3 in the histones was found to interact with PrPc . This study suggested H3 could be the target molecule of PrPC in nuclei, which might be useful for understanding the prion disease. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In eukaryotic cells, the tails of histone H2a, H2b, H3 and H4 pass through to the outside of the core particle of the nucleosome in their own manners [1]. Thus the histone proteins tails are exposed and many covalent modifications happened [2]. The modified histones not only alter the twist of the DNA double helix associated with the core particle and consequently alter the structural properties of the chromatin, also alter the binding affinity of the factors to chromatin, and these features are crucial for the role of nucleosome in transcriptional regulation [3]. These factors involved in the transcriptional regulation are very important for the illustration of function of histone post-translational modifications. As the effects of these modifications to transcription activation and repression, DNA repairs and other important biological process [4] being found, nucleosome is gradually considered to be the focus of transcriptional regulation [5–7]. Obviously, the proteins bound with histones in cell deserve to be studied. Studies on the downstream of histone post-translational modifications have pointed out that histone interacting proteins are supposed to be some kind of potential anticancer drugs [8]. For instance, HAMLET (human–lactalbumin made lethal to tumor cells), a folding variant of human–lactalbumin in an active complex with oleic acid, was discovered to be able to kill tumor cells by mechanism resembling apoptosis, and many different types of tumor cells were susceptible to this effect while healthy
differentiated cells were resistant. HAMLET combines with histone proteins and disorders nucleosome assembly [9,10]. Based on the principle of affinity chromatography, the purified histones were immobilized onto Sepharose 4B as ligands in this study to aim to find out whether HAMLET or the other histone interacting proteins existed in cow milk casein. It is interesting that two isoforms of cellular prion protein (PrPc ) instead of HAMLET in cow milk casein were found to be bound with histones. As we known, PrPc is the precursor of prions (PrPsc ) that is known to cause transmissible spongiform encephalopathies (TSE) while the transformation of PrPc to PrPsc is still unclear. In the past few years, absolute amount of PrPc is also found in milk and differs between the species [11]. In addition, this glycosylated protein is also abundantly expressed in many different tissues and cells [12], and localizes to nucleus of endocrine and neuronal cells and interacts with structure chromatin components [13]. So the study on interaction between PrPc and histones, especially in details on which one of histones interacts with PrPc , is of great significance. In the following process, the histone-Sepharose and PrPc -Sepharose column were altogether prepared and used to confirm the interaction of PrPc with H3, which may be useful for exploring the mechanism of TSE and the role of PrPc in transcriptional regulation in the nucleus. 2. Materials and methods 2.1. Materials
∗ Corresponding author. Tel.: +86 20 85220219; fax: +86 20 85220219. E-mail address: [email protected] (R. Li). 1 These authors contributed equally to this work. 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.04.003
Pig liver was from the local market. Fresh cow milk was obtained from healthy individuals and transported at 4 ◦ C. Sepharose 4B polymer beads with 45–165 m diameter was from
H. Cai et al. / J. Chromatogr. B 929 (2013) 40–44
Amersham. Epichlorohydrin, 1,4-dioxane and all other chemicals used in SDS-PAGE or for buffers were of analytical or chemical grade and were purchased from Guangzhou Chemical Co. Ltd. (Guangzhou, China). Markers for SDS-PAGE and BCA kit were supplied by Shanghai Sheng Zheng Biotechnology Co. Ltd. (Shanghai, China). The other reagents: grinding medium containing 0.2 M sucrose–10 mM MgCl2 –10 mM Tris–50 mM NaHSO3 (pH 7.5), washing medium A containing all the components of grinding medium and 1.0% emulsifying agent OP-10, washing medium B containing 10 mM Tris–20 mM Na2 EDTA–50 mM NaHSO3 –1 mM -mercaptoethanol–HCl (pH 7.5), equilibrium solution containing 10 mM Tris–0.05 M NaCl–1 mM Na2 EDTA–1 mM -mercaptoethanol–HCl (pH 7.5), eluting solution containing all the components of equilibrium solution and 1.0 M NaCl, 0.02 M PBS (pH 8.0). 2.2. Extraction of the chromatin All procedures were performed at 4 ◦ C unless specified. The pieces of fresh pig liver tissue were repeatedly frozen and thawed three times after having been frozen at −18 ◦ C for three days, 30 g of them mixed with 500 mL cold grinding medium was homogenized and filtered through 4 layers of gauze on funnel. The filtrate was centrifuged at 2500 rpm for 10 min to collect the sediment. The sediment was completely suspended with 500 mL washing medium A and stirred slowly for 5 min and centrifuged at 2500 rpm for 10 min to discard the supernatant. The process was repeated until the supernatant was clear. The sediment was continuously suspended with 200 mL washing medium B and stirred slowly for 8 min and centrifuged at 8000 rpm for 10 min to collect the sediment. The sediment pellets were swollen to a gel by fast homogenization in 80 mL deionized, glass-distilled water for 2 h and then centrifuged at 10,000 rpm for 10 min. The supernatant was sucked out gently using pipette in case the gray–black swollen pellet appeared. The chromatins were extracted and determined by microscope. 2.3. Purification of the core histones Sonication was adopted to crush the swollen pellet until the solution was clear and no more viscous. The solution was centrifuged at 8000 rpm for 5 min to remove those small pellets or fragments. DNA was precipitated by adding 1/9 volumes of 4 N H2 SO4 to the supernatant and stirring in 4 ◦ C cabinet for 2 h, then centrifuged at 12,000 rpm for 15 min to discard the DNA sediment. The histones and non-histone proteins in supernatant could be differentially separated with different concentration of NaCl salt-extracted and over 1 h standing by. Based on the preliminary tests, the final NaCl concentrations with 1.6, 3.0 M and saturated (approximately 5.6 M) were used, respectively, for separating histone and non-histone proteins. After adding NaCl, centrifugations at 12,000 rpm for 10 min were adopted to collect the protein sediment. These proteins were dialyzed against deionized water and PBS and concentrated by vacuum cold drying apparatus, then determined by SDS-PAGE. 2.4. Immobilization of core histones onto Sepharose 4B Fourteen grams of wet Sepharose 4B was washed using 1 M NaCl and deionized water at Buchner funnel. These polymer beads were mixed with 8.0 mL 2 M NaOH, 2.5 mL epichlorohydrin and 15 mL 56% 1,4-dioxane, shaken for 2 h at 37 ◦ C. After being washed at Buchner funnel by using deionized water, these activated Sepharose 4B beads were mixed with 10 mL of core histones solution (3.6 mg/mL), shaken at 40 ◦ C for about 24 h and washed using PBS to remove the histones that were not attached to polymer beads. Those protein contents before mixed with beads, and for
41
the part that were not attached to polymer beads were determined using BCA Kit. The data was used to calculate the mg histone fixed by the beads. Coupling rate between core histones and the beads was expressed using mg histones fixed per gram of wet Sepharose 4B beads. A column with 9 cm × 1.5 cm was made. For comparison, a control column of Sepharose 4B beads without histones coupled was also made. 2.5. Preparation of milk casein and its chromatography on histone-Sepharose column The preparation of milk casein was carried out according to the method of Malin Svensson [9]. The fresh milk incubated overnight at 4 ◦ C was centrifuged at 4000 rpm for 15 min to discard the upper fat layer. Casein was isolated by an overnight incubation at 4 ◦ C with 10% potassium oxalate followed by a second overnight incubation at 4 ◦ C after lowering the pH to 4.5 using 1 M HCl and heating the solution to 32 ◦ C for 2 h. The casein precipitated was harvested by 8000 rpm for 15 min. The precipitated casein was suspended with equilibrium solution and dialyzed against deionized water and equilibrium solution, respectively. Then the casein solution was filtered through one layer of filter paper at funnel in order to remove the large insoluble particles. The filtrate casein was the sample for affinity chromatography. The histone-Sepharose column was washed fully with eluting solution and equilibrated with equilibrium solution respectively. Then 15 mL of casein extraction was loaded on the column at a flow rate of 12 mL per hour. After being washed fully with equilibrium solution, eluting solution was used to elute the column. The chromatography process was under real-time monitored and the peak fractions at O.D280 were collected and detected by SDS-PAGE. 2.6. Preparation of PrPc -Sepharose column and chromatography of histones on this column Fourteen grams of wet Sepharose 4B beads was activated as same as that described in above Section 2.4. These activated Sepharose 4B beads were mixed with those PrPc pulled down by histone-Sepharose column and shaken at 40 ◦ C for about 24 h and washed at Buchner funnel using PBS. The concentration of proteins before mixing with beads and the part that were not attached to polymer beads was determined by BCA Kit respectively and the coupling rate between PrPc and the beads was calculated. A PrPc Sepharose column with 9 cm × 1.5 cm was made. Then 10 mL of native core histones extracted as describing in Section 2.3 was applied to the column at a flow rate of 12 mL per hour. After being washed fully with equilibrium solution, eluting solution was applied to elute the column. The chromatography process was under real-time monitored and a number of fractions at penetration peak and eluting peak were collected respectively and detected by SDS-PAGE. 2.7. SDS-PAGE All samples for SDS-PAGE were desalted by dialyzing against deionized water before mixed with the sample loading buffer (25% 0.5 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 10% mercaptoethanol and 0.05% bromphenol blue). The mixture was heated in 100 ◦ C boiling water for 3 min. Proteins (8–15 L) were subjected to electrophoresis in 12% separation gel and 3% stacking gel containing 0.1% SDS. After running in electrode buffer containing 0.25 M Tris–1.9 M glycine and 0.1% SDS (pH 8.3) at 20 mA constant current for 1 h or more, proteins were stained by immersing the gel in 0.25% Coomassie Blue solution for 2 h. Destaining was by several changes in 25% ethanol, 15% acetic acid until a clear
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H. Cai et al. / J. Chromatogr. B 929 (2013) 40–44
Fig. 1. SDS-PAGE analysis of the salt-extracted histone fractions. Lane 1, 1.6 M NaCl fraction; lane 2, 3.0 M NaCl; lane 3, 5.6 M (saturated) NaCl. The H3 and H2a bands overlapped on this gel because of their close mass. The molecular mass of markers and the core histone proteins H3, H2a, H2b, H4 (position of arrows) are indicated.
background was obtained. The analysis of SDS-PAGE pattern was performed using Electrophoresis Image Analysis System. 3. Results 3.1. Purification of histones The detection of histone and non-histone proteins after the NaCl salt-extraction was shown in Fig. 1. Most large molecular weight non-histone proteins were precipitated when NaCl was added to a concentration of 1.6 M. Although in 3.0 M, almost all of the nonhistone proteins were precipitated, a large amount of histones were precipitated as well probably because the swollen chromatin could not be precisely cut by sonication and leaved behind large amount of nucleosome chains. Results over 10 salt-extractions demonstrated that the order for biomolecules to precipitate was related very closely to their molecular sizes, as a result, the larger was tended to precipitate easier. After 3.0 M, the remainders were those small molecular proteins like H2a, H2b, H3 and H4. In order to harvest more core histones, NaCl was added into the solution until saturated. Generally, most of the works for histone extraction have been based on the solubility of histones in acid to separate histones. Considering the histones were purified for the use of ligands, it would not be wise to adopt either acid extraction or salt extraction alone because the residual DNA would greatly affect the result of chromatography. In the salt extraction for histones, almost all monomeric core histones were differentially separated from those large compounds including histone multimers through 1.6 and 3.0 M sediment.
Fig. 2. Chromatography process of milk casein on the histone-Sepharose column. Big peak refers to those proteins haven’t bound to the histone-Sepharose column and the second peak to the proteins captured by histones. The histone-Sepharose column was equilibrated with equilibrium solution containing 10 mM Tris–0.05 M NaCl–1 mM Na2 EDTA–1 mM -mercaptoethanol–HCl (pH 7.5), and eluted with eluting solution containing all the components of equilibrium solution and 1.0 M NaCl. The flow rate was about 12 mL per hour, and the experiments were performed at room temperature.
without the histones as the ligands was meanwhile performed, but no eluting peak was found after the eluting solution was applied into the column (data not shown). SDS-PAGE analysis of those proteins captured by histones was shown in Fig. 3. Electrophoresis result of PrPc in cow milk enriched by Alicon PrioTrap® indicated the molecular mass of the three isoforms of PrPc are between 25 and 37 kDa [11], while Fig. 3 indicated all of proteins corresponding to the nearly mass as between 25 and 37 kDa were found to bind with histone ligands. So the proteins captured by histones as shown in Fig. 3 were PrPc undoubtedly. Moreover, according to the electrophoresis result of PrPc by digestion with PNGase, an enzyme that cuts off oligosaccharides from N-linked glycoproteins, the PrPc with largest and larger mass were diglycosylated and monoglycosylated PrPc respectively. So the two proteins bound with histone ligands in Fig. 3 were determined as diglycosylated and monoglycosylated PrPc respectively. Almost all of the PrPc have been captured by the ligands according to lane 1 and 2, which demonstrated the affinity between core histones and PrPc was very convincing.
3.2. Interaction of core histones with PrPc In order to determine the coupling rate, BCA kit was used to calculate the histone proteins fixed with Sepharose 4B beads. More than 1.1 mg histone proteins were fixed by 1 g beads. The chromatography process of milk casein on the histone-Sepharose column was shown in Fig. 2. The first big peak represented the proteins haven’t bound to histones and the second peak was those proteins captured by histones. For comparison, the control column
Fig. 3. SDS-PAGE analysis of milk casein separated by histone-Sepharose column. Lane 1, proteins captured by histones; lane 2, proteins were not captured by the column; lane 3, total milk casein.
H. Cai et al. / J. Chromatogr. B 929 (2013) 40–44
Fig. 4. Chromatography process of histones on the PrPc -Sepharose column. Big peak refers to those proteins haven’t bound to PrPc -Sepharose column and the second peak to the proteins captured by PrPc . Position of arrows show that the sample fraction at this time was collected for doing SDS-PAGE analysis. The PrPc -Sepharose column was equilibrated with equilibrium solution containing 10 mM Tris–0.05 M NaCl–1 mM Na2 EDTA–1 mM -mercaptoethanol–HCl (pH 7.5), and eluted with eluting solution containing all the components of equilibrium solution and 1.0 M NaCl. The flow rate was about 12 mL per hour, and the experiments were performed at room temperature.
Fig. 5. SDS-PAGE analysis of histones during chromatography process by PrPc Sepharose column. Lane 1, histone proteins; lane 2, corresponding to 30 min collection (penetration peak); lane 3, 42 min collection (penetration peak); lane 4, 80 min collection (penetration peak); lane 5, 172 min collection (eluate); lane 6, 174 min collection (eluate); lane 7, 176 min collection (eluate).
3.3. PrPc interacts with H3 The coupling rate between PrPc and activated Sepharose 4B was about 1.0 mg PrPc per 1 g beads. When the histones passed through the PrPc -Sepharose column, the protein peak captured by PrPc was appeared as shown in Fig. 4. However, this peak was much smaller than expected, which indicated that many of histones were not captured by PrPc . A small number of fractions were collected at 30 min, 40 min and 80 min of the penetration peak and at 172 min, 174 min and 176 min of eluting peak during chromatography process (as shown in Fig. 4). Their SDS-PAGE analysis was shown in Fig. 5, which demonstrated that the histone captured by PrPc was H3 with the molecular mass of 17 kDa.
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of PrPc using Alicon PrioTrap® technology. These PrPc isoforms were later confirmed by Western Blot with anti- PrPc monoclonal antibodies which recognized the same proteins. Compared with the native PrPc enriched by Alicon PrioTrap® , the two proteins captured by histone-Sepharose column in this study were extremely consistent with PrPc , since the molecular mass of these two proteins were between 25 and 37 kDa. Moreover, these two proteins were identified as diglycosylated and monoglycosylated PrPc by further comparison to the electrophoresis results of PrPc in the reference. However, the Alicon PrioTrap® technology not only enriched diglycosylated and monoglycosylated PrPc , also nonglycosylated PrPc which was not appeared in the proteins captured by histone-Sepharose column. This is because Alicon PrioTrap® results from hydrophilic and hydrophobic surface cluster, while histone-Sepharose column focus on the biomolecular interaction between histone and PrPc . In another aspect, the absence of nonglycosylated PrPc by histone-Sepharose column suggests the necessity of glycosyl to the interaction between histone and PrPc . Although the affinity between histones and PrPc is very convincing according to SDS-PAGE analysis (Fig. 3), it is necessary to confirm which one of the histones actually interacted with PrPc . So the PrPc -Sepharose column was made. However, when the histones passed through the PrPc -Sepharose column, those proteins captured by PrPc were not as much as expected although H3 has been determined as the histone fraction to interact with PrPc . Comparing to the chromatography of cow milk casein by histoneSepharose column, the protein peak captured by ligands on the PrPc -Sepharose column was very small (comparing Fig. 4 with Fig. 2). This phenomenon is probably due to that the histone fractions used on the PrPc -Sepharose column were easy to form histone polymers, either dimers or tetramers, which reduced the touching and combination efficiency between PrPc and H3. Naturally when mixed with each other, this kind of combination among histones is believed to be stubborn [1]. But when immobilized onto Sepharose 4B, the histone polymers would not be existed, which explain the high affinity between PrPc and histones on the histone-Sepharose column. PrPsc is known to cause TSE after accumulation in the central nervous system. Prion diseases are neurodegenerative, infectious disorders characterized by the aggregation of a misfolded isoform of the cellular PrPC [14]. In addition, PrPc is also crucial in the role of Cu2+ regulation, blood glucose regulation, neural degeneration and cell signaling and cognitive functioning in human and transcriptional regulation in the nucleus [13,15,16]. Researches have indicated that prion expressed and colonized in central nervous system and secondary lymphoid organs and inflammatory foci [17]. However, the mechanism for the conversion of PrPc to PrPsc , which triggered occurrence of prion disease are still unclear. The results of this study suggest H3 could be the target molecule of PrPC in nuclei due to the existence of the interaction between PrPC and H3, which concluded by performing in vitro albeit. Actually, with the enrichment of the functions of PrPc being found, other molecules associated with PrPc appear to be necessary for the function of PrPc . The research for these additional molecules, like SiRT1 [18], one of the histone deacetylase, are involved in the function of prion and could be that these molecules will provide us with the key for understanding the prion disease.
4. Discussion
5. Conclusions
During chromatography of cow milk casein by histoneSepharose column, two proteins in milk casein were found to have high affinity to histones. According to Franscini [11], fresh milk from health cow individual was enriched for three isoforms
Two affinity chromatographies by using the histone-Sepharose column and the PrPc -Sepharose column respectively have demonstrated that histone H3 is interacted with two isoforms of PrPc with 34 and 30 kDa molecular mass corresponding to diglycosylated and
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H. Cai et al. / J. Chromatogr. B 929 (2013) 40–44
monoglycosylated PrPc respectively, which suggest H3 could be the target molecule of PrPC in nuclei. References [1] [2] [3] [4] [5] [6] [7] [8]
R.D. Kornberg, Y. Lorch, Cell 98 (1999) 285. C. Martin, Y. Zhang, Nat. Rev. Mol. Cell. Biol. 6 (2005) 838. B.D. Strahl, C.D. Allis, Nature 403 (2000) 41. A.J. Ruthenburg, H. Li, D.J. Patel, C.D. Allis, Nat. Rev. Mol. Cell. Biol. 8 (2007) 983. B. Lewin, Cell 79 (1994) 397. M. Grunstein, Nature 389 (1997) 349. J.T. Kadonaga, Cell 92 (1998) 307. J.B. Ruan, J.Y. Zang, J. Univ. Sci. Technol. China 38 (2008) 930.
[9] M. Svensson, H. Sabharwal, A. Håkansson, A.K. Mossberg, P. Lipniunas, H. Leffler, C. Svanborg, S. Linse, J. Biol. Chem. 274 (1999) 6388. [10] C. Düringer, A. Hamiche, L. Gustafsson, H. Kimura, J. Biol. Chem. 278 (2003) 42131. [11] N. Franscini, A.E.I. Gedaily, U. Matthey, S. Franitza, M.S. Sy, A. Buekle, M. Groschup, U. Braun, R. Zahn, PLoS ONE 1 (2006) e71. [12] M.J. Ford, L.J. Burton, R.J. Morris, S.M. Hall, Neuroscience 113 (2002) 177. [13] A. Strom, G.S. Wang, D.J. Picketts, R. Reimer, A.W. Stuke, F.W. Scott, Eur. J. Cell. Biol. 90 (2011) 414. [14] A.L. Horwich, J.S. Weissman, Cell 89 (1997) 499. [15] D.R. Brown, Trends Neurosci. 24 (2001) 85. [16] O. Nicolas, R. Gavín, J.A. del Río, Brain Res. Rev. 61 (2009) 170. [17] M. Nuvolone, A. Aguzzi, M. Heikenwalder, FEBS Lett. 583 (2009) 2674. [18] J.S. Seo, M.H. Moon, J.K. Jeong, J.W. Seol, Y.J. Lee, B.H. Park, S.Y. Park, Neurobiol. Aging 33 (2012) 1110.
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Prion protein (PrPc ) interacts with histone H3 confirmed by affinity chromatography Hanning Cai 1 , Ying Xie 1 , Lingyin Hu, Jingjing Fan, Renqiang Li ∗ Department of Biotechnology, Jinan University, Shipai, Guangzhou 510632, China
a r t i c l e
i n f o
Article history: Received 14 February 2013 Accepted 6 April 2013 Available online 12 April 2013 Keywords: Prion protein Histone H3 Interaction Affinity chromatography
a b s t r a c t The histones including H2a, H2b, H3 and H4 purified from pig liver tissue were immobilized onto Sepharose 4B to create a histone-Sepharose column. During chromatography of cow milk casein by histone-Sepharose column, two isoforms of prion protein (PrPc ) with 34 and 30 kDa molecular mass corresponding to diglycosylated and monoglycosylated PrPc respectively were found to be captured by histone ligands. To further verify the interaction between histones and PrPc , the PrPc -Sepharose column was prepared and used to separate the histones. Two chromatography processes and SDS-PAGE demonstrated that only H3 in the histones was found to interact with PrPc . This study suggested H3 could be the target molecule of PrPC in nuclei, which might be useful for understanding the prion disease. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In eukaryotic cells, the tails of histone H2a, H2b, H3 and H4 pass through to the outside of the core particle of the nucleosome in their own manners [1]. Thus the histone proteins tails are exposed and many covalent modifications happened [2]. The modified histones not only alter the twist of the DNA double helix associated with the core particle and consequently alter the structural properties of the chromatin, also alter the binding affinity of the factors to chromatin, and these features are crucial for the role of nucleosome in transcriptional regulation [3]. These factors involved in the transcriptional regulation are very important for the illustration of function of histone post-translational modifications. As the effects of these modifications to transcription activation and repression, DNA repairs and other important biological process [4] being found, nucleosome is gradually considered to be the focus of transcriptional regulation [5–7]. Obviously, the proteins bound with histones in cell deserve to be studied. Studies on the downstream of histone post-translational modifications have pointed out that histone interacting proteins are supposed to be some kind of potential anticancer drugs [8]. For instance, HAMLET (human–lactalbumin made lethal to tumor cells), a folding variant of human–lactalbumin in an active complex with oleic acid, was discovered to be able to kill tumor cells by mechanism resembling apoptosis, and many different types of tumor cells were susceptible to this effect while healthy
differentiated cells were resistant. HAMLET combines with histone proteins and disorders nucleosome assembly [9,10]. Based on the principle of affinity chromatography, the purified histones were immobilized onto Sepharose 4B as ligands in this study to aim to find out whether HAMLET or the other histone interacting proteins existed in cow milk casein. It is interesting that two isoforms of cellular prion protein (PrPc ) instead of HAMLET in cow milk casein were found to be bound with histones. As we known, PrPc is the precursor of prions (PrPsc ) that is known to cause transmissible spongiform encephalopathies (TSE) while the transformation of PrPc to PrPsc is still unclear. In the past few years, absolute amount of PrPc is also found in milk and differs between the species [11]. In addition, this glycosylated protein is also abundantly expressed in many different tissues and cells [12], and localizes to nucleus of endocrine and neuronal cells and interacts with structure chromatin components [13]. So the study on interaction between PrPc and histones, especially in details on which one of histones interacts with PrPc , is of great significance. In the following process, the histone-Sepharose and PrPc -Sepharose column were altogether prepared and used to confirm the interaction of PrPc with H3, which may be useful for exploring the mechanism of TSE and the role of PrPc in transcriptional regulation in the nucleus. 2. Materials and methods 2.1. Materials
∗ Corresponding author. Tel.: +86 20 85220219; fax: +86 20 85220219. E-mail address: [email protected] (R. Li). 1 These authors contributed equally to this work. 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.04.003
Pig liver was from the local market. Fresh cow milk was obtained from healthy individuals and transported at 4 ◦ C. Sepharose 4B polymer beads with 45–165 m diameter was from
H. Cai et al. / J. Chromatogr. B 929 (2013) 40–44
Amersham. Epichlorohydrin, 1,4-dioxane and all other chemicals used in SDS-PAGE or for buffers were of analytical or chemical grade and were purchased from Guangzhou Chemical Co. Ltd. (Guangzhou, China). Markers for SDS-PAGE and BCA kit were supplied by Shanghai Sheng Zheng Biotechnology Co. Ltd. (Shanghai, China). The other reagents: grinding medium containing 0.2 M sucrose–10 mM MgCl2 –10 mM Tris–50 mM NaHSO3 (pH 7.5), washing medium A containing all the components of grinding medium and 1.0% emulsifying agent OP-10, washing medium B containing 10 mM Tris–20 mM Na2 EDTA–50 mM NaHSO3 –1 mM -mercaptoethanol–HCl (pH 7.5), equilibrium solution containing 10 mM Tris–0.05 M NaCl–1 mM Na2 EDTA–1 mM -mercaptoethanol–HCl (pH 7.5), eluting solution containing all the components of equilibrium solution and 1.0 M NaCl, 0.02 M PBS (pH 8.0). 2.2. Extraction of the chromatin All procedures were performed at 4 ◦ C unless specified. The pieces of fresh pig liver tissue were repeatedly frozen and thawed three times after having been frozen at −18 ◦ C for three days, 30 g of them mixed with 500 mL cold grinding medium was homogenized and filtered through 4 layers of gauze on funnel. The filtrate was centrifuged at 2500 rpm for 10 min to collect the sediment. The sediment was completely suspended with 500 mL washing medium A and stirred slowly for 5 min and centrifuged at 2500 rpm for 10 min to discard the supernatant. The process was repeated until the supernatant was clear. The sediment was continuously suspended with 200 mL washing medium B and stirred slowly for 8 min and centrifuged at 8000 rpm for 10 min to collect the sediment. The sediment pellets were swollen to a gel by fast homogenization in 80 mL deionized, glass-distilled water for 2 h and then centrifuged at 10,000 rpm for 10 min. The supernatant was sucked out gently using pipette in case the gray–black swollen pellet appeared. The chromatins were extracted and determined by microscope. 2.3. Purification of the core histones Sonication was adopted to crush the swollen pellet until the solution was clear and no more viscous. The solution was centrifuged at 8000 rpm for 5 min to remove those small pellets or fragments. DNA was precipitated by adding 1/9 volumes of 4 N H2 SO4 to the supernatant and stirring in 4 ◦ C cabinet for 2 h, then centrifuged at 12,000 rpm for 15 min to discard the DNA sediment. The histones and non-histone proteins in supernatant could be differentially separated with different concentration of NaCl salt-extracted and over 1 h standing by. Based on the preliminary tests, the final NaCl concentrations with 1.6, 3.0 M and saturated (approximately 5.6 M) were used, respectively, for separating histone and non-histone proteins. After adding NaCl, centrifugations at 12,000 rpm for 10 min were adopted to collect the protein sediment. These proteins were dialyzed against deionized water and PBS and concentrated by vacuum cold drying apparatus, then determined by SDS-PAGE. 2.4. Immobilization of core histones onto Sepharose 4B Fourteen grams of wet Sepharose 4B was washed using 1 M NaCl and deionized water at Buchner funnel. These polymer beads were mixed with 8.0 mL 2 M NaOH, 2.5 mL epichlorohydrin and 15 mL 56% 1,4-dioxane, shaken for 2 h at 37 ◦ C. After being washed at Buchner funnel by using deionized water, these activated Sepharose 4B beads were mixed with 10 mL of core histones solution (3.6 mg/mL), shaken at 40 ◦ C for about 24 h and washed using PBS to remove the histones that were not attached to polymer beads. Those protein contents before mixed with beads, and for
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the part that were not attached to polymer beads were determined using BCA Kit. The data was used to calculate the mg histone fixed by the beads. Coupling rate between core histones and the beads was expressed using mg histones fixed per gram of wet Sepharose 4B beads. A column with 9 cm × 1.5 cm was made. For comparison, a control column of Sepharose 4B beads without histones coupled was also made. 2.5. Preparation of milk casein and its chromatography on histone-Sepharose column The preparation of milk casein was carried out according to the method of Malin Svensson [9]. The fresh milk incubated overnight at 4 ◦ C was centrifuged at 4000 rpm for 15 min to discard the upper fat layer. Casein was isolated by an overnight incubation at 4 ◦ C with 10% potassium oxalate followed by a second overnight incubation at 4 ◦ C after lowering the pH to 4.5 using 1 M HCl and heating the solution to 32 ◦ C for 2 h. The casein precipitated was harvested by 8000 rpm for 15 min. The precipitated casein was suspended with equilibrium solution and dialyzed against deionized water and equilibrium solution, respectively. Then the casein solution was filtered through one layer of filter paper at funnel in order to remove the large insoluble particles. The filtrate casein was the sample for affinity chromatography. The histone-Sepharose column was washed fully with eluting solution and equilibrated with equilibrium solution respectively. Then 15 mL of casein extraction was loaded on the column at a flow rate of 12 mL per hour. After being washed fully with equilibrium solution, eluting solution was used to elute the column. The chromatography process was under real-time monitored and the peak fractions at O.D280 were collected and detected by SDS-PAGE. 2.6. Preparation of PrPc -Sepharose column and chromatography of histones on this column Fourteen grams of wet Sepharose 4B beads was activated as same as that described in above Section 2.4. These activated Sepharose 4B beads were mixed with those PrPc pulled down by histone-Sepharose column and shaken at 40 ◦ C for about 24 h and washed at Buchner funnel using PBS. The concentration of proteins before mixing with beads and the part that were not attached to polymer beads was determined by BCA Kit respectively and the coupling rate between PrPc and the beads was calculated. A PrPc Sepharose column with 9 cm × 1.5 cm was made. Then 10 mL of native core histones extracted as describing in Section 2.3 was applied to the column at a flow rate of 12 mL per hour. After being washed fully with equilibrium solution, eluting solution was applied to elute the column. The chromatography process was under real-time monitored and a number of fractions at penetration peak and eluting peak were collected respectively and detected by SDS-PAGE. 2.7. SDS-PAGE All samples for SDS-PAGE were desalted by dialyzing against deionized water before mixed with the sample loading buffer (25% 0.5 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 10% mercaptoethanol and 0.05% bromphenol blue). The mixture was heated in 100 ◦ C boiling water for 3 min. Proteins (8–15 L) were subjected to electrophoresis in 12% separation gel and 3% stacking gel containing 0.1% SDS. After running in electrode buffer containing 0.25 M Tris–1.9 M glycine and 0.1% SDS (pH 8.3) at 20 mA constant current for 1 h or more, proteins were stained by immersing the gel in 0.25% Coomassie Blue solution for 2 h. Destaining was by several changes in 25% ethanol, 15% acetic acid until a clear
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Fig. 1. SDS-PAGE analysis of the salt-extracted histone fractions. Lane 1, 1.6 M NaCl fraction; lane 2, 3.0 M NaCl; lane 3, 5.6 M (saturated) NaCl. The H3 and H2a bands overlapped on this gel because of their close mass. The molecular mass of markers and the core histone proteins H3, H2a, H2b, H4 (position of arrows) are indicated.
background was obtained. The analysis of SDS-PAGE pattern was performed using Electrophoresis Image Analysis System. 3. Results 3.1. Purification of histones The detection of histone and non-histone proteins after the NaCl salt-extraction was shown in Fig. 1. Most large molecular weight non-histone proteins were precipitated when NaCl was added to a concentration of 1.6 M. Although in 3.0 M, almost all of the nonhistone proteins were precipitated, a large amount of histones were precipitated as well probably because the swollen chromatin could not be precisely cut by sonication and leaved behind large amount of nucleosome chains. Results over 10 salt-extractions demonstrated that the order for biomolecules to precipitate was related very closely to their molecular sizes, as a result, the larger was tended to precipitate easier. After 3.0 M, the remainders were those small molecular proteins like H2a, H2b, H3 and H4. In order to harvest more core histones, NaCl was added into the solution until saturated. Generally, most of the works for histone extraction have been based on the solubility of histones in acid to separate histones. Considering the histones were purified for the use of ligands, it would not be wise to adopt either acid extraction or salt extraction alone because the residual DNA would greatly affect the result of chromatography. In the salt extraction for histones, almost all monomeric core histones were differentially separated from those large compounds including histone multimers through 1.6 and 3.0 M sediment.
Fig. 2. Chromatography process of milk casein on the histone-Sepharose column. Big peak refers to those proteins haven’t bound to the histone-Sepharose column and the second peak to the proteins captured by histones. The histone-Sepharose column was equilibrated with equilibrium solution containing 10 mM Tris–0.05 M NaCl–1 mM Na2 EDTA–1 mM -mercaptoethanol–HCl (pH 7.5), and eluted with eluting solution containing all the components of equilibrium solution and 1.0 M NaCl. The flow rate was about 12 mL per hour, and the experiments were performed at room temperature.
without the histones as the ligands was meanwhile performed, but no eluting peak was found after the eluting solution was applied into the column (data not shown). SDS-PAGE analysis of those proteins captured by histones was shown in Fig. 3. Electrophoresis result of PrPc in cow milk enriched by Alicon PrioTrap® indicated the molecular mass of the three isoforms of PrPc are between 25 and 37 kDa [11], while Fig. 3 indicated all of proteins corresponding to the nearly mass as between 25 and 37 kDa were found to bind with histone ligands. So the proteins captured by histones as shown in Fig. 3 were PrPc undoubtedly. Moreover, according to the electrophoresis result of PrPc by digestion with PNGase, an enzyme that cuts off oligosaccharides from N-linked glycoproteins, the PrPc with largest and larger mass were diglycosylated and monoglycosylated PrPc respectively. So the two proteins bound with histone ligands in Fig. 3 were determined as diglycosylated and monoglycosylated PrPc respectively. Almost all of the PrPc have been captured by the ligands according to lane 1 and 2, which demonstrated the affinity between core histones and PrPc was very convincing.
3.2. Interaction of core histones with PrPc In order to determine the coupling rate, BCA kit was used to calculate the histone proteins fixed with Sepharose 4B beads. More than 1.1 mg histone proteins were fixed by 1 g beads. The chromatography process of milk casein on the histone-Sepharose column was shown in Fig. 2. The first big peak represented the proteins haven’t bound to histones and the second peak was those proteins captured by histones. For comparison, the control column
Fig. 3. SDS-PAGE analysis of milk casein separated by histone-Sepharose column. Lane 1, proteins captured by histones; lane 2, proteins were not captured by the column; lane 3, total milk casein.
H. Cai et al. / J. Chromatogr. B 929 (2013) 40–44
Fig. 4. Chromatography process of histones on the PrPc -Sepharose column. Big peak refers to those proteins haven’t bound to PrPc -Sepharose column and the second peak to the proteins captured by PrPc . Position of arrows show that the sample fraction at this time was collected for doing SDS-PAGE analysis. The PrPc -Sepharose column was equilibrated with equilibrium solution containing 10 mM Tris–0.05 M NaCl–1 mM Na2 EDTA–1 mM -mercaptoethanol–HCl (pH 7.5), and eluted with eluting solution containing all the components of equilibrium solution and 1.0 M NaCl. The flow rate was about 12 mL per hour, and the experiments were performed at room temperature.
Fig. 5. SDS-PAGE analysis of histones during chromatography process by PrPc Sepharose column. Lane 1, histone proteins; lane 2, corresponding to 30 min collection (penetration peak); lane 3, 42 min collection (penetration peak); lane 4, 80 min collection (penetration peak); lane 5, 172 min collection (eluate); lane 6, 174 min collection (eluate); lane 7, 176 min collection (eluate).
3.3. PrPc interacts with H3 The coupling rate between PrPc and activated Sepharose 4B was about 1.0 mg PrPc per 1 g beads. When the histones passed through the PrPc -Sepharose column, the protein peak captured by PrPc was appeared as shown in Fig. 4. However, this peak was much smaller than expected, which indicated that many of histones were not captured by PrPc . A small number of fractions were collected at 30 min, 40 min and 80 min of the penetration peak and at 172 min, 174 min and 176 min of eluting peak during chromatography process (as shown in Fig. 4). Their SDS-PAGE analysis was shown in Fig. 5, which demonstrated that the histone captured by PrPc was H3 with the molecular mass of 17 kDa.
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of PrPc using Alicon PrioTrap® technology. These PrPc isoforms were later confirmed by Western Blot with anti- PrPc monoclonal antibodies which recognized the same proteins. Compared with the native PrPc enriched by Alicon PrioTrap® , the two proteins captured by histone-Sepharose column in this study were extremely consistent with PrPc , since the molecular mass of these two proteins were between 25 and 37 kDa. Moreover, these two proteins were identified as diglycosylated and monoglycosylated PrPc by further comparison to the electrophoresis results of PrPc in the reference. However, the Alicon PrioTrap® technology not only enriched diglycosylated and monoglycosylated PrPc , also nonglycosylated PrPc which was not appeared in the proteins captured by histone-Sepharose column. This is because Alicon PrioTrap® results from hydrophilic and hydrophobic surface cluster, while histone-Sepharose column focus on the biomolecular interaction between histone and PrPc . In another aspect, the absence of nonglycosylated PrPc by histone-Sepharose column suggests the necessity of glycosyl to the interaction between histone and PrPc . Although the affinity between histones and PrPc is very convincing according to SDS-PAGE analysis (Fig. 3), it is necessary to confirm which one of the histones actually interacted with PrPc . So the PrPc -Sepharose column was made. However, when the histones passed through the PrPc -Sepharose column, those proteins captured by PrPc were not as much as expected although H3 has been determined as the histone fraction to interact with PrPc . Comparing to the chromatography of cow milk casein by histoneSepharose column, the protein peak captured by ligands on the PrPc -Sepharose column was very small (comparing Fig. 4 with Fig. 2). This phenomenon is probably due to that the histone fractions used on the PrPc -Sepharose column were easy to form histone polymers, either dimers or tetramers, which reduced the touching and combination efficiency between PrPc and H3. Naturally when mixed with each other, this kind of combination among histones is believed to be stubborn [1]. But when immobilized onto Sepharose 4B, the histone polymers would not be existed, which explain the high affinity between PrPc and histones on the histone-Sepharose column. PrPsc is known to cause TSE after accumulation in the central nervous system. Prion diseases are neurodegenerative, infectious disorders characterized by the aggregation of a misfolded isoform of the cellular PrPC [14]. In addition, PrPc is also crucial in the role of Cu2+ regulation, blood glucose regulation, neural degeneration and cell signaling and cognitive functioning in human and transcriptional regulation in the nucleus [13,15,16]. Researches have indicated that prion expressed and colonized in central nervous system and secondary lymphoid organs and inflammatory foci [17]. However, the mechanism for the conversion of PrPc to PrPsc , which triggered occurrence of prion disease are still unclear. The results of this study suggest H3 could be the target molecule of PrPC in nuclei due to the existence of the interaction between PrPC and H3, which concluded by performing in vitro albeit. Actually, with the enrichment of the functions of PrPc being found, other molecules associated with PrPc appear to be necessary for the function of PrPc . The research for these additional molecules, like SiRT1 [18], one of the histone deacetylase, are involved in the function of prion and could be that these molecules will provide us with the key for understanding the prion disease.
4. Discussion
5. Conclusions
During chromatography of cow milk casein by histoneSepharose column, two proteins in milk casein were found to have high affinity to histones. According to Franscini [11], fresh milk from health cow individual was enriched for three isoforms
Two affinity chromatographies by using the histone-Sepharose column and the PrPc -Sepharose column respectively have demonstrated that histone H3 is interacted with two isoforms of PrPc with 34 and 30 kDa molecular mass corresponding to diglycosylated and
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monoglycosylated PrPc respectively, which suggest H3 could be the target molecule of PrPC in nuclei. References [1] [2] [3] [4] [5] [6] [7] [8]
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