An Altered Pattern of Liver Apolipoprotein A-I Isoforms Is Implicated in Male Chronic Hepatitis B Progression Fu Yang,† Yixuan Yin,† Fang Wang,* Ling Zhang, Yuqi Wang, and Shuhan Sun* Department of Medical Genetics, Second Military Medical University, Shanghai, China Received July 06, 2009
Abstract: Chronic hepatitis B (CHB) appears to progress more rapidly in males than in females, and CHB-related hepatic cirrhosis and hepatocellular carcinoma are predominately diseases that tend to occur in men and postmenopausal women. To obtain more insight into the underlying mechanisms of gender disparity of CHB progress, two-dimensional difference gel electrophoresis was employed to compare liver proteome of C57BL/6 and HBV transgenic (HBV-Tg) mice both in male and female groups. We identified 8 differently expressed proteins in male HBV-Tg mice and 12 in female HBV-Tg mice. Apolipoprotein A-I (Apo A-I) was found to be downregulated in male and female HBV-Tg mouse liver. It is more interesting that the pattern of liver Apo A-I isoforms was altered in male HBV-Tg mice but not in female HBVTg mice. Our further results indicated that the basic Apo A-I isoform, based on pI positions from serum 2-dimensional Western blotting, increased in male CHB patient sera but not in female CHB patient sera. Finally, we identified that the oxidative modification Apo A-I mainly reside in basic isoform. This pattern of selectively modified Apo A-I isoforms may be considered as a pathological hallmark that may extend our knowledge of the molecular pathogenesis of CHB progression. Keywords: Chronic hepatitis B • Apolipoprotein A-I • gender disparity • DIGE • HBV transgenic mice
Introduction Chronic hepatitis B (CHB) is a major risk factor for hepatocellular carcinoma (HCC).1 One interesting feature of CHBrelated HCC is the male predominance,2-4 with a male-tofemale ratio of 5-7:1.5 There is similar or even more pronounced gender disparity in hepatitis B virus (HBV) transgenic mouse models.6,7 All these facts indicate that HBV influences the function of male and female host cells in different ways. Many researchers focus on androgen and estrogen. Some researchers’ data provide strong evidence for CHB-related HCC risk among men who have higher levels of androgen signaling * To whom correspondence should be addressed. Professor Shuhan Sun, e-mail,
[email protected];ProfessorFangWang,e-mail,
[email protected], Department of Medical Genetics, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P. R. China. Tel: +86-021-81871053. Fax: +86-021-81871053. † Both authors contributed equally to the work.
134 Journal of Proteome Research 2010, 9, 134–143 Published on Web 09/29/2009
and increased androgen receptor-mediated transcriptional activity.8,9 However, many other studies suggest that the greater progression of hepatic fibrosis and HCC in males may be due to a lower production of estradiol and a lessened response to the action of estradiol.10,11 Willscot E. Naugler and his colleagues think that estrogen-mediated restraint of IL-6 production by Kupffer cells reduces the risk of chemically induced liver carcinogenesis in females.12 In addition to host sex hormone levels, the virus itself may also affect the course of this disease. Hepatitis B virus x protein (HBx), one of the important antigens of HBV, likely augments androgen receptor activity by increasing the phosphorylation of the androgen receptor, which provides an explanation for the male predominance of HCC in HBV-infected individuals.13 But in another study, the authors suggest that hepatitis B virus surface antigen (HBsAg) may be the major risk factor affecting the gender difference in the causes of HCC because estrogen receptor-β was extremely up-regulated in tumor tissues of male HBsAg transgenic mice. It seems likely that overexpression of estrogen receptor-β could cause changes in the levels and in the expression of target genes that affect genomic stability, eventually contributing to the development of HCC.7 Overall, the mechanisms that account for this gender disparity remain unclear. We need some new points of view and methods to study this problem. Recently, many researchers have conducted systematic-omics studies using proteomics, transcriptomics, genomics and metabolomics to elucidate deregulated proteins, mRNAs, genes and metabolites involved in the progression of CHB.14-17 In this study, we focused on 6-8-week-old male and female HBV-transgenic (HBV-Tg) mouse liver, which represents the typical age at which early pathological lesions are detectable.17 Because of the great sensitivity and dynamic range that are afforded by fluorescent tags Cy2, Cy3 and Cy5, two-dimensional difference gel electrophoresis (2-D DIGE) can give much greater accuracy of quantization.18 To obtain more insight into the underlying mechanisms of the gender disparity of CHB progression, 2-D DIGE was employed to compare the liver proteome of C57BL/6 and HBV-Tg mice in males and females. Our objective here was to systematically identify relevant biomarkers and to pinpoint pathways that are differently affected by HBV transgene in male and female mice.
Methods and Materials Animals and Sample Collection. The previously reported HBV-Tg and C57BL/6 mice were used for this study.17,19 All mice used in this experiment received humane care. Mouse 10.1021/pr900593r
2010 American Chemical Society
CHB and Pattern of Apo A-I Isoforms liver sample collection and sample preparation for DIGE analysis were performed as described previously.17 DIGE Experimental Design and Protein Labeling. Considering that there are some protein expression differences between the two sexes,20 we performed DIGE analysis to compare the liver proteomes for HBV-Tg mice and C57BL/6 mice in the same sex (male HBV-Tg vs male C57BL/6 mice, and female HBV-Tg vs female C57BL/6 mice). Proteins in each sample were fluorescently tagged with a set of matched fluorescent dyes according to the manufacturer’s protocol for minimal labeling. In order to eliminate any dye-specific labeling artifacts, two samples of each group were labeled with Cy3 and the other two with Cy5. The pooled-sample internal standard was always labeled with Cy2. In every case, 400 pmol of dye was used for 50 µg of protein. Briefly, labeling was performed for 30 min on ice in darkness, and the reaction was quenched with 1 µL of 10 mM L-lysine for 10 min under the same conditions. DIGE and Imaging of Cy-Labeled Proteins. The eight pairs of Cy3- and Cy5-labeled samples (each containing 50 µg of protein) were combined and mixed with a 50 µg aliquot of the Cy2-labeled pooled standard. The mixtures containing 150 µg of protein were diluted 1:1 with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 4% ampholytes pH 4-7 and 200 mM DTT). Then, the samples were applied to IPG strips (13 cm, pH 4-7, GE Healthcare) using an active rehydration method (30 V for 3 h and 60 V for 8 h). After 11 h of rehydration, the strips were transferred to an Ettan IPGphor 3 Isoelectric Focusing System (GE Healthcare). IEF was performed as follows: 200 V for 2.5 h, 500 V for 1 h, 1000 V for 1 h, and 80 000 V for 5 h. The second dimension was performed using 12% SDS polyacrylamide gel (SDS-PAGE) at 20 mA constant current per gel after equilibration. The differentially labeled coresolved proteins within each gel were imaged using a Typhoon 9400 laser scanner (GE Healthcare). Cy2-, Cy3- and Cy5-labeled images of each gel were acquired at excitation/emission values of 488/520, 523/580 and 633/670 nm, respectively. DIGE Data Analysis. Gel files were analyzed using the Batch Processor, Difference In-gel Analysis (DIA) and Biological Variation Analysis (BVA) modules within DeCyder version 6.5 software (GE Healthcare). Batch Processing was accomplished setting 3500 spots as the upper limit. Results were obtained as abundance ratios for each protein spot (HBV-Tg/C57BL/6 mice). Statistical significance was assessed using the Student’s t test on the logs of the ratios. Spots that showed a statistically significant (p e 0.05) difference were processed for further analysis. Once significantly different spots were determined, 2-D gels with the same parameters as the originals were loaded with unlabeled 300 µg of protein (37.5 µg from each of the 8 samples) and run under the identical conditions as the DIGE gels, except that they were visualized with silver stain. Of the 30 spots previously determined to be the most significantly different (p e 0.05), 28 were detected on the silver stained gels (13 and 15 in male and female groups, respectively). These spots were excised and identified using MALDI-TOF/TOF. In-gel digestion and protein identification were performed as described previously.21 Patient Sample Collection. CHB and chronic hepatitis C (CHC) patient sera were obtained from Changhai Hospital. Patient samples were collected according to a standard operating procedure by a single research nurse from patients who had consented to participation in the study. Ethical consent
technical notes was granted from the Second Military Medical University ethics committee. CHB patient sera were obtained at the time of diagnosis from 35 male and 32 female patients with CHB. And CHC patient sera were obtained from 9 male and 7 female patients. Twenty male and 25 female serum samples, as controls, were obtained from healthy subjects who were negative for HBs antigen and HCV-RNA. Clinical characteristics of these CHB patients, CHC patients and health controls are presented in Supporting Information Tables 1-3. Blood samples (1.0 mL) were collected into anticoagulant-free tubes. Samples were coded and transported on ice to the laboratory. The tubes were centrifuged at 3000 rpm and 4 °C for 10 min within a 2-h time frame. Serum from each patient sample was then collected, aliquoted and stored at -80 °C until analysis. Each serum sample underwent no more than 2 freeze/thaw cycles prior to analysis. Quantitative Real-Time PCR. The same quantity of mouse liver total RNA was reverse-transcribed with M-MLV Transcriptase (Promega) in the presence of oligo-dT primers. The products were subjected to the real-time PCR analysis using a SYBR Green I PCR Master Mix (Tiangen, China) with an ABI PRISM 7000 sequence detection system (Applied Biosystems). PCR primers specific for Apo A-I (sense, 5′-CGTATGGCAGCAAGATG-3′; antisense, 5′-CCAGAAGTCCCGAGTCA-3′) and internal control gene β-actin (sense, 5′-GCACCACACCTTCTACAATGAG-3′; antisense, 5′-ACAGCCTGGATGGCTACGT-3′) were designed with the help of Primer Premier 5.0 (Primer, Canada). 1-D and 2-D Western Blotting. For 1-D Western blotting, liver lysates of HBV-Tg and C57BL/6 mouse livers were prepared in lysis buffer (1% Triton-X-100, 50 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 2 mM Na orthovanadate, 0.2 mM PMSF, 1 mM HEPES, 1 mg/mL leupeptin, and 1 mg/mL aprotinin). Briefly, 80 µg of lysate was resolved by electrophoresis on a 12% SDS-PAGE (120 V) in a running gel buffer containing 25 mM Tris, pH 8.3, 162 mM glycine, and 0.1% SDS. The samples were transferred to a sheet of nitrocellulose membrane for Western blotting. For 2-D Western blotting, firstdimension separation of liver, CHB or CHC patient serum proteins (250 µg, prepared as described before)17 was performed on 13 cm pH 4-7 IPG strips followed by SDS-PAGE using 12% SDS-PAGE gels and then transferred to a sheet of nitrocellulose membrane for Western blotting. The antibody dilutions were 1:500 for a goat polyclonal antibody against Apo A-I (Santa Cruz Biotechnology, Inc.); 1:5000 for a mouse monoclonal antibody against β-actin (Sigma-Aldrich); 1:10 000 for an IRdye800-conjugated rabbit anti-goat IgG (Li-Cor; Lincoln, NE); and 1:5000 for an IRdye800-conjugated goat antimouse IgG (Li-Cor; Lincoln, NE). Antibody binding was detected using an Odyssey infrared scanner (Li-Cor; Lincoln, NE). Determination of Serum 8-OHdG in Male HBV-Tg and C57BL/6 Mice. Levels of mouse sera 8-OHdG were measured with a DNA damage ELISA kit (Stressgen, Japan) following the manufacturer’s protocol. Immunoprecipitation. The immunoprecipitation was performed as described previously.22 The antibodies for Apo A-I were added directly to tissue lysates with NP-40 (Bioyuntian China), and the mixture was incubated on a rotary mixer overnight at 4 °C. The antigen/antibody complexes were precipitated with protein-A-conjugated agarose beads (Santa Cruz). Beads were then centrifuged and washed with PBS three times. Proteins were solubilized in sample buffer (1% TritonJournal of Proteome Research • Vol. 9, No. 1, 2010 135
technical notes X-100, 50 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA) for further analysis. Immunochemical Detection of Protein Carbonyl Levels. The carbonyl levels of Apo A-I were detected by postWestern blot derivatization after immunoprecipitation using OxiSelect Protein Carbonyl Immunoblot Kit (Cell Biolabs). Following the electro-blotting step, the nitrocellulose membranes were equilibrated in 20% methanol for 5 min. Membranes was then washed in 2 N HCl for another 5 min and incubated in 1× DNPH solution for exactly 5 min. The membranes were then washed three times in 2 N HCl and 5 times in 50% methanol (5 min each wash). After postWestern blot derivatization, the 2, 4-dinitrophenyl hydrazone (DNP) adducts of the carbonyls of the proteins were detected on the nitrocellulose paper using a primary rabbit antibody (1:1000) specific for DNP-protein adduct, followed by an IRdye800-conjugated goat anti-rabbit IgG (1:8000) incubation. The result was detected using an Odyssey infrared scanner. Two-D Western blot detection of protein carbonyl levels was performed as described before.23 The same amount of protein (250 µg) was incubated with 4 vol of 20 mM 2,4-dinitrophenyl hydrazine (DNPH) at 24 °C for 20 min. The electrophoresis was carried out in the same way as described above. The proteins were transferred to nitrocellulose paper from the seconddimension electrophoresis gels at 100 V for 2.5 h. The immunochemical detection and measurement of carbonyl levels of total proteins was similar to that for Apo A-I carbonyl level detection described above. Then, the membrane was incubated with antibody dilutions of a goat polyclonal antibody against Apo A-I (1:500) to detect the pattern of Apo A-I as described above. Statistical Analysis. The differences between two groups were compared using Student’s t test. Statistical significance was set at p e 0.05. Statistical analysis for DIGE expression ratios of proteins was evaluated using DeCyder version 6.5 software (GE Healthcare).
Results Analysis of Differentially Expressed Proteins. DIGE was performed for the male (male HBV-Tg vs male C57BL/6) and female (female HBV-Tg vs female C57BL/6) groups. Panels a and b of Figure 1 are representative DIGE images with proteins from male and female groups. In these gels, approximately 1800-2000 spots were detected by the software. Of these spots, only the spots that were successfully identified were marked. Differentially Expressed Protein Identification in the Male HBV-Tg Group. To identify the proteins deregulated in male HBV-Tg mouse liver, DIGE gels of male C57BL/6 and HBV-Tg mice liver were analyzed. Analysis of the resulting images showed 12 protein spots to be statistically different between the two groups. Of these, five protein spots were found to be down-regulated, and seven protein spots were upregulated. By MALDI-TOF/TOF, two of the down-regulated and six of the up-regulated protein spots were identified both from the control and the experimental group. The spots identified are listed in Table 1. Differentially Expressed Proteins Identification in Female HBV-Tg Group. To identify the proteins deregulated in the female HBV-Tg group, DIGE gels of female C57BL/6 and HBV-Tg mice were analyzed. Analysis of the resulting images showed 18 protein spots to be statistically different between the two groups. Of these, 7 spots were found to be up-regulated and 11 spots down-regulated. MALDI-TOF/TOF analysis iden136
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Yang et al.
Figure 1. Differentially expressed protein spots (marked with master numbers) displayed in DIGE images. (a) The significantly regulated protein spots in male HBV-Tg mouse liver. (b) The significantly regulated protein spots in female HBV-Tg mouse liver. Each gel contains 50 µg of protein lysate from C57BL/6 mice, 50 µg from HBV-Tg mice (labeled with Cy3 or Cy5) and 50 µg of pooled internal standard. One representative experiment out of four is shown.
tified 4 up-regulated and 8 down-regulated spots. The spots identified are listed in Table 2. Contradistinction of the Different Proteins in Male and Female Mice. In order to explain the basis for higher susceptibility of male mice, the differentially expressed proteins of mouse livers from both male and female groups were analyzed, as shown in Figure 2. Apolipoprotein A-I (Apo A-I) precursor was found to be up-regulated in male HBV-Tg mice and downregulated in female HBV-Tg mice, so we choose this protein for further analysis. Detection of Mouse Liver Apo A-I by Western Blotting and Real-Time PCR. Since proteomic data indicated that Apo A-I was up-regulated in male HBV-Tg mice and down-regulated in female HBV-Tg mice, we further characterized the Apo A-I expression in male and female groups by Western blotting. As indicated by the protein blot (Figures 3a and 4a), it is clear that the total Apo A-I expression in male and female HBV-Tg mouse liver is down-regulated. To examine whether the downregulation of Apo A-I could be attributed to the transcriptional level, real-time PCR analysis of Apo A-I was carried out. The results indicated that the mRNA expression of Apo A-I was down-regulated in male and female HBV-Tg mice (Figures 3b and 4b) in accordance with their total protein levels. The total Apo A-I was down-regulated in male HBV-Tg mice (Figure 3a), which was not consistent with our proteomic data (Figure 2, Table 1). Apo A-I can be modificated at the posttranslational level and present in several isoforms.24-27 Therefore, we assumed that HBV transgenes might up-regulate some specific Apo A-I isoforms in male HBV-Tg mice. To further explore the different production of HBV-specific Apo A-I isoforms, we utilized 2-D Western blotting to analyze mouse liver proteins. Two-dimensional Western blotting analysis showed the down-regulation of isoform 2 and the up-regulation of isoform 3 in male HBV-Tg mice (Figure 3c,d and Supporting Information Figure 1a). The pattern of Apo A-I isoforms in female HBV- Tg mice was not disturbed (Figure 4c,d, and Supporting Information Figure 1b). Changes of Pattern of Apo A-I Isoforms in CHB Patient Sera Were in Accordance with the Results of Analysis on HBV-Tg Mouse Liver. Using 2-D Western blotting analysis on serum of chronic HBV-infected patients in comparison with
technical notes
CHB and Pattern of Apo A-I Isoforms
Table 1. List of the Differentially Expressed Protein Spots in Male HBV-Tg Mice Identified by MALDI-TOF/TOF spot no.a
protein name
accession no.b
Mr
pI
abundance ratio (means ( SD) c, d
protein score/total ion C.I.% e
399
Predicated: similar to eukaryotic translation initiation factor 4A, isoform 1
gi|73966207
44463.9
5.4
1.46 ( 0.23
154/100
380
Methionine adenosyltransferase I, alpha
gi|19526790
43481
5.51
1.52 ( 0.15
423/100
366
selenium binding protein 1
gi|22164798
52480.4
5.87
1.45 ( 0.16
222/100
286
2-hydroxyacyl-CoA lyase
gi|31560355
63618.9
5.89
0.65 ( 0.08
44/95.877
742
apolipoprotein A-I precursor
gi|109571
30358.5
5.52
1.50 ( 0.12
67/99.984
394
selenophosphate synthetase 2
gi|15011843
47804
5.75
1.75 ( 0.25
86/100
304
Epoxide Hydrolase
gi|563510
62504.6
5.85
1.58 ( 0.14
85/100
297
carboxylesterase 6
gi|19527178
61900.2
5.74
0.26 ( 0.09
99/100
peptide identifiedf
VFDMLNR GIYAYGFEKPSAIQQR GRIDVQQVSLVINYDLPT TACYGHFGR NFDLRPGVIVR SGVLPWLRPDSK FVIGGPQGDAGVTGR YLDEDTVYHLQPSGR TRQVTVQYMQDNGAVIPV TQGRMVLLCGEITSVAMVDY VCHNLDVRLVALEQQSPDIAQC QFYPDLIR LILPGLISSR IFVWDWQR LAGQIFLGGSIVR EEIVYLPCIYR HEIIQTLQMTDGLIPLEIR FSLNHIQDRPSATQGFVGCALS LANHPENLRFLVDFGKEPLGPAL ALQSADVIVLFGAR NRQEAMGAFQEFPQVEAC ARPALEDLR LSPVAEEFR VAPLGAELQESAR FLPGRLLQGTSAETSGGLLIC ENERVELAYQEAMFNMATL SWFRSNYRPFEPQTLGFSPS YQIPALAQAGFR VFRAAFDLDGVLALPSIAGA AAKRPNEVVFLDDFGSNLKP GNWGYLDQVAALR FAPPEDPEPWSGVR LGVLGFFSTGDQHAR LAATEEIVIVAIQYR
a Spot no. is the unique master number which refers to the labels in Figure 1a. b Accession no. is the MASCOT results of MALDI-TOF/TOF searched from the NCBInr database. c Mean, the average protein abundance ratio for paired samples in which one certain spot could be detected. SD means the standard deviation of protein abundance ratios of one certain spot. d The p values of t test p e 0.05. e Protein score (based on combined MS and MS/MS spectra) was from MALDITOF/ TOF identification. The proteins had statistically significant total ion C.I.% greater than 95 (P e 0.05) were considered successfully identified. f The peptides identified by MALDI-TOF/TOF with statistically significant ion score.
healthy controls, we found that the pattern of the Apo A-I isoform was disturbed in male patients (Figure 5) and that it was not disturbed in female patients (Figure 6). All these results were in accordance with analysis on HBV-Tg mouse livers. In order to demonstrate those changes of Apo A-I pattern were specific to CHB; we detected the serum Apo A-I pattern in CHC patients, and we did not find any changes of Apo A-I pattern in male or female CHC patients’ sera (Supporting Information Figure 2). Excessive Oxidative DNA Damage Marker Existed in Serum of Male and Female HBV-Tg Mice. The levels of serum 8-OHdG of female HBV-Tg and C57BL/6 mice were compared in our previous study, in which the results indicated that the average serum 8-OHdG excretion in female HBV-Tg mice was markedly increased compared to that of female C57BL/6.17 In this study, we only determined the serum 8-OHdG in male HBV-Tg and C57BL/6 mice. We found that the levels of serum 8-OHdG were dramatically increased in male HBV-Tg mice (72.35 ( 6.2 ng/mL) compared to male C57BL/6 mice (43.62 ( 5.9 ng/mL) (p ) 0.0002, n ) 6). Altogether, these results indicate that HBV expression evokes oxidative DNA damage in HBV-Tg mice. Apo A-I Carbonyl Level. The most common products of protein oxidation in biological samples are the protein carbonyl
derivation of Pro, Arg, Lys, and Thr. Since the protein-bound carbonyl groups react with DNPH and can be recognized by antibodies to the protein hydrazones, oxidized proteins can be detected by Western blotting. Figure 7a shows the carbonyl levels of Apo A-I were significantly increased by about 60% in male HBV-Tg mice liver compared to controls. Nevertheless, we did not find any changes in female HBV-Tg mice. To further confirm which isoforms contain oxidized Apo A-I, we performed 2-D Western blotting to detect the carbonyl level of Apo A-I. As shown in Figure 7b, the oxidized Apo A-I mainly resides in basic isoform (isoform 3).
Discussion Although a variety of mechanisms have been proposed to explain the male gender susceptibility in CHB progress, none of them adequately clarify the basis for such a difference. Here we found a down-regulation of total Apo A-I expression in male and female HBV-Tg mouse liver. Further analysis revealed a different pattern of Apo A-I isoform expression in male HBVTg mouse liver and CHB patient serum compared to that of controls. Many studies have shown that the level of circulating Apo A-I correlates with CHB and CHB-related HCC.27-29 A low Journal of Proteome Research • Vol. 9, No. 1, 2010 137
technical notes
Yang et al.
Table 2. List of the Differentially Expressed Protein Spots in Female HBV-Tg Mice Identified by MALDI-TOF/TOF spot no.a
protein name
accession no.b
Mr
pI
protein abundance ratio (means score/total c, d ion C.I.% e ( SD)
648 ketohexokinase
gi|31982229
32719.5
5.81
0.25 ( 0.11
228/100
635 3-hydroxyisobutyrate dehydrogenase precursor
gi|21704140
35416.6
8.37
2.63 ( 0.24
46/97.682
619 apolipoprotein E
gi|6753102
35830.3
5.56
1.66 ( 0.36
114/100
612 hypothetical protein LOC70984
gi|19526926
34973.3
5.86
0.34 ( 0.15
252/100
555 alpha-fetoprotein
gi|191765
47194.9
5.47
0.64 ( 0.13
70/99.994
488 phosphoglycerate kinase 1
gi|70778976
44522
8.02
1.42 ( 0.37
54/99.736
422 Suclg2 protein
gi|32484342
40144
5.15
0.64 ( 0.25
170/100
400 Cytochrome b-c1 complex subunit gi|14548301 1
52735.4
5.75
0.40 ( 0.15
259/100
399 thioredoxin domain containing 4
gi|19072792
46823.3
5.09
1.56 ( 0.24
149/100
381 Proteasome (prosome, macropain) gi|33859604 26S subunit, ATPase 2
52833.2
5.97
0.16 ( 0.12
91/100
735 NADH dehydrogenase (ubiquinone) gi|58037117 Fe-S protein 3
30130.5
6.67
0.49 ( 0.23
108/100
787 apolipoprotein A-I precursor
30358.5
5.52
0.48 ( 0.12
60/99.924
gi|109571
peptide identifiedf
WIHIEGR HLGFQSAVEALR GGNASNSCTVLSLLGAR CADAFFMRGSLAPGHVADFLV GCSVCDCVSIVQNVNTSWNQGSSQRGDTP GPDAGTLQVLCLAHWSDAAEFEPGPAPDRALG DFSSVFQYLR KDFSSVFQYLR MARGAVFMDAPVSGGVGA FWDYLR LGPLVEQGR LQAEIFQAR GWFEPIVEDMHR ELEEQLGPVAEETR EPFTFPVR TGELNFVSCMR APLVCLPVFVSK IAEVGGVPYLLPLVNK YQHPDGFKGCALLANLFASEG LGEYGFQNAILVR DVFLGTFLYEYSR LGDVYVNDAFGTAHR ALESPERPFLAILGGAK DTVCIVLLGEPNELVRENACANPAAG ETYLAILMDR ETYLAILMDR VVGELAQQMIGYNLATK IVDVACTFQDVAEKVNPFGETPEGQ SEHVANAGSPSVPIVEGLISFPKQGGVDIE SGMFWLR RIPLAEWESR RIPLAEWESR VYEEDAVPGLTPCR NRALVSHLDGTTPVCEDIG YPFDYYDNQRCPAVAGYGPIEQL TPADCPVIAIDSFR DDTESLEIFQNEVAR VSAKSPIELRHDDCAFLSAFGDL KIEFSLPDLEGR ACLIFFDEIDAIGGAR TMLELINQLDGFDPR FGVTNGLKGIEPPKGVLLFGPP FEIVYNLLSLR VVAEPVELAQEFR ILTDYGFEGHPFR LSPVAEEFR VAPLGAELQESAR
a Spot no. is the unique master number which refers to the labels in Figure 1a. b Accession no. is the MASCOT results of MALDI-TOF/TOF searched from the NCBInr database. c Mean, the average protein abundance ratio for paired samples in which one certain spot could be detected. SD means the standard deviation of protein abundance ratios of one certain spot. d The p values of t test p e 0.05. e Protein score (based on combined MS and MS/MS spectra) was from MALDITOF/ TOF identification. The proteins had statistically significant total ion C.I.% greater than 95 (P e 0.05) were considered successfully identified. f The peptides identified by MALDI-TOF/TOF with statistically significant ion score.
level of this protein implies severe liver cell injury. We found that Apo A-I down-regulation occurs in male and female HBV-Tg mouse liver, which indicated that HBV antigen expression in vivo might cause liver cell injury and disturb liver cell lipid metabolism. In addition to Apo A-I, another protein in which we identified a difference of expression, apolipoprotein E, is also related to liver cell lipid metabolism (Table 2). The derangement of lipid metabolism in host liver cells was also identified by cDNA microarray analysis of HBVTg mouse liver.19 In a previous proteomic and metabolomic study by our group on HBV-Tg mouse liver, we also found that the host cell lipid metabolism was impaired.17 HBx was shown to cause lipid accumulation in hepatic cells, mediated 138
Journal of Proteome Research • Vol. 9, No. 1, 2010
by the activation of SREBP-1 and peroxisome proliferatoractivated receptor γ.30,31 In a recent study, it was proven that HBx increases lipogenesis predominantly through the liver X receptor and its lipogenic downstream target genes.32 Therefore, our study provides more useful information to further understand the mechanisms that underlie HBVmediated derangement of liver cell lipid metabolism. HBV can affect liver cell lipid metabolism in male and female HBV-Tg mice, but there are some interesting differences between the two sexes. Our results indicated that HBV antigen expressionmediated Apo A-I alteration occurred not only in its total protein expression level, but also in the pattern of Apo A-I isoform expression in male HBV-Tg mouse liver (Figure 3 and Supporting
CHB and Pattern of Apo A-I Isoforms
technical notes
Figure 2. A Venn diagram summary of differentially expressed proteins in HBV-Tg mouse liver (v, up-regulation in HBV-Tg mice; V, down-regulation in HBV-Tg mice).
Figure 3. Results of Western blotting analysis. (a) Expression of total Apo A-I in male groups was detected using 1-D Western blotting. Representative blots are shown (left). The volumes of individual bands are shown as the mean ( SD of values derived from all six blots (right). (b) Messenger RNA (mRNA) expression of Apo A-I in male HBV-Tg and C57BL/6 mouse liver, as detected by specific real-time PCR, is in agreement with the expression levels of protein. All data represent mean values ( SD from experiments performed at least seven times. (c) 2-D Western blotting analysis of 250 µg of liver total proteins from male HBV-Tg (up) and C57BL/6 (down) mice. Acidic (isoform 1) and basic (isoform 3) Apo A-I isoforms, based on pI positions from liver 2-D Western blotting, are indicated as shown. (d) Data represent the alteration of the isoforms of Apo A-I in male HBV-Tg mice liver compared to corresponding controls using 2-D Western blot. Error bars indicate mean ( SD for 4 samples in each group.
Information Figure 1a), whereas we did not find any changes in the pattern of Apo A-I isoform expression in female HBVTg mouse liver (Figure 4 and Supporting Information Figure 1b).
Furthermore, we obtained accordant results in male and female CHB patient sera (Figures 5 and 6) but did not in CHC patient sera (Supporting Information Figure 2). Journal of Proteome Research • Vol. 9, No. 1, 2010 139
technical notes
Yang et al.
Figure 4. Results of Western blotting analysis. (a) Expression of total Apo A-I in female groups was detected using 1-D Western blotting. Representative blots are shown (left). The volumes of individual bands are shown as the mean ( SD of values derived from all six blots (right). (b) Messenger RNA (mRNA) expression of Apo A-I in female HBV-Tg and C57BL/6 mouse liver, as detected by specific real-time PCR, is in agreement with the expression levels of protein. All data represent mean values ( SD from experiments performed at least seven times. (c) 2-D Western blot analysis of 250 µg of liver total proteins from female HBV-Tg (up) and C57BL/6 (down) mouse liver. Acidic (isoform 1) and basic (isoform 3) Apo A-I isoforms, based on pI positions from liver 2-D Western blotting, are indicated as shown. (d) Data represent the alteration of the isoforms of Apo A-I in female HBV-Tg mice liver compared to corresponding controls using 2-D Western blot. Error bars indicate mean values ( SD for 4 samples in each group.
Apo A-I is initially synthesized as a preproprotein. It contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The 18-amino acid prepeptide is cleaved during translocation of the protein into the endoplasmic reticulum, and conversion to the mature protein occurs after secretion by removal of the 6 residue propeptide in the blood or lymph. It is believed that Apo A-I can be modificated at the post-translational level.25 Fatty acid acylation and phosphorylation of Apo A-I are the reported methods of post-translational modification,24,33 and they may play an important role in lipoprotein assembly, intracellular transport as well as processing, and lipoprotein secretion. The heterogeneous alteration of the pattern of Apo A-I isoform expression in male HBV-Tg mouse liver reflects different post-translational regulation of different isoforms of Apo A-I, which may be correlated with specific features or functions of the isoforms. It is interesting that a recent study reported the up-regulation in serum of a highly oxidized Apo A-I isoform both in a genetic mouse model of hepatocarcinogenesis and in CHB patients who developed HCC.34 Many groups have revealed the fact that HBV can induce oxidative stress using HBV transgenic mice or HBV DNA transfection models.35,36 Our results also indicated that there was an increase in 8-OHdG in male and female HBV140
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Tg mouse serum. Reactive oxygen species such as hydrogen peroxide, hydroxyl radical and superoxide may damage cellular macromolecules. We should also note that estradiol and its derivatives can reduce lipid peroxide levels in the liver and serum and are strong endogenous antioxidants.37,38 Some findings suggest that estradiol could protect hepatocytes from oxidative damage by NF-kB activation and induction of Bcl-2 expression.39,40 In our results, the oxidized Apo A-I was increased in male HBV-Tg mouse liver (Figure 7a) and the increase in the Apo A-I isoform 3 in liver of male HBV-Tg mice may represent a highly oxidized Apo A-I isoform (Figure 7b). This process of overoxidizing Apo A-I may be blocked or deduced by estradiol or its derivatives in female HBV-Tg mouse liver. The different pattern of Apo A-I isoforms observed in male and female transgenic mouse liver may be the result of an interaction between oxidative stress induced by HBV antigen expression and sex hormones in vivo. The increase in Apo A-I isoform 3 in male HBV-Tg mouse liver and CHB patient serum may provide a new clue to explain the male gender susceptibility to CHB progression. Moreover, Apo A-I is the major protein component of high density lipoprotein in plasma. The protein promotes cholesterol efflux from tissues to the liver for excretion, and it is a cofactor
CHB and Pattern of Apo A-I Isoforms
technical notes
Figure 5. Western blotting analysis of male CHB patient sera. (a) 2-D Western blot analysis of 250 µg serum total proteins from male CHB patients (up) and healthy controls (down). Acidic (isoform 1) and basic (isoform 3) Apo A-I isoforms, based on pI positions from serum 2-D Western blotting, are indicated as shown. (b) Component proportion ratio of serum Apo A-I isoforms in male HBV-infected patients and healthy controls is shown. (c) Data represent the alteration of the isoforms of Apo A-I in male CHB patient sera compared to corresponding controls using 2-D Western blot. Error bars indicate mean values ( SD for thrice.
Figure 6. Western blotting analysis of female CHB patient sera. (a) 2-D Western blot analysis of 250 µg serum total proteins from female CHB patients (up) and healthy controls (down). Acidic (isoform 1) and basic (isoform 3) Apo A-I isoforms, based on pI positions from serum 2-D Western blotting, are indicated as shown. (b) Component proportion ratio of serum Apo A-I isoforms in female CHB patients and healthy controls is shown. (c) Data represent the alteration of the isoforms of Apo A-I in female CHB patient sera compared to corresponding controls using 2-D Western blot. Error bars indicate mean values ( SD for thrice.
for lecithin cholesterolacyltransferase, which is responsible for the formation of most plasma cholesterol esters. Some findings suggest that there is a possible role of chronic hepatitis B infection in the pathogenesis of carotid arteriosclerosis41 or early atherosclerosis.42 The derangement of the normal Apo A-I isoform pattern caused by chronic HBV infection may hamper Apo A-I functions, which may be important to explain these previous findings.
Apo A-I is implicated in many diseases such as infection, metabolic diseases, inflammation, and so forth.43-45 It is possible that the seen effect is only a secondary effect with low specificity for CHB. Although we did not find any changes of Apo A-I in CHC patient sera, we still cannot rule out other diseases. A larger scale clinical study on Apo A-I isoforms in CHB and other diseases patient liver and serum may provide more useful information of CHB progression. Also, we identified Journal of Proteome Research • Vol. 9, No. 1, 2010 141
technical notes
Yang et al. CHC patient sera Apo A1 pattern. Tables of clinical characteristics of CHB patients, CHC patients, and healthy controls. This material is available free of charge via the Internet at http:// pubs.acs.org.
References
Figure 7. Carbonyl levels of Apo A-I. (a) Increased carbonyl level of Apo A-I in male HBV-Tg mice. The production of immunoprecipitation was used for detection of carbonyl levels of Apo A-I (n ) 4). (b) Oxidized Apo A-I mainly reside in basic isoform. The transferred nitrocellulose papers were incubated with antibodies against DNP (left) and then Apo A-I (right) as described in Methods and Materials. The arrowheads indicate the site of basic isoform of Apo A-I.
overoxidized Apo A-I mainly resides in basic isoform (isoform 3), but the detail information of Apo A-I isoforms in structure and function is still an open question. In summary, in this study, we have reported that Apo A-I expression is down-regulated in male and female HBV-Tg mouse liver and that there is a disordered expression pattern of Apo A-I isoforms in male HBV-Tg mouse liver. We also verified this finding in CHB patient serum. Furthermore, we identified overoxidized Apo A-I mainly resides in basic isoform (isoform 3). Although it is not clear at present whether the occurrence of these modifications has a causal role or simply reflects secondary epiphenomena, the selectively modified Apo A-I isoforms may be considered to be a pathological hallmark that could extend our knowledge of the molecular pathogenesis of CHB. Development of antibodies that specifically recognize the isoforms of Apo A-I may prove to be useful, in combination with other traditional markers, as a more efficient way to evaluate the prognosis of CHB. Abbreviation: CHB, chronic hepatitis B; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HBx, Hepatitis B virus x protein; HBV-Tg mice, HBV-trangenic mice; 2-D DIGE, twodimensional difference gel electrophoresis; Apo A-I, apolipoprotein A-I.
Acknowledgment. This work was supported by National Key Basic Research Program of China (Grant No. 30530660) and National Natural Science Foundation of China (Grant No. 30671829, 30671920). We thank Miss Hong Lei and Xiu-hua Zhang for help with DIGE data analysis. Supporting Information Available: Figures of component proportion ratio of Apo A1 isoforms and analysis of 142
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