Anal. Chem. 2006, 78, 3571-3576
Altered Levels of Acute Phase Proteins in the Plasma of Patients with Schizophrenia Yifeng Yang,†,‡,⊥ Chunling Wan,†,‡,⊥ Huafang Li,§ Hui Zhu,†,‡ Yujuan La,†,‡ Zhengrui Xi,†,‡ Yongshuo Chen,†,‡ Lei Jiang,†,‡ Guoyin Feng,§ and Lin He*,†,‡
Bio-X Center, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China, Institute for Nutritional Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China, and Shanghai Institute of Mental Health, 600 South Wan Ping Road, Shanghai, China
Schizophrenia is a relatively common psychiatric syndrome that affects virtually all brain functions. We investigated the plasma proteome of 22 schizophrenia male patients and 20 healthy male controls using two-dimensional gel electrophoresis and mass spectrometry. In total, we have identified 66 protein spots in human plasma and found that seven of them showed altered changes in schizophrenia patients, as compared to healthy controls, which mainly were acute phase proteins (APPs). Among these APPs, haptoglobin r2 chain (p < 0.001), haptoglobin β chain (p < 0.001), r1-antitrypsin (p ) 0.001), and complement factor B precursor (p ) 0.022) showed overexpression in schizophrenia patients, whereas apolipoprotein A-I (p ) 0.034) and transthyretin (p ) 0.035) were found to be significantly decreased in patients. In addition, the expression of apolipoprotein A-IV(p ) 0.018) was significantly up-regulated in schizophrenia patients, as compared to controls. We also found these APP genes, which were differentially expressed in this study, overlap in the schizophrenia susceptibility loci. Our findings further support the hypothesis that the inflammatory response system is linked to the pathophysiology of schizophrenia. Schizophrenia is a relatively common psychiatric syndrome that affects virtually all brain functions. Linkage studies, particularly those examining concordance rates in twin pairs and siblings, strongly implicate genetic susceptibility in the pathogenesis of schizophrenia. The inheritance pattern of schizophrenia suggests multiple susceptibility genes, none of which has any great individual significance, but which interacting with one another and with environmental factors are able to influence susceptibility to the disease.1 Despite major research effort, the etiology of schizophrenia is still unknown. Research into schizophrenia has focused on neurons and, especially, the role of presumed excess dopamine neurotransmis* To whom correspondence should be addressed. Phone and fax: 86-2162822491. E-mail:
[email protected]. † Shanghai Jiao Tong University. ‡ Shanghai Institutes of Biological Sciences. § Shanghai Institute of Mental Health. ⊥ These authors contributed equally to this work. (1) Harrison, P. J.; Owen, M. J. Lancet 2003, 361, 417-419. 10.1021/ac051916x CCC: $33.50 Published on Web 05/04/2006
© 2006 American Chemical Society
sion.2 However, neurotransmitter-based hypotheses have so far led to only moderate success in predicting new pathogenetic findings in the etiology of schizophrenia. Recently, dysregulation of the inflammatory response system has been linked to the pathophysiology of schizophrenia.3,4 In this regard, an important role has been suggested for proinflammatory cytokines produced by activated glial cells, neurons, and immune cells that invade brain tissue. Within the central nervous system (CNS), cytokines stimulate inflammatory processes that may impair blood-brain barrier permeability and may also promote apoptosis of neurons and oligodendrocytes and induce myelin damage.5 These proinflammatory cytokines, such as interleukin (IL)-1, (IL)-6, and tumor necrosis factor R (TNF-R), are found at varied levels in peripheral blood or cerebrospinal fluid of schizophrenic patients.6-9 Proteomics studies the products of genes, the functional translation of the genomic information, and in particular, the characterization of the proteins themselves. Owing to its high throughput and the possibility of showing the whole protein profile in one gel simultaneously, the proteomic technique has recently been widely used in the study of complex diseases. This methodology not only reveals posttranslational modifications, such as phosphorylation, sulfation, and glycosylation, but also gives insights into protein-protein interactions and subcellular localization, thus providing clues as to their function.10 In this study, we used the proteomic technique to search for schizophreniaassociated proteins in human plasma. EXPERIMENTAL SECTION Materials. All the proteomics technology apparatus was sourced from Amersham Biosciences (now part of GE Healthcare, (2) Weinberger, D. R.; McClure, R. K. Arch. Gen. Psychiatry 2002, 59, 553558. (3) Hanson, D. R.; Gottesman, I. I. BMC Med. Genet. 2005, 6, 7. (4) Lin, A.; Kenis, G.; Bignotti, S.; Tura, G. J.; De Jong, R.; Bosmans, E.; Pioli, R.; Altamura, C.; Scharpe, S.; Maes, M. Schizophr. Res. 1998, 32, 9-15. (5) Czlonkowska, A.; Ciesielska, A.; Gromadzka, G.; Kurkowska-Jastrzebska, I. Curr. Pharm. Des. 2005, 11, 1017-1030. (6) Akiyama, K. Schizophr. Res. 1999, 37, 97-106. (7) Boin, F.; Zanardini, R.; Pioli, R.; Altamura, C. A.; Maes, M.; Gennarelli, M. Mol. Psychiatry 2001, 6, 79-82. (8) Erbagci, A. B.; Herken, H.; Koyluoglu, O.; Yilmaz, N.; Tarakcioglu, M. Mediators Inflammation 2001, 10, 109-115. (9) Gilmore, J. H.; Jarskog, L. F.; Vadlamudi, S.; Lauder, J. M. Neuropsychopharmacology 2004, 29, 1221-1229. (10) Van Oostrum, J.; Voshol, H. Am. J. Psychiatry 2002, 159, 208.
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USA), including immobilized pH gradient (IPG) strips, IPG buffer, urea, glycerol, glycine, and dithiothreitol (DTT). Acrylamide and the other reagents for the polyacrylamide gel preparation were obtained from Bio-Rad (U.S.A.). CHAPS, iodoacetamide (IAA), ortho-phosphoric acid, and ammonium sulfate were obtained from Fluka (Switzerland). Thiourea, acetonitrile, trifluoroacetic acid, and R-cyano-4-hydroxycinnamic acid were sourced from Sigma (U.S.A.), and methanol, from Merck (Germany). Subjects. Plasma samples from schizophrenic male patients undergoing clozapine treatment (n ) 22) and healthy male controls (n ) 20) were recruited from the Shanghai Institute of Mental Health. The mean age of patients was 40.2 years (SD ) 9.8), whereas the mean age for the controls was 35.4 years with a SD of 13.3. All subjects were Han Chinese. To exclude the effect of drug treatment, we also collected plasma from 11 male schizophrenia patients (45.2 ( 10.6) before medication and after 2 months of medical treatment (clozapine treatment). Written informed consent was obtained from all participating subjects after the procedure had been fully explained. The diagnosis of schizophrenia was defined according to CCMD-II-R (a counterpart diagnostic criterion of DSM-III-R in China) and DSM-III11 Two-Dimensional Gel Electrophoresis. All the experiments were performed in a blind manner. The plasma samples were stored at -80 °C until assay by 2-D gel electrophoresis. Protein concentration determined by the Coomassie blue method in the plasma from controls and patients was ∼60 mg/mL. After thawing, 13 µL of plasma was add to the 440 µL of IEF sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris pH 8.8, 65 mM 1,4dithioerythritol) with 0.5% IPG buffer and then centrifuged at 49 000 rpm and 4 °C for 30 min. The supernatant sample for IEF was applied on immobilized pH 3-7 nonlinear gradient strips (24 cm). IEF was performed under the following conditions: 100 V (rehydration, 1400 Vhr), 500 V (500 Vhr), 1000 V (1000 Vhr), 4000 V (4000 Vhr) and 8000 V (80 000 Vhr). After IEF, each strip was incubated for 15 min in an equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 2% DTT and for another 15 min in an equilibration buffer containing 2.5% IAA before loading onto SDS-PAGE gels. The second-dimensional separation was performed in 12.5% SDS polyacrylamide gels. The gels (260 × 200 × 1.5 mm) were run at 50 mA per gel for ∼4.5 h. After the second-dimensional separation, gels were fixed with 50% methanol containing 5% ortho-phosphoric acid for 2 h and then exposed overnight to staining solution (containing 8% ammonium sulfate, 5% ortho-phosphoric acid, 0.1% Coomassie blue G-250, and 20% methanol). Excess dye was washed out from the gels with 20% ammonium sulfate until the background was completely clear. Gels were scanned in a UMAX PowerLook III scanner (resolution 300). ImageMaster 2D Platinum software was used for the analysis of the Coomassie-stained gels. The protein spots from different gels were matched, and the percentage of the volume of the spots representing a certain protein was determined in comparison with the total proteins present in the 2-D gel. MALDI-TOF-MS. Mass spectrometer (MS) sample preparation was performed automatically in an Ettan Spot Handling Workstation on the basis of the protocol set out in the Workstation (11) Spitzer, R. L.; Williams, J. B.; Gibbon, M.; First, M. B. Arch. Gen. Psychiatry 1992, 49, 624-629.
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manual. The dried peptide fragments were resuspended with 3 µL of matrix solution consisting of 50% acetonitrile, 0.03% trifluoroacetic acid, and semisaturated R-cyano-4-hydroxycinnamic acid. Samples were analyzed in a time-of-flight mass spectrometer (Ettan MALDI-ToF/Pro, Amersham Biosciences). Peptide matching and protein searching were performed using the Ettan MALDI-ToF/ Pro instrument’s software. The peptide masses were compared with the theoretical peptide masses of all available proteins from all species. Monoisotopic masses were used, and a mass tolerance of 1 Da was allowed. Statistical Analysis. We used an independent-sample t-test (two-tailed) to detect differences in plasma protein expression levels between schizophrenia and the controls. Statistical analysis was performed using the Statistical Package of Social Science (SPSS) for Windows, version 11.0. All the above tests were twotailed, and significance was accepted at p < 0.05. RESULTS AND DISCUSSIONS Protein levels in human plasma were quantified and identified from 2-DE gels using the 2D ImageMaster Platinum software and MALDI-ToF/Pro MS. Approximately 1500 protein spots were detected by the 2D ImageMaster software on the Coomassie bulestained gels. We compared 66 clear plasma protein spots in 2-DE gel maps, which were identified using the PMF (peptide-mass fingerprinting) method of MALDI-ToF/Pro MS (Figure 1). Table 1 lists the Swiss-Prot accession numbers as well as the full names of the 66 protein spots, the molecular mass and pI values, the number of matching peptides, the expectation level, and the protein amino acid sequence coverage by matching peptides. A comparison of the 2DE gels from the patients with those of healthy controls indicated that some chains of spots possibly representing different degrees of APP modification, or degradation products were significantly varied. Among these proteins, haptoglobin (Hp)R2 chain (p < 0.001), Hp-β chain (p < 0.001), R1-antitrypsin (p ) 0.001), and complement factor B precursor (p ) 0.022) were overexpressed, whereas ApoA-I (p ) 0.034) and transthyretin (p ) 0.035) were found to be significantly decreased in patients. In addition, the schizophrenia patients exhibited a significantly high content of ApoA-IV (p ) 0.018) than those in normal controls (Table 2). We can see the representative gels of schizophrenia and the controls in Figure 2. Especially, the expression of R1antitrypsin, Hp-R2 chain, and Hp-β chain displayed remarkably significant differences (p < 0.001) between the schizophrenia patients and the control subjects (see Figure 3). In our study, the frequencies for Hp 1/1, Hp 1/2, and Hp 2/2 were 4.5, 36.4, and 59.1% and 10, 25, and 65% in the schizophrenia patients and the controls, respectively. The distribution of the haptoglobin R-1/R-2 alleles showed no association with schizophrenia (X2 ) 0.000618, df ) 1, P ) 0.98). Figure 4 illustrates how we genotype haptoglobin R-1/R-2 polymorphism with the images obtained from the control and case plasmas. To evaluate the effects of drug treatment, we also investigated the expression levels of those protein spots in plasma between patients who had had no medication and patients who had been receiving medication for two months. We found that none of the investigated protein spots showed significant differences. (Table 2)
Figure 1. 2-DE gel map of human plasma. The gel was separated on a 26- × 20-cm plate and Coomassie blue-stained. The horizontal axis represents the IEF dimension, which stretches from pH 3 to 7 (nonlinear). The vertical axis represents 12.5% SDS-PAGE gel.
DISCUSSION This study demonstrates the use of proteomic analysis of human plasma in investigating the nature of schizophrenia. In the study, we found higher plasma levels of APPs, such as Hp-R2 chain, Hp-β chain, R1-antitrypsin, and complement factor B precursor, in schizophrenia patients. Wong et al.12 observed increased levels of serum R1-antitrypsin, R1-microglobulin, haptoglobin, and ceruloplasmin in two series of schizophrenic patients. Maes et al.13 found that schizophrenic patients had significantly higher plasma haptoglobin, complement C3 and C4, R1-acid glycoprotein, and hemopexin levels than the controls. Our findings are in agreement with these previous reports. These proteins may provide a fast and effective control of inflammatory damage until the subsequent defensive mechanisms can begin to operate.14 Hp, one of the positive acute phase proteins, shows a molecular variation or polymorphism, with three common phenotypes, designated Hp 1-1, Hp 2-1, and Hp 2-2. On the basis of the length of R chain, there are two kinds of Hp-R in the population, Hp-1
and Hp-2. Rudduck et al. reported a significant departure from Hardy-Weinberg equilibrium of the Hp allelic distribution in schizophrenic patients.15 In Mae’s study, the frequency of the Hp-2 gene was significantly higher in schizophrenic patients, as compared with the norm for the population in northwest Italy.16 The frequencies for Hp-1/1, Hp-1/2, and Hp-2/2 were 4.5, 36.4, and 59.1 and 10, 25, and 65% in schizophrenia patients and controls, respectively. The distribution of haptoglobin R1/R2 alleles showed no association with schizophrenia (X2 ) 0.000618, df ) 1, P ) 0.98). Furthermore, in our own study, we found that the three positive APP genes, which were differentially expressed, are localized in or nearby schizophrenia positive linkage chromosomal regions previously identified from genome-wide linkage studies, 16q22.1 for haptoglobin,17 14q32.1 for R1-antitrypsin,18,19 and 6p21.3 for complement factor B precursor20,21 (see Table 3). In the research of Lewis et al., rank-based genome scan meta-analysis (GSMA) was applied to data from 20 schizophrenia genome scans. They
(12) Wong, C. T.; Tsoi, W. F.; Saha, N. Schizophr. Res. 1996, 22, 165-171. (13) Maes, M.; Delange, J.; Ranjan, R.; Meltzer, H. Y.; Desnyder, R.; Cooremans, W.; Scharpe, S. Psychiatry Res. 1997, 66, 1-11. (14) Moshage, H. J. Pathol. 1997, 181 Suppl, 257-266.
(15) Rudduck, C.; Franzen, G.; Frohlander, N.; Lindstrom, L. Hum. Hered. 1985, 35, 65-68. (16) Maes, M.; Delanghe, J.; Bocchio Chiavetto, L.; Bignotti, S.; Tura, G. B.; Pioli, R.; Zanardini, R.; Altamura, C. A. Psychiatry Res. 2001, 104, 1-9.
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Table 1. List of Proteins Identified in Human Plasmaa spot
protein name
Swiss-prot no.
matched peptide
coverage (%)
pI
mass
exp
1-3 4 5 6 7 8-10 11-12 13 14-15 16-17 18 19 20-21 22-24 25 26-30b 31 32-36b 37-38 39-41 42-46 47-49 50 51-53 54 55-58 59-63 64-66
apolipoprotein A-I albumin proapo A-I serum amyloid P-component albumin haptoglobin R2 chain plasma retinol binding protein transthyretin haptoglobin R1 chain apolipoprotein A-IV human albumin ficolin 3 albumin apolipoprotein E transthyretin haptoglobin-β chain apolipoprotein A-IV haptoglobin-β chain R1-microglobulin apolipoprotein J R-1-antitrypsin vitamin D-binding protein antithrombin III R1-B-glycoprotein transferrin R-1-antitrypsin precursor ceruloplasmin complement factor B precursor
P02647 P02768 P02647 P02743 P02768 P00738 P02753 P02766 P00738 P06727 P02768 O75636 P02768 P02649 P02766 P00738 P06727 P00738 P02760 P10909 P01009 P02774 P01008 P04127 P02787 P01009 P00450 P00751
12/15 7/12 9/14 5/10 7/12 5/7 8/13 8/16 8/15 10/13 12/16 6/10 7/12 19/25 12/15 5/9 10/14 7/11 7/10 5/8 7/12 18/25 10/15 7/10 19/24 13/16 13/20 7/12
47.4 43.1 36.9 24.7 43.1 16.9 67.0 51.3 32.6 34.7 11.9 25.2 43.1 56.8 26.9 16.9 34.7 30.2 28.1 16.1 41.6 50.4 27.4 23.2 28.2 25.4 13.6 13.9
5.4 6.2 5.4 6.1 6.2 6.3 5.3 5.3 6.6 5.6 5.7 5.7 6.2 5.6 6.0 6.3 5.6 6.3 6.0 6.3 5.4 5.2 6.3 5.6 7.1 5.5 5.4 6.6
28.94 71.32 28.94 25.37 71.32 41.51 20.94 12.83 38.94 30.76 52.06 32.89 71.32 36.14 53.74 41.51 30.76 41.51 38.98 48.79 74.23 51.21 52.5 54.24 77.06 46.69 115.45 83.27
