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Antipsychotic Treatment of Acute Paranoid Schizophrenia Patients with Olanzapine Results in Altered Glycosylation of Serum Glycoproteins Jayne E. Telford,† Jonathan Bones,† Ciara McManus,† Radka Saldova,† Gwen Manning,‡ Margaret Doherty,† F. Markus Leweke,§,∥ Matthias Rothermundt,⊥ Paul C. Guest,# Hassan Rahmoune,# Sabine Bahn,# and Pauline M. Rudd*,† †

NIBRT Dublin-Oxford Glycobiology Laboratory, National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Co., Dublin, Ireland ‡ Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland § Department of Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany ∥ Department of Psychiatry and Psychotherapy, University of Cologne, Cologne, Germany ⊥ Department of Psychiatry, University of Muenster, Muenster, Germany # Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom S Supporting Information *

ABSTRACT: Atypical antipsychotic drugs, such as olanzapine, have been shown to alleviate the positive, negative and, to a lesser degree, the cognitive symptoms of schizophrenia in many patients. However, the detailed mechanisms of action of these drugs have yet to be elucidated. We have carried out the first investigation aimed at evaluating the effects of olanzapine treatment on the glycosylation of serum proteins in schizophrenia patients. Olanzapine treatment resulted in increased levels of a disialylated biantennary glycan and reduced levels of a number of disialylated bi- and triantennary glycans on whole serum glycoproteins. These changes were not observed on a low-abundance serum protein fraction. α1 acid glycoprotein was identified as a carrier of some of the detected altered oligosaccharides. In addition, glycan analysis of haptoglobin, transferrin, and α1 antitrypsin reported similar findings, although these changes did not reach significance. Exoglycosidase digestion analysis showed that olanzapine treatment increased galactosylation and sialylation of whole serum proteins, suggesting increased activity of specific galactosyltransferases and increased availability of galactose residues for sialylation. Taken together, these findings indicate that olanzapine treatment results in altered glycosylation of serum proteins. KEYWORDS: glycosylation, schizophrenia, olanzapine, α1 acid glycoprotein

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INTRODUCTION Schizophrenia is a complex debilitating neuropsychiatric disorder that affects 1% of the population and is manifested by positive symptoms such as hallucinations and delusions, negative symptoms including apathy and loss of motivation, and cognitive symptoms such as memory impairment. The pathophysiology of the disease is not understood. However, aberrant dopaminergic and glutamatergic neurotransmission are thought to be associated with the disease, along with neurodevelopmental alterations. To gain a deeper understanding of the physiology of the disease and to identify disease biomarkers, numerous proteomic profiling studies of serum, plasma, cerebrospinal fluid (CSF) and urine from © 2012 American Chemical Society

schizophrenic patients and many disease-specific proteomic differences have been performed. Decreased apolipoprotein A1 levels were found in serum, brain, CSF and liver from schizophrenic patients.1 Levin et al. reported that the concentrations of 10 proteins, including transferrin, α2-HS glycoprotein and a number of apolipoproteins, were altered in serum from schizophrenic patients compared to healthy controls.2 Other studies have shown that acute phase reactants including haptoglobin, hemopexin, fibrinogen, complement component 3 and 4, α1 acid glycoprotein (AGP) and α1 Received: March 6, 2012 Published: May 17, 2012 3743

dx.doi.org/10.1021/pr300218h | J. Proteome Res. 2012, 11, 3743−3752

Journal of Proteome Research

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antitrypsin were increased in plasma from schizophrenic patients, as determined by laser nephelometry3 and matrixassisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry analysis.4 The concentration of acute phase proteins increases dramatically in inflammation, and there are many reports of increased inflammation in schizophrenia.5−8 Studies focusing on alterations in post-translational modifications, such as glycosylation, have also been reported in schizophrenia.9−12 Glycosylation is the most abundant posttranslational modification and plays crucial roles in cellular processes, such as cell signaling, cellular recognition processes and protein folding. Oligosaccharides, synthesized in the rough endoplasmic reticulum and in the Golgi apparatus through the action of glycosidases and glycosyltransferases, are highly heterogeneous structures that can have variable lengths and complexities. Glycan changes, including altered levels of A3F1G3S3 and A4G4LacS4, have previously been found in serum samples from schizophrenia patients when compared to healthy subjects and may therefore have potential as biomarkers for the disease.9 However, there are no studies to date that investigate the effects of antipsychotic drug treatment on the glycosylation of serum proteins in schizophrenia. A recent study from our laboratory highlighted the importance of considering the effects of medication on glycosylation with glycan changes reported as a result of treatment with nonsteroidal antiinflammatory drugs and oral contraceptives.13 Atypical antipsychotics such as olanzapine are widely used in the treatment of schizophrenia. Olanzapine has been shown to have beneficial effects on the positive, negative and, to a lesser extent, the cognitive symptoms of schizophrenia in many patients.14−16 Previous studies suggest that olanzapine has enhanced therapeutic properties when compared with other antipsychotic treatments. Olanzapine was more effective at reducing positive and negative symptoms, depression and agitation compared to typical antipsychotics such as haloperidol.17 A more recent study showed that olanzapine treatment resulted in greater improvements in the general mental state of patients compared with other atypical drugs such aripiprazole, risperidone, ziprasidone and quetiapine.18 Alvarez et al. found that olanzapine reduced negative symptoms more effectively than risperidone in schizophrenia patients.19 The mode of action of olanzapine is not completely understood. However, it is known to bind with high affinity to serotonin, dopamine, histamine and adrenergic α1 receptors.20,21 After dosing, olanzapine is extensively distributed throughout the body and shows 93% binding to plasma proteins, primarily albumin and AGP.22 Studies have been carried out on the effects of olanzapine on gene expression23,24 and on protein levels,25,26 but the effects of olanzapine on post-translational modifications such as glycosylation have not been investigated to date. In the present study, we have investigated the glycosylation profile of glycoproteins in serum from acute paranoid schizophrenia patients before and after 6 weeks of treatment with olanzapine by using high-throughput hydrophilic interaction chromatography (HILIC)-based glycoanalytical technology. Subsequently, we attempted to identify the proteins bearing the identified changes. In addition, we used exoglycosidase digestion analyses and weak anion exchange chromatography to determine which stages of the glycan processing pathway were potentially affected.

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EXPERIMENTAL SECTION

Sample Selection and Collection

Blood samples were collected from acute paranoid schizophrenia patients (pretreatment) and after 6 weeks of treatment with olanzapine (post-treatment) at the Department of Psychiatry and Psychotherapy of the University of Cologne under protocols for sample collection and analysis approved by the Ethics Committee of the University of Cologne. Informed consent was provided in writing by all participants, and clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Diagnosis was determined according to the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) and the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD 10) by experienced psychiatrists using the Structured Clinical Interview for DSMIV (SCID). The subjects included 23 antipsychotic-naive patients diagnosed with acute paranoid schizophrenia (DSMIV 295.3). The group consisted of 7 females and 16 males with an age of 30.3 ± 10.5 years and body mass index (BMI) of 22.6 ± 3.1 kg/m2, and included 6 smokers and 17 nonsmokers. Blood samples were obtained by venous puncture and collected into S-Monovette 7.5 mL serum tubes (Sarstedt; Numbrecht, Germany). The blood was kept at room temperature for 2 h to allow a consistent time for clot formation, followed by centrifugation at 4000g for 5 min to pellet clotted material and other cellular debris. The supernatants (sera) were collected and stored at −80 °C in Low Binding Eppendorf tubes (Hamburg, Germany) until analysis. Serum Depletion with MARS-14 Column

Serum samples were depleted of the 14 most abundant proteins (albumin, IgG, transferrin, fibrinogen, IgA, haptoglobin, α1 antitrypsin, α2 macroglobulin, IgM, apolipoprotein AI, AGP, complement C3, apolipoprotein AII and transthyretin) using the Multiple Affinity Removal System (MARS) Human 14 (Agilent Technologies; Wilmington, DE, USA). Samples (40 μL) were diluted with 120 μL of MARS Buffer A and centrifuged at 16000g for 1 min through a 0.22 μm spin filter. The filtered samples were injected onto the MARS-14 column according to the manufacturer’s instructions using a 1200 LC system (Agilent Technologies). The unbound (depleted) serum fractions were collected and stored at −80 °C. Preparation and Analysis of N-Glycans

N-Glycans were released, derivatized and analyzed using highthroughput HILIC fluorescence-based glycoanalytical technology as described previously by Royle et al.27 Glycan structures were assigned using GlycoBase (http://glycobase.nibrt.ie). As required, specific exoglycosidase digestions were used in the assignment of glycan structures and in the further investigation of the effects of olanzapine on glycosylation as outlined in Royle et al.27 Arthrobacter ureafaciens sialidase (ABS), bovine testes β-galactosidase (BTG), bovine kidney α-fucosidase (BKF), almond meal α-fucosidase (AMF), β-N-acetylglucosaminidase cloned from Streptococcus pneumonia, expressed in Escherichia coli (GUH) were used in these experiments. The glycan nomenclature applied is illustrated in Figure 1.28 Isolation of Proteins by Two-Dimensional Gel Electrophoresis (2DE)

The protein concentrations of whole serum samples from 6 patients before and after treatment with olanzapine were determined using the method of Bradford29 with bovine serum 3744



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MALDI-TOF Mass Spectrometry Identification of Isolated Proteins

Following removal of glycans, the gel pieces were prepared for MALDI-TOF mass spectrometry (MS) analysis. In-gel digestion was carried out overnight by incubation with trypsin (Promega; Madison, WI, USA) at 37 °C. The supernatants were harvested and further extraction was carried out by washing the gel pieces with 70% acetonitrile containing 4% formic acid. The subsequent supernatants were added to the previously collected supernatants, dried down and resuspended in 2 μL of 1% formic acid. The samples were analyzed using a 4800 plus MALDI TOF/TOF Analyzer (Applied Biosystems; Foster City, CA, USA). Peptide masses were acquired over a range from 800 to 4000 m/z, with a focus mass of 2000 m/z. MS spectra were summed from 2500 shots using an Nd:YAG laser (355 nm, 200 Hz). Automated plate calibration was performed using five peptide standards (masses 900−2400 m/ z; Applied Biosystems) to update the instrument default mass calibration, which was applied to all MS and MS/MS spectra. A maximum of 12 precursors per sample well with a signal-tonoise ratio of >20 were selected automatically for subsequent fragmentation by collision induced dissociation, and MS/MS spectra were summed from 4000 laser shots. Spectra were processed and analyzed by the Global Protein Server Workstation (Version 3.6, Applied Biosystems) and searched using MASCOT (Matrix Science; London, U.K.) against the UniProt knowledgebase with the human taxonomic filter specified (release 2011_01; http://ftp.ebi.ac.uk/pub/ databases). The search parameters included a maximum of one missed cleavage, a maximum of two variable posttranslational modifications (oxidation of methionines and deamidation of asparagines), a precursor tolerance of 100

Figure 1. The glycan symbols, linkages, and linkage position information. The Oxford nomenclature was used in this paper to represent glycan structures.

albumin as a reference standard. 2DE was performed as described in Bones et al.30 using pH 3−7 nonlinear (NL) for isoelectric focusing followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels. The protein load was 1 mg in all instances. Protein spots were visualized by Coomassie Brilliant Blue staining, and the spots were manually excised from the gels. The migration positions of the proteins of interest were cross-validated with the SwissProt 2D PAGE Human plasma reference gel to confirm protein identifications (http://world-2dpage.expasy.org/swiss-2dpage/ ). Glycans were released from proteins in the excised gel pieces with PNGase F and were prepared for glycan analysis by HILIC-fluorescence as described above. Chromatographic peaks were assigned with the same numbers as those in the whole serum profile allowing comparisons to be made.

Figure 2. Olanzapine treatment significantly altered the relative percentage areas of HILIC peaks 16 and 20 from whole serum proteins but did not alter the relative percentage areas of peaks in the HILIC profile of low-abundance serum proteins in schizophrenia samples. Typical HILIC chromatograms for (a) whole serum N-glycans and the assigned peaks (1−24) and (b) low-abundance serum protein N-glycans and the assigned peaks. The relative percentage areas of peaks pretreatment and post-treatment are shown in black and gray, respectively, for whole serum (c) and for low-abundance serum protein fraction (d). The glycans contained in the peaks 16 and 20 are illustrated. GLM repeated measures ANOVA with posthoc paired sample t-tests were performed. Significance levels of posthoc tests were corrected for multiple comparisons (α/n), with p-values ≤ 0.0022 found to be significant and shown with ***. Samples were analyzed in triplicate, and results are expressed as mean ± SE (error bars). 3745



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contain three disialylated bi- and triantennary glycans, A3G3S2, A2F1G2S2 and A3BG3S2. The same analysis was also performed on the low-abundance serum protein fraction of serum, obtained after the depletion step, and peaks were assigned with the equivalent numbers as those in whole serum. This showed that olanzapine had no effect on the relative size of any of the 15 peaks detected, as all p-values were higher than 0.0022 (Figure 2). However, peaks 16 and 20 did show the same directional changes as those seen in whole serum (peak 16 was increased and peak 20 was decreased after treatment). Characterization of the glycans in each of the peaks showed that peak 16 contained the most abundant glycan in serum, A2G2S2, and peak 20 contained A3G3S2, A2F1G2S2 and A3BG3S2 (Supporting Information Table S1).

ppm, MS/MS tolerance of 0.25 Da and peptide charge of +1. Peptides were filtered for subsequent identification and analysis with a confidence interval of 95% for top ranked proteins using the Mowse scoring algorithm. ELISA Determination of AGP Concentration

An AssayMax quantitative competitive enzyme-linked immunosorbent assay (ELISA) kit for human AGP (EG5001-1) was purchased from AssayPro (St Charles, MO, USA). Serial dilutions of AGP standard were carried out to generate a standard curve. Bradford protein assays29 were used to determine protein concentrations of serum samples, and all samples were adjusted to 70 mg/mL using phosphate buffered saline. Serum samples were diluted 1:1000 with the mix diluent, and all samples and standards were incubated for 2 h with biotinylated AGP on a 96-well plate, precoated with polyclonal antibody specific for human AGP. The wells were washed and incubated subsequently with streptavidin-peroxidase conjugate for 30 min. The wells were washed again, and chromogen substrate was added, followed by addition of the stop solution. The absorbance values were determined on a Perkin-Elmer Victor X3 multilabel plate reader at 450 nm. The results were analyzed by regression analysis using four-parameter logistic curve-fit.

Identification of Serum Proteins Associated with Olanzapine-Induced Glycan Changes

The subsequent phase of the study was aimed at identification of serum proteins that were associated with the olanzapineinduced glycan changes. 2DE of whole serum from 6 patients before and after treatment was performed as described in the Experimental Section. These 6 samples were chosen as they showed the highest increase in A2G2S2 in whole serum after the olanzapine treatment. Protein spots corresponding to 4 glycoproteins (AGP, haptoglobin β-chain, transferrin and α1 antitrypsin) were isolated using a Gilson p1000 pipet and tips and the SwissProt 2D PAGE Human plasma image as a reference guide (Figure 3). AGP was selected because it has been shown that olanzapine can bind to this protein.22 In addition, AGP and the other selected proteins (haptoglobin βchain, α1 antitrypsin, transferrin and α1 antitrypsin) are known to carry A2G2S2, and some also carry A3G3S2, A2F1G2S2 and A3BG3S2.30−33 MALDI-TOF analysis was performed on the isolated protein spots to confirm the protein identifications. The resulting data showed that spot 1 contained AGP, spots 2− 6 contained haptoglobin β-chain, spots 7−9 contained α1 antitrypsin and spot 10 contained transferrin (Table 1). The isolated proteins were subjected to glycan analysis, and examples of the HILIC-fluorescence profiles are shown in Figure 3. Peak numbers were assigned as above for whole serum to allow direct comparisons. The low intensity HILICfluorescence profiles from analysis of spots 2 and 9 from some samples resulted in numbers that were too small for statistical analyses. However, analysis of the AGP spots resulted in a significantly different post-treatment profile (Figure 4). The results show that peak 16 increased from 22.4 ± 1.2% to 28.1 ± 1.2% (p = 0.027) and peak 24 decreased from 11.6 ± 0.6% to 9.2 ± 0.5% (p = 0.007) with treatment. Peak 20 was not significantly altered as seen above for the whole serum analysis, although the same directional change was observed with a decrease in peak area. Analysis of the glycan structures associated with AGP showed that peak 16 contained A2G2S2 as above, peak 20 contained A3G3S2, and peak 24 contained multiple tetra-antennary structures, including A4G4S4, A4F1G4S4 and A4F2G4S4 (Supporting Information Table S1). Even though olanzapine did not significantly alter the glycosylation of haptoglobin, α1 antitrypsin and transferrin, the same directional changes were observed for peak 16 (increased) and peak 20 (decreased) as found in the case of whole serum proteins (Figures 2 and 4).

Statistical Analysis

General linear model (GLM), repeated measures ANOVA tests were carried out to determine if there were significant differences between the glycosylation profiles of serum from patients before and after olanzapine treatment using SPSS statistical analysis software version 18 (Dublin, Ireland). When significant interactions were found, posthoc paired sample ttests were performed to identify peaks that were altered significantly after treatment. In experiments that tested the effects of olanzapine treatment on glycosylation of whole serum proteins and low-abundance serum proteins (n = 23), the significance levels of posthoc tests were corrected for multiple comparisons (α/n; 0.05/23 = 0.0022). Therefore, only p-values ≤ 0.0022 were considered to show significant differences in these studies. The significance levels of the paired sample t-tests on glycosylation of isolated high-abundance proteins and on the levels of branching, galactosylation and sialylation in whole serum were not corrected for multiple comparisons, as only 6 patients were considered in these tests. A paired sample t-test was carried out on the AGP ELISA results.

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RESULTS

Effects of Olanzapine Treatment on Serum Glycome

HILIC fluorescence was used to analyze released N-glycan pools from proteins in whole serum obtained from patients before and after treatment with olanzapine (Figure 2). This figure shows that two glycan peaks were altered significantly in response to olanzapine treatment, on the basis of average relative peak area measurements (n = 23). Peak 16 was increased from 32.7 ± 0.6% to 35.1 ± 0.7% (increase of 7.3%; p = 0.0018), and peak 20 was decreased from 2.17 ± 0.07% to 1.98 ± 0.06% (decrease of 8.8%; p = 0.0016). Glycan assignments were made with the aid of exoglycosidase digestions as described by Royle et al.27 and comparisons with GlycoBase (Supporting Information Table S1). The glycan identified in peak 16 was A2G2S2, a disialylated digalactosylated biantennary structure that is the most abundant glycan in human serum. Peak 20 was found to 3746



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Table 1. Identification of the Peptides in Excised Gel Spots 1 to 10 by MALDI-TOF/TOF Mass Spectrometry Analysis spot

identification

1 2

AGP haptoglobin βchain haptoglobin βchain haptoglobin βchain haptoglobin βchain haptoglobin βchain α1 antitrypsin α1 antitrypsin α1 antitrypsin transferrin

3 4 5 6 7 8 9 10 a

accession number

protein ion score

sequence coveragea (%)

peptides matcheda

P02763 P00738

122 124

23 6

3 2

P00738

407

20

8

P00738

459

20

8

P00738

380

15

6

P00738

442

17

7

P01009 P01009 P01009 P02787

96 482 594 491

9 27 31 21

3 8 10 12

Combined results from identical gel spots from up to 12 samples.

level of 0.50 ± 0.02 mg/mL and a post-treatment level of 0.44 ± 0.03 mg/mL (p = 0.069). Effects of Olanzapine Treatment on Branching, Galactosylation and Sialylation of Whole Serum Glycans

Glycans are synthesized by the actions of a number of glycosyltransferases and glycosidases in the endoplasmic reticulum and the Golgi apparatus, which bring about removal and addition of monosaccharides to the trimannosyl core in a stepwise manner. To investigate the olanzapine-induced glycan alterations found in whole serum, experiments were carried out on specific sections of the glycan processing pathway to determine if the identified changes were due to altered branching, galactosylation and/or sialylation. Analysis of samples after specific exoglycosidase digestions examined the degree of glycan branching and the levels of galactosylation. These analyses showed that the olanzapine treatment did not affect the levels of mono- (A1), di- (A2), tri- (A3) and tetraantennary (A4) glycans, although significant alterations were identified in the degree of galactosylation (Figure 5). Specifically, the levels of non- (G0) and monogalactosylated (G1) glycans were significantly lower after treatment (p = 0.025 and 0.027, respectively), whereas the levels of digalactosylated (G2) glycans were significantly increased (p = 0.022). G0 and G1 forms decreased from 7.5 ± 1.1% to 5.6 ± 0.7% and from 20.9 ± 1.6% to 17.6 ± 1.1%, respectively, and G2 increased from 64.9 ± 1.8% to 68.7 ± 1.4%. Similarly, the levels of sialylated glycans were affected by olanzapine treatment with monosialylated (S1) glycans decreased from 23.5 ± 1.5% to 20.3 ± 1.0% (p = 0.038), and disialylated (S2) glycans increased from 60.7 ± 0.6% to 63.5 ± 0.5% (p = 0.040) (Figure 5).

Figure 3. Isolation of high-abundance serum proteins by 2DE and typical HILIC profiles. (a) 2DE separation of whole serum from a drug-naive schizophrenia patient. High-abundance serum glycoproteins, AGP (spot 1), haptoglobin β-chain (spots 2−6), α1 antitrypsin (spots 7−9), and transferrin (spot 10), were excised from the gels and were prepared for N-glycan analysis and for identification of proteins by MALDI-TOF/TOF mass spectrometry. (b) Typical HILIC profiles for AGP, haptoglobin β-chain, α1 antitrypsin and transferrin, with the assigned peak numbers shown for AGP and relevant peaks illustrated for haptoglobin β-chain, α1 antitrypsin, and transferrin.

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DISCUSSION This is the first study to investigate the effects of antipsychotic treatment on serum glycosylation profiles in schizophrenia patients. Serum samples were obtained from paranoid schizophrenia patients before and after 6 weeks of treatment with olanzapine. This protocol served to minimize differences in factors such as age, smoking, BMI and cholesterol levels, which have been reported previously to affect the glycosylation of proteins.34,35 The pharmacokinetics of olanzapine show linearity and are dose-proportional, with an average half-life of

Effects of Olanzapine Treatment on the Concentration of AGP in Serum Samples

To determine whether AGP levels were altered after olanzapine treatment, we performed an ELISA analysis to determine the concentrations of this protein in the serum samples from schizophrenia patients before and after treatment with olanzapine. This showed that the concentration of AGP was not significantly altered by the treatment with a pretreatment 3747



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Figure 4. Effects of olanzapine treatment on glycosylation of AGP and on disease-related peaks associated with haptoglobin, transferrin, and α1 antitrypsin. (a) Olanzapine treatment significantly altered the relative percentage areas of HILIC peaks 16 and 24 from serum AGP from schizophrenia patients. (b) There is a trend of olanzapineinduced increases in the relative percentage areas of peak 16 (A2G2S2) on haptoglobin, α1 antitrypsin, and transferrin, but the increases were not found to be significant. (c) There is a trend of olanzapine-induced reductions in the relative percentage areas of peak 20 (A3G3S2, A2F1G2S2, A3BG3S2) on haptoglobin and α1 antitrypsin, but the decreases were not found to be significant. The relative percentage areas of peaks pretreatment and post-treatment are shown in black and gray, respectively. GLM repeated measures ANOVA with posthoc paired sample t-tests were performed. Results that were significantly different post-treatment are shown with * for p ≤ 0.05 and ** for p ≤ 0.01. Results are expressed as mean ± SE (error bars).

Figure 5. Olanzapine treatment alters the galactosylation and sialylation of whole serum proteins but does not affect their levels of branching. (a) Exoglycosidase digestions (ABS, BTG, AMF and BKF) were carried out on samples from 6 patients, and the relative percentage areas of peaks containing mono- (A1), di- (A2), tri- (A3), and tetra-antennary (A4) glycans pretreatment and post-treatment are shown in black and gray, respectively. (b) Exoglycosidase digestions (ABS, AMF and BKF) were carried out on serum samples taken from 6 patients, and the relative percentage areas of peaks containing non(G0), mono- (G1), di- (G2), tri- (G3), and tetragalactosylated (G4) glycans pretreatment and post-treatment are shown in black and gray, respectively. (c) Weak anion exchange chromatography was carried out on serum samples from 6 patients, and the relative percentage areas of peaks containing mono- (S1), di- (S2), tri- (S3), and tetrasialylated (S4) glycans were determined and are shown in black and gray, respectively. GLM repeated measures ANOVA with posthoc paired sample t-tests were performed. Results that were significantly different post-treatment are shown with * for p ≤ 0.05. Results are expressed as mean ± SE (error bars).

33 h and an average clearance rate of 26 L/h.22 Administration of olanzapine once daily is known to result in a steady state concentration after approximately one week.36 3748



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glycosylation state, with the presence of branched glycans leading to more effective inhibition.44 Also, the nonsialylated form of AGP has been shown to have greater platelet aggregation inhibitory capacity than other forms.45 AGP has the ability to bind to several basic and neutral drugs,46 such as olanzapine. The drug interaction sites on AGP overlap spatially, and amino acids such as tryptophan, tyrosine, lysine and histidine are crucial for binding.47 Previous studies have suggested that the glycans carried on AGP are not involved in the binding of drugs to the protein, with reports showing that the removal of glycan structures such as sialic acids from AGP did not reduce the binding of drugs to this protein.48−50 This is also supported by the finding that a recombinant form of AGP containing high mannose glycans had similar binding capacity as human AGP, even though the glycans are different.51 These findings suggest that it is unlikely that the binding of olanzapine to AGP is responsible for the olanzapine-induced glycan alterations found in the current study. In addition, the changes were not likely to be due to changes in overall AGP levels, as we found that this protein was not significantly altered by the olanzapine treatment. Instead, these findings suggest that the observed changes were due to alterations in the glycan processing pathway. To confirm this, we examined the effects of olanzapine on specific stages of glycan processing, including branching, galactosylation and sialylation. Extension of oligosaccharide chains takes place in the Golgi apparatus where glycosyltransferases facilitate transfer of mononucleotide sugars onto carbohydrate backbone structures in a specific order. The existing carbohydrate structure can affect and determine the position and addition of further mononucleotides to the chain. Exoglycosidase digestion analyses showed that olanzapine did not alter the degree of branching (A1−A4) of the whole serum glycans, suggesting that there was no effect on the Nacetylglucosaminyltransferase enzymes that add N-acetylglucosamine (GlcNAc) residues to core mannose residues or on the expression of the genes encoding these enzymes. However, the analyses identified alterations in galactosylation of glycans. The levels of G2 glycans were increased post-treatment, whereas the levels of G0 and G1 glycans were decreased. This increase in digalactosylated structures could occur as a result of increased expression of genes that encode for specific galactosyltransferases, or it could result from increased activation of the enzymes. In addition, other factors that can affect the level of galactosylation include the availability of the UDP-α-galactose substrate, the availability of cofactors required by the enzymes, and the residence times of proteins in the Golgi apparatus and endoplasmic reticulum where glycosylation processing takes place. A previous study showed that olanzapine treatment increased the expression of the B4GALT1 gene in the liver of schizophrenia patients. 23 This gene encodes for β1,4galactosyltransferase I (Gal-T1), which adds galactose from UDP-α-galactose to a GlcNAc residue at the nonreducing end of a carbohydrate chain in a β1−4-linkage. Gal-T1 is a type II transmembrane protein that is localized to the trans-Golgi network and has a secondary function of converting αlactalbumin and glucose to lactose in the presence of αlactalbumin.52 Increased expression and activity of this enzyme could lead to increased levels of galactosylation of GlcNAc residues in glycans. Sialylation occurs on galactose residues at the nonreducing ends of glycans. Weak anion exchange chromatography was used in the current study to determine the levels of mono-

The olanzapine treatment in the current study brought about alterations in the glycosylation profile of whole serum glycoproteins. The levels of the disialylated biantennary glycan, A2G2S2, were significantly increased, whereas other disialylated bi- and triantennary oligosaccharides, A3G3S2, A2F1G2S2 and A3BG3S2, were significantly decreased post-treatment. A2G2S2 is the most abundant glycan in human serum and is expressed on the surface of many glycoproteins, including a number of abundant serum proteins such as haptoglobin, transferrin, AGP, α1 antitrypsin and IgG. A2F1G2S2 contains the sialyl lewis X (sLex) epitope, which is a ligand for the selectins (E-selectin, P-selectin and L-selectin) and is involved in the process of leukocyte extravasation and possibly in metastasis, making it an interesting target in cancer research.30,31,37 In a previous study, we showed that the levels of another sLex-containing glycan, A3F1G3S3, were altered in serum from schizophrenia patients compared to healthy controls.9 However, sLex-carrying glycans did not appear to be altered by the olanzapine treatment in the current study. In the current study, we identified alterations on whole serum glycoproteins but did not identify any significant alterations in glycosylation profiles through analysis of an immunodepleted serum fraction, comprising proteins of lower abundance. This suggested that olanzapine treatment only affects glycosylation of the most abundant serum proteins. Alternatively, it is also possible that significant changes in the low-abundance fraction were not detected because of limitations in the sensitivity of the method. This is consistent with the finding that increased levels of A2G2S2 and decreased A3G3S2, A2F1G2S2 and A3BG3S2 were observed in the lowabundance protein fraction, although these alterations did reach the level of significance. Four high-abundance serum proteins were purified from 2DE gels to determine whether any of these could account for the observed changes in the glycosylation profile in whole serum. This included selection of AGP based on studies that have shown that olanzapine can bind to this protein.22 In addition, AGP and the other selected proteins (haptoglobin β-chain, transferrin and α1 antitrypsin) are known to carry A2G2S2, and some also carry A3G3S2, A2F1G2S2 and A3BG3S2.30−33 The IPG strips used for the 2DE gels had a pH range from 3 to 7, which was chosen to maximize detection of targeted sialylated glycoproteins AGP, haptoglobin β-chain, transferrin and α1 antitrypsin. This analysis revealed that AGP carried significantly altered glycans, with increased levels of A2G2S2 and reduced levels of tetra-antennary glycans. However, nonsignificant changes were also observed in the levels of other glycan structures on haptoglobin β-chain, transferrin and α1 antitrypsin. These findings suggested that the olanzapineinduced glycan alterations result from modification of specific aspects of the glycan processing machinery (e.g., branching, galactosylation and/or sialylation) rather than solely as a result of altered glycans on AGP. AGP is a 41−43 kDa acute phase protein that has 5 sites of N-glycosylation,38 and the oligosaccharides account for 40% of the molecular weight. Alterations in microheterogeneity of AGP glycosylation can occur in diseases such as in pancreatic cancer,39 ovarian cancer,32 inflammation40−42 and diabetes,41 and can result in modification of certain immunomodulatory and binding properties of the protein.43 Such alterations in AGP glycosylation can have downstream effects on biological pathways. For example, it has been shown that the ability of AGP to inhibit lymphocyte proliferation is affected by its 3749



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genicity, their involvement in cell−cell interactions and functions and/or their clearance rate. A previous study showed that the glycans on the surface of AGP regulated the clearance rate of the protein with high mannose structures resulting in the fastest rate of clearance and complex-type glycans showing the slowest clearance rate.58 Previous studies have shown that the levels of A2G2S2 on AGP are increased in acute inflammation and the levels of triand tetra-antennary glycans are increased in chronic inflammation.40−42 There is evidence of inflammation occurring in schizophrenia,5−7,59,60 but there are conflicting reports on whether olanzapine has pro- or anti-inflammatory effects.61,62 Sugino et al. showed that olanzapine decreased the levels of TNFα and IL-6 and increased the levels of anti-inflammatory IL-10,62 implying a drug-induced decrease in inflammation. However, Meyer et al. reported an olanzapine-induced increased level of c-reactive protein, a marker of inflammation.63

(S1), di- (S2), tri- (S3), and tetrasialylated (S4) glycans in whole serum samples. This analysis showed that the levels of S2 glycans were increased post-treatment and the levels of S1 glycans were decreased. The concurrent increase of S2 and G2 suggests that sialylation levels may have been increased as a result of increased levels of digalactosylated glycans available for sialylation. Taken together, these results suggest that olanzapine alters the overall glycosylation of whole serum proteins by modifying the levels of galactosylation and sialylation of glycans. The source of the olanzapine-induced glycan alterations in the current study remains unclear; however, two possible sources are discussed here. One possible source could be related to olanzapine-induced changes to the central nervous system (CNS). Approximately 80% of proteins in the CSF originate from the blood and 20% from the CNS.53 There is bidirectional transfer of proteins from the plasma to CSF by size dependent diffusion through the blood CSF barrier. Because the concentration of proteins in the CSF is much lower than that of plasma, it is unlikely that many proteins transfer from the CSF to the plasma, suggesting that the olanzapineinduced changes observed in the current study in serum may not come about as a result of olanzapine-induced alterations to the CNS, as these effects would most likely not be evident on serum proteins. A more likely possible source of the glycan alterations is that olanzapine acts on the periphery, altering the activity of hepatic enzymes, resulting in altered glycosylation of proteins, particularly acute phase proteins. Olanzapine is primarily metabolized in the liver to its inactive compound by cytochrome P450 CYP1A2, a monooxygenase that is located in the endoplasmic reticulum.22 Cytochrome P450 enzymes are concentrated in the liver and are also present in lower levels in tissues of other organs including lungs and kidneys. Previous studies have investigated the effects of olanzapine on the levels of liver enzymes such as alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase during treatment with the drug. Olanzapine-induced elevations in liver enzymes in a transient, asymptomatic and dose-independent manner were reported in a number of patients.54−56 To a lesser degree, increased liver enzymes in a considerable and enduring manner were also reported in a small number of patients.57 Acute phase proteins such as AGP, haptoglobin and α1 antitrypsin are produced in the liver, with their production dramatically increasing during inflammatory processes. Glycosylation of these acute phase proteins takes places in the endoplasmic reticulum and Golgi apparatus in hepatic cells by the actions of glycosyltransferases and glycosidases. Because of the reported effects of olanzapine on hepatic enzymes, it may be possible that the olanzapine-induced glycosylation alterations reported in the current study may occur as a result of altered activity of hepatic glycosylation processing enzymes. The current study examined the glycosylation profiles of serum samples from schizophrenia patients who had been treated with olanzapine. Because it is possible that the druginduced alterations may occur as a result of alterations to hepatic enzyme function as discussed above, it is possible that these reported drug-induced glycan alterations could be seen in a population of healthy controls if treated with olanzapine in the same manner as the schizophrenia patients. This provides scope for a future study to examine this further. Such induced changes by olanzapine to the glycosylation of biologically active glycoproteins may influence their immuno-

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CONCLUSIONS This study has shown that olanzapine treatment of schizophrenia patients resulted in changes in the glycosylation machinery associated with the biosynthesis of abundant serum proteins. Specifically, olanzapine appeared to affect the extent of digalactosylation and disialylation of serum proteins. As glycosylation impacts on many important cellular processes including cellular recognition, protein function, protein stability, immunogenicity and protein clearance rates, olanzapine-induced glycosylation changes may induce a number of downstream effects.

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ASSOCIATED CONTENT

* Supporting Information S

Table S1: Predominant glycan structures that are present in whole serum, low-abundance serum protein fraction, and AGP and the GU values of the peaks in each sample. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +353-12158142. Fax: +353-12158116. E-mail: pauline. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the European Commission under the Seventh Framework Programme (FP7) SchizDX [Grant No. 223427]. R.S. acknowledges the European Union Seventh Framework Programme (FP7) under Grant Agreement No. 260600 (GlycoHIT).

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ABBREVIATIONS A1, A2, A3, A4, mono-, bi-, tri-, tetra-antennary glycans; 2AB, 2amino benzamide; ABS, Arthrobacter ureafaciens sialidase; AGP, α1 acid glycoprotein; AMF, almond meal α-fucosidase; BKF, bovine kidney α-fucosidase; BTG, bovine testes β-galactosidase; CSF, cerebrospinal fluid; 2DE, 2D electrophoresis; G0, G1, G2, G3, G4, non-, mono-, di-, tri-, tetragalactosylated glycans; GalT1, β1,4-galactosyltransferase I; GlcNAc, Nacetylglucosamine; GLM, general linear model; GU, glucose 3750



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units; GUH, β-N-acetylglucosaminidase cloned from Streptococcus pneumonia, expressed in Escherichia coli; HILIC, hydrophilic interaction liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; S1, S2, S3, S4, mono-, di-, tri-, tetrasialylated glycans; sLex, sialyl-Lewis X

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