Quantitative Glycome Analysis of N-Glycan Patterns in Bladder Cancer

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Quantitative glycome analysis of N-glycan patterns in bladder cancer vs. normal bladder cells using an integrated strategy Feng Guan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr5006026 • Publication Date (Web): 23 Dec 2014 Downloaded from http://pubs.acs.org on January 8, 2015

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Quantitative glycome analysis of N-glycan patterns in bladder cancer vs. normal bladder cells using an integrated strategy

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

Journal of Proteome Research pr-2014-006026.R2 Article 16-Dec-2014 Yang, Ganglong; The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University Tan, Zengqi; The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University Lu, Wei; The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University Guo, Jia; The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University Yu, Hanjie; Laboratory for Functional Glycomics, College of Life Sciences Yu, Jingmin; Laboratory for Functional Glycomics, College of Life Sciences Sun, Chengwen; Affiliated Hospital of Jiangnan University, Department of Urology Qi, Xiaowei; Affiliated Hospital of Jiangnan University, Department of Pathology Li, Zheng; Laboratory for Functional Glycomics, College of Life Sciences Guan, Feng; The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology,Jiangnan University

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Quantitative glycome analysis of N-glycan patterns in bladder cancer vs. normal bladder cells using an integrated strategy

Ganglong Yang1, Zengqi Tan1, Wei Lu1, Jia Guo1, Hanjie Yu2, Jingmin Yu2, Chengwen Sun3, Xiaowei Qi4, Zheng Li2,*, Feng Guan1,*

1

The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education; School of

Biotechnology, Jiangnan University, Wuxi, China. 2

Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, Xi’an, China.

3

Department of Urology, Affiliated Hospital of Jiangnan University, Wuxi, China.

4

Department of Pathology, Affiliated Hospital of Jiangnan University, Wuxi, China.



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ABSTRACT Diagnosis of bladder cancer, one of the most common types of human cancer, at an early (non-muscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy in general is closely associated with alterations of glycan expression. Glycosylation status, particularly global glycomes, in bladder cancer have not been well studied. We integrated lectin microarray and mass spectrometry (MS) methods to quantitatively analyze and compare glycan expression in four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed using lectin microarray analysis and confirmed by lectin staining and lectin blotting. Associations of glycopatterns with diverging clinical courses were evaluated by lectin histochemistry on tissue microarrays. N-glycans were derivatized by amidation of sialylated glycans with acetohydrazide and reductive amination with the stable isotope tags [12C6]- and [13C6]-aniline, and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF-MS). N-glycan biosynthesis-associated proteins were quantitatively analyzed by a Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) proteomics method, which revealed significant differences in expression of 13 glycosyltransferases and 4 glycosidases. Our findings indicate that sialyl Lewis X (sLex), terminal GalNAc and Gal, and high mannose-type N-glycans were more highly expressed in bladder cancer cells and tissues than in normal cells. Bladder cancer cells showed high expression of core-fucosylated N-glycans but low expression of terminally fucosylated N-glycans. Each of these glycome changes may be directly related to bladder cancer progression.

KEYWORDS: Bladder cancer, mass spectrometry, lectin microarray analysis, N-glycans, quantitative glycomics, SILAC method.


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Introduction Bladder cancer is the fifth most common type of human cancer, and its incidence has increased steadily during the past decade. There were an estimated 74,690 newly diagnosed cases and 15,580 deaths from this disease in the United States in 20131. Of total bladder cancer patients, >70% have non-muscle-invasive disease and ~25% present initially with muscle invasion. Patients with the muscle-invasive form have a 50% risk of distant metastases, and a poor prognosis2. The commonly used diagnostic techniques for bladder cancer are cystoscopy and urine cytology. The cystoscopy procedure is painful for most patients. A number of molecular biomarkers for bladder cancer diagnosis have been FDA-approved. These include soluble proteins such as bladder tumor associated antigen (BTA), nuclear matrix protein 22 (NMP22), proteins detected on fixed urothelial cells (ImmunoCyt), and chromosomal aberrations detected by fluorescence in situ hybridization (UroVysion)3, 4. The ability of these biomarkers to detect bladder cancer at an early (non-muscle-invasive) stage is poor, and development of novel biomarkers for early diagnosis of bladder cancer is therefore a high priority. Glycosylation is one of the most frequent post-translational protein modifications, and is estimated to occur in >70% of all human proteins5. It plays crucial roles in molecular recognition and cell-cell adhesion, and glycosylation disorders are associated with dysmorphic features, failure to thrive, and various types of mortality. Aberrant protein glycosylation often occurs during malignant transformation and leads to expression of specific tumor-associated glycans. The appearance of aberrant glycans on cancer cells is typically associated with tumor grade, invasiveness, and metastasis, and is correlated with overall poor prognosis6. Alterations in glycosylation appear very early during carcinogenesis, before it is possible to detect changes in cell proliferation or differentiation7. Attempts to utilize glycans as biomarkers for various types of cancer have had only moderate success to date, because of (i) technical difficulties in glycan purification and analysis, and (ii) variations in accuracy depending on analytical method and type of equipment8, 9.



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Several studies indicate that bladder cancer is associated with aberrant expression of glycans. Ishimura et al. demonstrated an inverse correlation between N-acetylglucosaminyltransferase V (GnT-V) expression and tumor grade/stage10. In a glycoblotting analysis of patient N- and O-glycomes of serum and urine, Nishimura et al. showed significant alteration of several N- and O-glycans11. However, there have been few quantitative glycomic analyses of human bladder cancer. Quantitative glycome analysis is the key to detecting changes in glycoproteins and their associated glycans, for elucidating the functions of glycans in biological pathways and disease development. The past decade has seen major progress in this area, including effective methods for high-throughput glycan analysis: liquid chromatography (LC), mass spectrometry (MS), and capillary electrophoresis (CE)12. MS is a powerful tool for quantitative glycan analysis. Mass differences of a given glycan from different samples can be quantitatively analyzed in one-step MS detection based on permethylation and reductive amination, and on techniques for mass shift of isotopic tag labels, e.g., glycan reductive isotope labeling (GRIL)13, isotopic labeling through permethylation14, isobaric aldehyde reactive tags (iARTs), isotopic glycan hydrazide tags (IGHT), and isotopic detection of aminosugars with glutamine (IDAWG)15-17. Thousands of different glycans attached to human proteins are of clinical or scientific interest, but no analytical method currently available can simultaneously quantify all of them. In this study, we integrated lectin microarray and MS methods for quantitative analysis of glycan expression levels of bladder cancer (KK47, YTS1, J82, and T24) vs. normal bladder mucosa (HCV29) cell lines, which have been used in several previous studies of the functional roles of glycans and glycoconjugates in bladder cancer progression18-20. We analyzed the glycopattern of glycoproteins using lectin microarrays, and confirmed the differential glycopatterns by lectin staining, lectin blotting, and immunohistochemical staining. To characterize the sialic acid and quantitatively analyze the N-glycans in one step, we developed a GRIL labeling method to derivatize the acidic and reducing termini of N-glycans. N-glycans linked to glycoproteins were amidated by acetohydrazide and released by PNGase F. The reducing termini of released glycans from different cells were derivatized using [12C6]-aniline or [13C6]-aniline, and the derivatized glycans were quantitatively analyzed by MALDI-TOF/TOF-MS.


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Expression levels of glycosyltransferases and glycosidases associated with N-glycan biosynthesis were quantitatively analyzed by the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) method. This integrated strategy (summarized schematically in Fig. 1) allowed quantitative comparison of altered N-glycan patterns in bladder cancer progression, and provided important information on bladder cancer glycomes.

Materials and Methods

Cell lines and culture Non-muscle-invasive bladder cancer KK47, normal bladder mucosa HCV29, and highly invasive/ metastatic YTS1 cell lines were kindly provided by Dr. S. Hakomori (The Biomembrane Institute/ Pacific Northwest Diabetes Research Institute; Seattle, WA, USA). Transitional carcinoma cell lines T24 and J82 were from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 or MEM medium (HyClone; Logan, UT, USA) containing 10% fetal bovine serum (HyClone) and 1× penicillin/streptomycin (Gibco; Carlsbad, CA, USA) at 37 °C in a 5% CO2 atmosphere.

SILAC labeling Arginine and lysine were added in either light form (Arg0; Lys0) or heavy form (Arg6; Lys4) (Thermo Scientific; San Jose, CA, USA) to a final concentration of 100 µg/mL. To prevent arginine-to-proline conversion, 200 µg/mL L-proline was added to the medium. Prior to treatment, cells were grown for 5 or 6 passages in SILAC medium. Labeling efficiency was checked to ensure that all labeled cell lines attained >95% incorporation rate.

Total protein extraction



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Total proteins of KK47 and HCV29 cells were lysed and extracted with T-PER Reagent (Thermo Scientific) according to the manufacturer's instructions. In brief, cells (~1×107) were detached with trypsin, washed twice with ice-cold 1× PBS (0.01 M phosphate buffer containing 0.15 M NaCl, pH 7.4), lysed with 1 mL T-PER Reagent containing protease inhibitor (1 mM PMSF and 0.1% aprotinin), incubated for 30 min on ice, homogenized, and centrifuged at 12,000 rpm for 15 min. The supernatant was collected and stored at -80 °C. Protein concentration was determined by BCA assay (Beyotime; Haimen, China).

Lectin microarray analysis Lectin microarrays were constructed and analyzed as described previously21, 22. In brief, 37 commercial lectins from Vector Laboratories (Burlingame, CA, USA), Sigma-Aldrich (St. Louis, MO, USA), or Merck (Darmstadt, Germany), in the manufacturers' recommended buffers, were immobilized on epoxysilane-coated solid support at high spatial density. Protein samples were labeled with fluorescent dye Cy3 (GE Healthcare; Buckinghamshire, UK) and applied to the lectin microarrays. After incubation, the slides were scanned with a GenePix 4000B confocal scanner (Axon Instruments; Union City, CA, USA).

Microarray data acquisition and analysis The GenePix Pro 3.0 software program was used to extract numerical data from the scanned images. The average background was subtracted, and values that were less than the average background ± 2 SDs were removed from each data point. The medians of the three repeats for each lectin in one block were obtained. The data for each lectin consisted of the average of the normalized medians resulting from the ratio of medians to the sum of the medians of the data points in one block. Each sample was observed consistently from three repeated slides, and the normalized medians and SD of each lectin were averaged from nine repeated blocks. Aijk was defined as the median of the three repeats for each lectin in one block and one experiment, where i is the number of the lectin, j is the number of blocks in one experiment, and k is the number of the experiment. The normalized relative intensity (NRI) of a lectin was calculated by the following equation:

=

A ijk 9 × 100 ∑


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The normalized data from KK47 and HCV29 cells were compared to assess relative changes in protein glycosylation levels. The generated data were further analyzed by the HCE software program (V3.0). Differences between the two data sets were evaluated by Student's t-test applied to each lectin signal.

Lectin staining Cells were cultured in 24-well plates with sterilized coverslips to obtain monolayers with 70-80% confluence. Cells were washed with cold PBS, immobilized with 2% fresh paraformaldehyde for 15 min at room temperature (RT), permeabilized with 0.2% Triton X-100 in 1× PBS for 10 min at RT, and blocked with 5% BSA in 1× PBS overnight at 4 °C. The fixed cells were incubated with 15-20 µg/mL Cy3 fluorescein-labeled lectins (LCA, SJA, and Con A) in 5% BSA for 3 hr in the dark at RT. The cells were washed with PBS, stained with 20 µg/mL DAPI in 1× PBS for 10 min at RT, washed again with 1×PBS, and photographed with a fluorescence microscope (model Eclipse E600; Nikon; Tokyo, Japan).

Lectin blotting Isolated proteins from KK47 and HCV29 cells were analyzed by SDS-PAGE and lectin blotting23. In brief, samples were boiled and mixed with 5× loading buffer and run on a 10% polyacrylamide resolving gel. Proteins in the gels were transferred to a PVDF membrane (Immobilon-P; Millipore; Bedford, MA, USA). The membranes were washed twice with 1× TTBS (150 mM NaCl, 10 mM Tris-HCl, 0.05% v/v Tween 20, pH 7.5) and blocked with blocking buffer (1× TTBS, 2% BSA) for 1 hr at RT. The membranes were incubated with Cy3-labeled lectins as above (2 µg/mL in blocking buffer) in the dark overnight at 4 °C with gentle shaking. The membranes were washed twice with TTBS for 10 min and scanned by red fluorescence channel (532 nm excitation/ 580 LP emission) with optional voltages for different lectins using a phosphorimager (Typhoon TRIO; Molecular Dynamics; Ramsey, MN, USA).

Tissue microarray analysis Bladder cancer tissue microarrays (TMAs) containing 17 cases of bladder carcinoma and 7 cases of cervical carcinoma (see detailed information in Fig. S1 and Table S1) were from Shanghai Outdo Biotech Co. The paraffinized slides were incubated for 1 hr at 63 °C, and then deparaffinized in xylene and graded concentrations of alcohol. Non-specific protein binding was blocked by incubating with blocking buffer (5% BSA in 1× PBS) at 4 °C overnight. Lectins (2 µg/mL) labeled with fluorescent dye Cy3 were applied to the



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slides and incubated for 3 hr in the dark. TMAs were then washed in 1× PBS, stained with 20 µg/mL DAPI in 1× PBS for 10 min at RT, and washed again with 1×PBS and 1× PBST. The slides were scanned with a confocal scanner and photographed with a fluorescence microscope as described above.

Amidation of sialylated N-linked glycans Sialylated N-linked glycans of glycoproteins were amidated by acetohydrazide and released in a size-exclusion spin ultrafiltration unit (Amicon Ultra-0.5 10 KD; Millipore) as described previously24, 25. In brief, proteins (1.5 mg) were concentrated and denatured with 8 M urea, reduced, and alkylated by addition of 10 mM DTT and 10 mM IAM (Sigma-Aldrich). The samples were washed with 40 mM NH4HCO3, and proteins were desalted by washing with deionized water. The desalted proteins were redissolved with 100 µL of 1 M acetohydrazide, 20 µL of 1 N HCl, and 20 µL of 2 M EDC. The mixture was incubated at RT for 4 hr. The amidated glycoproteins were washed with 40 mM NH4HCO3, added with 2 µL PNGase F in 40 mM NH4HCO3, and incubated overnight at 37 °C. The released amidated N-linked glycans were collected by centrifugation and lyophilized.

Clean-up of N-glycans Desalting was performed using Sepharose 4B (Sigma-Aldrich) as described previously23. Sepharose 4B in a microtube was washed with methanol/ water (MW; 1:1, v/v) and 1-butanol/ methanol/ water (BMW; 5:1:1, v/v) under centrifugation. Glycans were dissolved in 500 µL BMW and added to the microtube. The mixture was gently shaken for 45 min and washed three times with BMW. N-glycans were eluted with MW, and the eluent was collected and lyophilized.

Derivatization of released N-linked glycans with [12C6]- or [13C6]-aniline Ten µL [12C6]- or [13C6]-aniline and 25 µL fresh NaCNBH3 (1 M) prepared in DMSO/acetic acid (7:3, v/v) were added separately to dried amidated N-linked glycans from KK47 and HCV29 cells, and incubated at 75 °C for 10 min as described previously13. The reaction mixture was lyophilized under vacuum, redissolved in 500 µL BMW, and desalted with Sepharose 4B. The eluted glycan derivatives were dried and stored at -20 °C in the dark.

Characterization of N-glycans


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N-glycans were characterized by MALDI-TOF/TOF-MS (UltrafleXtreme; Bruker Daltonics; Bremen, Germany). Lyophilized N-glycans were resuspended in 10 µL MW. A 1-µL aliquot of the mixture was spotted onto an MTP AnchorChip sample target and air-dried. One µL of 20 mg/mL DHB in MW was spotted to recrystallize the glycans. Mass calibration was performed using peptide calibration standards (250 calibration points; Bruker). Measurements were taken in positive-ion mode, and m/z data were analyzed and annotated using the GlycoWorkbench software program (http://code.google.com/p/glycoworkbench/)26.

Quantitative analysis of proteins (glycosyltransferases and glycosidases) associated with N-glycan biosynthesis by the SILAC method SILAC-labeled cells were lysed by T-PER Reagent. Proteins were mixed at equivalent ratios and further processed as described previously 27. In brief, proteins were reduced with 10 mM DTT, alkylated with 30 mM IAM, and incubated with sequencing grade trypsin (Promega; Madison, WI, USA) at 1:100 (w/w) overnight at 37 °C. Two-dimensional LC-MS and data analysis were performed using an LTQ Orbitrap MS (Thermo Fisher; Waltham, MA, USA) and the MaxQuant software program (V. 1.4.1.2) as described previously28-30 (Supporting Information I). Proteins associated with N-glycan biosynthesis were analyzed by the DAVID software program (SAIC-Frederick; Frederick, MD, USA), based on the proteins identified in KK47 and HCV29 cells.

Results

Glycopattern analysis of KK47 and HCV29 cells HCV29 are human normal bladder urothelial cells, and KK47 are human non-muscle-invasive bladder cancer cells. The cell motility patterns of the two types of cells are similar. Lectin microarray analysis, including 37 lectins, 2 negative controls (BSA), and 1 positive control (Cy3-BSA), was performed to identify glycopatterns of the two types of cells. The results revealed significant differences in the glycopatterns, as indicated by white boxes in Fig. 2A. Normalized relative signal intensities greater than 1 were considered to be valid intensities (Table 1). To facilitate the analysis, lectin signal patterns were assigned to three groups: (i) stronger signals with KK47: HCV29 intensity ratios >1.5 (NRIs >1;



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fold-change >1.5; p 1.5 from glycans identified in both samples, based on three replicates; (ii) glycans identified from fewer than three replicates in one cell line, considered to be present in only one cell line. These stringent criteria were necessary to ensure that identification of a particular glycan in two samples was most likely due to differential abundance between the samples -- not because parent ions were present but not identified in the MS analysis. According to


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these criteria, seven of the glycans were up-regulated and one was down-regulated in KK47, two were present only in KK47, and two were present only in HCV29. N-linked glycans detected in KK47 were distinguishable from those in HCV29 by the following four characteristics: (i) Higher relative concentration of sLex N-glycan structures, e.g., m/z 2683.490/2689.533 (Neu5Ac)1(Fuc)2(Gal)2(GlcNAc)2(Man)2+(Man)3(GlcNAc)2 (1.68); 3311.888/3317.529 (Neu5Ac)2(Fuc)1(Gal)4(GlcNAc)4+(Man)3(GlcNAc)2 (1.61); 2930.801/2936.994 (Neu5Ac)2(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2 (only in KK47). (ii) Higher relative concentration of terminal GalNAc and Gal N-glycan structures, e.g., m/z 1997.269/2003.270 (Fuc)1(Gal)2 (GlcNAc)2(Xyl)1+(Man)3(GlcNAc)2 (2.13); 2145.216/2151.182 (Gal)2(GalNAc)1(GlcNAc)3+(Man)3(GlcNAc)2 (1.55); 1943.227/1949.139 (Gal)2(GlcNAc)3+(Man)3(GlcNAc)2 (only in KK47). (iii) Higher relative concentration of high mannose-type N-glycan structures, e.g., m/z 1496.833/1502.826 (Man)3+(Man)3(GlcNAc)2 (1.58); 1983.089/1989.102 (Man)6+(Man)3(GlcNAc)2 (2.03). Accumulation of these structures indicated a blocked step in N-glycan synthesis, resulting in incomplete glycosylation in KK47. (iv) Alternating relative concentrations of fucosylated complex-type N-glycan structures in the two cell lines, e.g., m/z 2033.125/2039.216 (Fuc)2(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2 (0.60); 1997.269/2003.270 (Fuc)1(Gal)2(GlcNAc)2(Xyl)1+(Man)3(GlcNAc)2 (2.13).

Expression levels of proteins (glycosyltransferases and glycosidases) associated with N-glycan biosynthesis, analyzed by the SILAC method Typical changes in carbohydrate expression associated with malignant transformation result from “aberrant glycosylation”, catalyzed primarily by specific glycosyltransferases and glycosidases. Differences in expression of these N-glycan biosynthesis-associated proteins in HCV29 vs. KK47 were investigated using the SILAC proteomics method. A total of 16168 peptides and 3792 proteins were identified from the two cell lines by LTQ Orbitrap MS (Supporting Information II). Among the proteins, 17 (13 glycosyltransferases and 4 glycosidases) were associated with N-glycan biosynthesis (Table S4). SILAC analysis showed notable differences between the two cell lines for 13 of these N-glycan biosynthesis-associated proteins. Three glycosyltransferases were up-regulated (SILAC ratio >1.50) in KK47: α1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGT5B), β1,4-galactosyltransferase 1 (B4GT1), and chitobiosyldiphosphodolichol β-mannosyltransferase (ALG1),



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involved respectively in assembly of GlcNAcβ1,6Man, Manβ1,4GlcNAc, and Galβ1,4GlcNAc intermediates on the cytoplasmic surface of the ER. Ten N-glycan biosynthesis-associated proteins were down-regulated (SILAC ratio

Quantitative Glycome Analysis of N-Glycan Patterns in Bladder Cancer

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