Comparative N-Glycan Profiling of Colorectal Cancer Cell Lines

Article pubs.acs.org/jpr

Comparative N‑Glycan Profiling of Colorectal Cancer Cell Lines Reveals Unique Bisecting GlcNAc and α‑2,3-Linked Sialic Acid Determinants Are Associated with Membrane Proteins of the More Metastatic/Aggressive Cell Lines Manveen K. Sethi,† Morten Thaysen-Andersen,† Joshua T. Smith,‡ Mark S. Baker,† Nicolle H. Packer,† William S. Hancock,†,‡ and Susan Fanayan*,† †

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States

‡

S Supporting Information *

ABSTRACT: Advances in colorectal cancer (CRC) diagnosis will be enhanced by development of more sensitive and reliable methods for early detection of the disease when treatment is more effective. Because many known disease biomarkers are membrane-bound glycoproteins with important biological functions, we chose to compare N-glycan profiles of membrane proteins from three phenotypically different CRC cell lines, LIM1215, LIM1899, and LIM2405, representing moderately differentiated metastatic, moderately differentiated primary, and poorly differentiated (aggressive) primary CRC cell lines, respectively. The N-glycan structures and their relative abundances were determined as their underivatized reduced forms, using porous graphitized carbon LC−ESI-MS/MS. A key observation was the similar N-glycan landscape in these cells with the dominance of high mannose type glycan structures (70−90%) in all three cell lines, suggesting an incomplete glycan processing. Importantly, unique glycan determinants such as bisecting Nacetylglucosamine were observed at a high level in the metastatic LIM1215 cells, with some expressed in the moderately differentiated LIM1899, while none were detected in the poorly differentiated LIM2405 cells. Conversely, α-2,3-sialylation was completely absent in LIM1215 and LIM1899 and present only in LIM2405. RNA-Seq and lectin immunofluorescence data correlated well with these data, showing the highest upregulation of Mgat3 and binding with PHA-E in LIM1215. Downregulation of Man1α1 and Mgat1 in LIM1215 also coincided with the higher degree of incomplete N-glycan processing and accumulation of high mannose type structures as well as bisecting N-glycans when compared to the other two cell lines. This study provides a comprehensive analysis of the membrane N-glycome in three CRC cell lines and identifies N-glycosylation differences that correlate with the histological and pathological features of the cell lines. The unique glycosylation phenotypes may therefore serve as a molecular feature to differentiate CRC disease stages. KEYWORDS: colorectal cancer, N-glycosylation, membrane proteins, mass spectrometry, bisecting GlcNAc, high mannose, sialylation, fucosylation

■

INTRODUCTION

methods suffer from low patient compliance due to their expensive and uncomfortable nature (colonoscopy) or low sensitivity and nonspecificity (fecal occult blood test).5,6 An ideal cancer screening program must be noninvasive, inexpensive, and sufficiently sensitive to detect tumors at an early stage when treatment is likely to succeed. Altered protein glycosylation is recognized as a cellular hallmark event during carcinogenesis.7−9 Both N- and O-linked glycoproteins have been shown to be altered in a wide range of

Colorectal cancer (CRC), commonly known as bowel cancer, is a major health problem worldwide, with global increases in incidence and death due to an expanding and aging population. There is an annual global incidence of approximately one million CRC cases and half a million deaths each year.1,2 If CRC is detected early, while tumors are still localized within the colon/bowel wall, the 5-year survival rate is >90% following surgical resection.3,4 Unfortunately, 30−50% of patients already have metastatic disease at presentation when prognosis is poor with a 5-year survival of less than 10%.2,3 Despite evidence that early detection can reduce CRC mortality, screening rates remain low. Current screening © 2013 American Chemical Society

Special Issue: Chromosome-centric Human Proteome Project Received: August 21, 2013 Published: December 2, 2013 277

dx.doi.org/10.1021/pr400861m | J. Proteome Res. 2014, 13, 277−288

Journal of Proteome Research

Article

cancers, including CRC. Examples include (1) increased β-1,6linked GlcNAc branching, yielding complex N-glycans with more than two antennas;10,11 (2) upregulation of GlcNAc transferase III coded by the Mgat3 gene, increasing the levels of structures containing bisecting β-1,4-linked GlcNAc residues;12 (3) elevation of the high mannose glycan content;13 and (4) increased core fucosylation and sialylation.14−18 Thus, it is not surprising that the emerging field of glycomics is gaining increasing attention in cancer research in addition to genomic and proteomic approaches. The use of glycoproteins as biomarkers for detection, staging, and monitoring of cancer has the advantage that both the protein and glycan moieties, which both contain valuable disease-related information suited for blood-based detection, can be studied, giving higher sensitivity and specificity of candidate biomarkers.8,9 Although the glycoproteome has only recently been targeted for potential biomarkers, due to technical complexity,8,19,20 several clinical glycoprotein biomarkers, e.g., Her2/neu in breast cancer, cancer antigen 125 (CA125) in ovarian cancer, prostate specific antigen (PSA) in prostate cancer, carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA19-9) are widely used in patient care.20 While our understanding of CRC has significantly advanced in recent years through the large amount of data generated by high-throughput genomic and proteomic studies, this knowledge has not yet translated into clinically validated biomarkers. An increased understanding of CRC biology could accelerate the search for improved methods for noninvasive detection and surveillance of the disease. In a recent study we employed a proteogenomic approach to identify a number of differentially expressed proteins, with known cancer associations, in LIM1215, LIM1899, and LIM2405 cell lines, representing moderately differentiated metastatic, moderately differentiated primary, and poorly differentiated (aggressive) primary CRC tumors, respectively. Using this approach, we also identified a number of protein interaction networks that were significantly perturbed in one or more of the CRC cell lines.21 As part of the Human Proteome Project, we also employed a novel chromosome-centric approach to organize the identified proteins from CRC proteomic studies on chromosome 7. Using this approach, we identified several gene clusters with known functional relevance to cancers, including CRC, as well as differential expression of N-glycosylated membrane proteins such as epidermal growth factor receptor (EGFR), carcinoembryonic antigen-related cell adhesion molecules (CEACAM5, CEACAM6), and spectrins (SPTBN1 and SPTAN1).22 In the present study we employed two ‘omics’ platforms, namely, glycomics and transcriptomics, to characterize the membrane protein N-glycome of these cell lines to identify glycan structure landscapes and the key enzymes that may explain the differences in the biology of these cells. Quantitative and qualitative comparison of the N-glycomes revealed strong similarities between the three cell lines by dominance of high mannose type glycans as a common feature. Importantly, presence of unique glycan determinants such as bisecting GlcNAc in metastatic LIM1215 and α-2,3-linked sialic acid residues in poorly differentiated LIM2405 cells were observed. RNA-seq data and lectin immunofluorescence studies were additionally employed to complement the N-glycan profiling, and these confirmed that the N-glycosylation phenotypes are altered in CRC disease development. Overall we established the differences and similarities in expression profiles of N-glycan

determinants between the CRC cell lines and correlated it with their histological characteristics.

■

MATERIALS AND METHODS

Materials

RPMI 1640 media, human recombinant insulin, glutamine, fetal bovine serums, and molecular weight standards were purchased from Invitrogen (Carlsbad, CA). Biotinylated lectins, i.e., SNA, AAL, ConA, and PHA-E, were purchased from Vector Laboratories (Burlingame, CA). AG50W-X8 action exchange resin was purchased from Biorad (Hercules, CA). Protease inhibitor cocktail tablet (EDTA-free) and peptide-N-glycosidase F (PNGase F) (Flavobacterium meningospeticum) were purchased from Roche Diagnostics (GmbH, Mannheim, Germany). PVDF membranes were purchased from Millipore and empty-top zip tips were purchased from Glygen (MD, USA). Pierce BCA protein assay kit was purchased from Thermo scientific (Victoria, Australia). FITC-streptavidin, Bradford reagent, hydrocortisone, sodium borohydroxide, Triton X-114, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) if not specifically stated otherwise. Cell Culture

Three colorectal cancer cell lines LIM1215, LIM1899, and LIM240523−25 were obtained from Ludwig Institute for Cancer Research Ltd. (Parkville, Victoria, Australia). Cells were cultured in RPMI 1640 media supplemented with 10% FBS, α-thioglycerol (10−6 M), insulin (25 U/L), hydrocortisone (1 mg/mL), and L-glutamine (5 mM) at 37 °C/5% CO2. Cells were grown to 90% confluency, washed 3 times in ice-cold PBS, and collected, using a cell scraper, in lysis buffer containing 200 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA.2H2O, and protease inhibitor cocktail. The suspended cells were incubated on ice for 30 min and ultrasonicated (Branson Sonifier 450, VWR, CA) at intervals of 15 s for 2 min, with 15 s pause between each treatment. This was followed by centrifugation at 17,000g for 1 h, and supernatant was collected into a separate tube and stored at −20 °C for membrane protein extraction. All of the above steps were performed at 4 °C. Two biological replicates (separate cell culture experiments) were performed for each cell line. Cell Proliferation and Concentration of Total Secreted Proteins

Cells were seeded in six-well plates at 1 × 104 cells/mL and grown overnight at 37 °C/5% CO2. Cells were counted daily for 4 days to determine the cell growth rate. The doubling time was calculated using data from the exponential growth phase of the plot for each cell line. To measure the concentration of total proteins secreted by each cell line, cells were plated in quadruplicates (in 100 mm tissue culture dish3w). When 80% confluent, serum -free media (25 mL) was added to each dish and incubated for 48 h at 37 °C/5% CO2. Conditioned media were then collected and concentrated using 10 kDa cutoff Amicon filters (Millipore, MA), followed by 4 washes with 1x PBS. Concentration of secreted proteins in each sample was determined using a BCA protein assay. RNA Extraction

RNA was isolated using Trizol reagent (Sigma-Aldrich, CA, Australia) according to manufacturer’s instructions. Briefly, Trizol (1 mL per 106 cells) was added, and lysing was 278



Article

The upper aqueous phase was removed and stored. The detergent phase was further diluted with 4 vol of binding buffer containing 1% (v/v) Triton X-114, and phase partitioning was repeated. The combined aqueous phases were mixed with 9 vol of ice-cold acetone, overnight at −20 °C, to precipitate proteins and remove any detergent. Precipitated membrane proteins were resolublized with 8 M urea, and protein concentrations were measured using a Bradford protein assay. The enriched proteins were stored at −80 °C if not used immediately.

performed using a 23/26 G needle by repeated shearing (20x) and incubation at room temperature for 5 min. For phase separation, 200 μL of chloroform was added to the cells and incubated for 3 min at room temperature, followed by centrifugation at 12,000g for 15 min at 4 °C. The upper aqueous layer was collected in a fresh tube, and RNA was precipitated using isopropyl alcohol. The RNA pellet was collected following centrifugation at 12,000g for 10 min at 4 °C and washed twice with 75% ethanol with centrifugation at 7500g after each wash. Finally, RNA pellet was air-dried and resuspended in RNase-free water. The RNA concentration was determined using Nanodrop 2000 spectrophotometer (260/ 280 nm) and was stored at −80 °C until further use.

Release of N-Glycans from Membrane Glycoproteins

N-Glycans were released from purified CRC membrane proteins using the protocol outlined by Jensen et al.28 Briefly, 20 μg of membrane proteins was spotted on PVDF membranes and stained with Direct Blue (Sigma-Aldrich, St. Louis, MO). The membrane spots were excised and washed in separate wells in a flat-bottom polypropylene 96-well plate (Corning Incorporated, Corning, NY, USA). N-Linked glycans were released from the membrane proteins using 3 U of PNGase F (Flavobacterium meningospeticum) with overnight incubation at 37 °C. Released N-glycans were reduced with 20 μL of 1 M NaBH4 in 50 mM KOH for 3 h at 50 °C. Reduced samples were quenched using 2 μL of glacial acetic acid, desalted using AG 50W-X8 cation exchange resins (Biorad, Hercules, CA), and packed in empty top zip-tips. The N-glycans were dried by vacuum centrifugation and further purified by washing (5x) with 100 μL of methanol and drying by vacuum centrifugation after every wash. The purified glycans were resuspended in 10 μL of water and subjected to PGC-LC−ESI-MS/MS.

cDNA Library Preparation and Transcriptome Sequencing (RNA-Seq)

RNA preparation and sequencing was performed following the protocol outlined by Nagalakshmi et al.26 Briefly, total RNA (7 μg) was subjected to two rounds of hybridization to oligo (dT) beads (Invitrogen, CA) to enrich mRNA. To evaluate rRNA contamination, RNA pico chip were employed using a BioAnalyzer. (Agilent, CA). The cDNA library was prepared using mRNA using a RNA sequencing preparation kit (Illumina, CA). Sample 1 and sample 2 were sequenced using an Illumina Genome Analyzer followed by Genome Analyzer II, which generated four data sets: S1-R1, S2-R1 and S1-R2, S2-R2. Lectin Fluorescence Assay

LIM1215, LIM1899, and LIM2405 cells were plated on 18-mm glass coverslips in a 12-well plate at a density of 4 × 104 cells/ mL and grown overnight at 37 °C/5% CO2. Cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. The reaction was quenched by rinsing the cells twice with 100 mM glycine in PBS, followed by rinsing with PBS. Cells were incubated with biotinylated lectins SNA (Sambucus nigra), AAL (Aleuria Aurantia), ConA (Concanavalin A), or PHA-E (Phaseolus vulgaris erthyroagglutinin) at 5, 10, 20, and 40 μg/ mL for 1 h at 4 °C. Cells were then washed 3x with PBS to remove unbound lectins, followed by incubation with FITCconjugated streptavidin (1:500) for 1 h in dark. Cells were counter-stained with Hoechst 33342 (1 μg/mL) for 5 min followed by three washes with PBS. Coverslips were mounted and sealed on microscopic slides and viewed under an Olympus BX63 fluorescent microscope. Controls included replacing biotinylated lectins or secondary antibody with PBS as well as inclusion of a sugar inhibition step in which each lectin was incubated overnight with its inhibitory sugar solution at 4 °C prior to addition to cells. Elution buffers used were 0.5 M lactose (SNA), 0.1 M L-fucose (AAL), 0.2 M methyl-α-Dmannopyranoside/0.2 M methyl-α-D-glucopyranoside (ConA), and 0.05 M glycine (PHA-E).

Porous Graphitized Carbon (PGC)-LC−ESI-MS/MS

Released N-glycans were separated on a Hypercarb porous graphitized carbon (PGC) column (5 μm particle size, 320 μm (i.d.) × 10 cm, Thermo Scientific) on an Agilent 1100 capillary LC (Agilent Technologies, Santa Clara, CA) and analyzed using an Agilent MSD three-dimensional ion-trap XCT Plus mass spectrometer coupled directly to the LC. Separation was carried out at a constant flow rate of 2 μL/min using a linear gradient with 2−16% (v/v) acetonitrile/10 mM NH4HCO3 for 45 min, followed by a gradient from 16% to 45% over 20 min before washing the column with 45% v/v acetonitrile/10 mM NH4HCO3 for 6 min and re-equilibrating in 10 mM NH4HCO3. MS/MS was set up with drying gas temperature of 325 °C with a drying gas low of 7 L/min and nebulizer gas at 18 psi. Skimmer, trap drive, and capillary exit were set at −40 V, −99.1 V, and −166 V, respectively. Smart fragmentation was used with 30% start and 200% end amplitude with maximum accumulation time of 200 ms and ICC (Ion target) of 100,000 ions. ESI-MS was performed in negative ion mode with two scan events: MS full scan (m/z 100−2200) with a scan speed of 8,100 m/z/s and data dependent MS/MS scan after collisioninduced fragmentation (CID) of the top two most intense precursor ions with threshold of 30,000 and relative threshold of 5% base peak. No dynamic exclusion was activated to enable MS/MS triggering of closely eluting yet separated N-glycan isoforms. All precursors were observed in charge state −1 and/ or −2. The instrument was calibrated prior to acquisition. The deviations of the observed molecular masses from the theoretical masses were generally below 0.8 Da. Few LC−MS data sets were further calibrated post acquisition using a multipoint calibration curve obtained from LC−MS analysis of the N-glycans released from bovine fetuin analyzed on the same day.

Enrichment of Membrane Proteins by Triton X-114 Phase Partitioning

Membrane proteins were prepared as previously described by Lee et al.27 Briefly, supernatant obtained from the previous step was diluted with buffer containing 20 mM Tris-HCl and 100 mM NaCl, followed by ultracentrifugation at 120,000g for 80 min. Supernatant was removed, and the pellet was resuspended in 20 mM Tris-HCl, 100 mM NaCl, phase partitioned using 1% (v/v) Triton X-114, and chilled on ice with intermittent vortexing for 20 min. The samples were heated at 37 °C for 20 min and phase partitioned by centrifugation at 1000g for 3 min. 279



Article

Table 1. Characteristics of CRC Cell Lines Used in This Studya

Information is obtained from published reports,23−25 online resources, and cell line databanks. ∗ signifies information based on data obtained in this study. The cellular doubling times and the protein secretion rates represent the mean values of two independent experiments.

a

Data Analysis

ployed to assist in structure design and in silico fragmentation analysis. The characterized N-glycans have been assigned with nonmetric confidence scores (low/medium/high) based on the above categories for characterization (i−iii) as well as their structural similarities (see Supplemental Table S1). Some structural ambiguity, in particular regarding linkage types, was still left on several N-glycan structures following PGC-LC−ESIMS/MS. These structures have been left without complete assignment or partially assigned using the knowledge of the biosynthetic machinery.

Raw MS data were viewed using ESI-compass 1.3 Bruker Daltonic software, and the glycan molecular masses were manually generated. Data were initially filtered for species for which no MS/MS spectra could be identified. A few other species, for which MS/MS spectra existed but were of low quality (either poor MS/MS quality or EIC peak quality), were further filtered out. Determination of the potential glycan monosaccharide compositions for the glycan precursors were assisted using the GlycoMod tool (http://www.expasy.ch/ tools/glycomod). Potential monosaccharide compositions were then imported into UnicarbKB (http://unicarbKB.org) to relate with previously experimentally validated N-linked glycan structures. From this, the glycan structures could be characterized from (i) molecular mass, (ii) MS/MS partial de novo sequencing, and (iii) LC retention time.29 MS/MS fragmentation profiles were matched to in silico fragments, and the fragmentation pattern was compared qualitatively to MS/MS profiles of identical structures using UniCarbDB (http://www.unicarb-db.org). GlycoWorkbench (http://www. eurocarbdb.org/applications/ms-tools) was additionally em-

■

RESULTS

Phenotypic Characterization of CRC Cell Lines

Three human colon cancer epithelial cell lines (LIM1215, LIM1899, and LIM2405) representing moderately and poorly differentiated states of CRC with or without metastases were employed for this study. Table 1 provides a brief description of the phenotypes and growth characteristics of the cell lines used in this study. To determine the cellular doubling time and concentration of secreted proteins, supporting experiments were performed for each cell line. The observed doubling times 280



Article

Figure 1. Mass profiles of membrane protein N-glycome of LIM1215, LIM1899, and LIM2405 (retention time 30−65 min) from negative ion PGCLC−ESI-MS/MS. The N-glycans released from membrane proteins derived from the three cell lines were confirmed by molecular mass, fragmentation, and PGC retention order. m/z values corresponding to N-glycans are represented by numbers in bold, and these structures are listed in Supplemental Table S1.

branching on the trimannosyl core structure, D-221 ion for bisecting glycans (m/z 508 and 670), and position of fucose residues at the core (m/z 350 and 553)34−37 (Supplemental Figure S1). In total, 34 monosaccharide compositions yielding 42 Nglycan structures, including the observed isomers, were identified in the combined data from all three cell lines as summarized in Supplemental Table S1. High mannose type Nglycans, Man7 (structure 3a) (7.8%, 6.3%, 7.8% relative abundance for LIM1215, LIM1899, and LIM2405, respectively), Man8 (structure 4) (23.7%, 19.4%, 21.7% relative abundance for LIM1215, LIM1899, and LIM2405, respectively), and Man9 (structure 5) (45.4%, 46%, 32.5% relative abundance for LIM1215, LIM1899, and LIM2405, respectively), were the most abundant structures observed for all three cell lines (Supplemental Table S1). Most of the observed monosaccharide compositions were in accordance with known mammalian glycan structures, including high mannose, complex, and hybrid type structures. In addition, some more unusual N-glycans, such as truncated paucimannosidic structures were also identified. A Venn diagram was constructed to visualize the common and unique N-glycans identified in these cell lines (Figure 2). Fifteen common N-glycan structures were identified, which mostly included high mannose type Nglycans (Man5-9) and some hybrid and complex N-glycans, showing relative abundances of 93% of the total N-glycomes in both LIM1215 and LIM1899 cell lines and 89% in LIM2405. Twelve structures were expressed exclusively in LIM1215 (metastatic) constituting mainly complex/hybrid bisecting Nglycans (5% relative abundance), while only one unique Nglycan structure was observed in LIM1899 with a relative

of 26, 42, and 25 h for LIM1215, LIM1899, and LIM2405, respectively, were consistent with the observation made by Whitehead et al.25 All three cell lines secrete similar levels of proteins: 160, 160, and 170 μg/100 mL serum free media/48 h for LIM1215, LIM1899, and LIM2405, respectively (Table1). Structural Characterization of N-Glycans of Membrane Proteins from CRC Cell Lines

The N-glycomes of membrane proteins originating from the CRC cell lines (LIM1215, LIM1899, and LIM2405) were quantitatively and qualitatively compared. N-Glycans from membrane proteins extracted from these cell lines were enzymatically released, purified, and analyzed in their free reduced forms, using negative ion PGC-LC−ESI-MS/MS. Figure 1 represents the N-glycan mass profiles for each cell line. While the overall mass profiles for all three cell lines appear similar, a more detailed analysis reveals some unique glycan features for each cell line. Supplemental Table S1 summarizes the characterized N-glycans released from the CRC membrane proteins with their relative abundances using area under curve analysis from extracted ion chromatogram (EIC) of all assigned charge states for all observed glycan masses, which has been shown to be an accurate approach for establishing the relative abundances of the N-glycan species. 30−33 The relative abundances shown here are based on the mean of two separate sets of LC−MS analyses. Elucidation of glycan structures was based on the known human N-glycan biosynthesis glycobiology, the precursor mass, PGC retention time, and MS/MS fragmentation pattern upon negative ion CID fragmentation of reduced native (underivatized) N-glycan species. The latter was based on the fragmentation patterns as well as the knowledge of diagnostic ions such as the presence of D and D-18 ions for 281



Article

Figure 2. Venn diagram of common and unique N-glycans characterized from membrane proteins derived from LIM1215, LIM1899, and LIM2405 cell lines. Monosaccharide symbols are based on the Consortium for Functional Glycomics (CFG) nomenclature: red triangle, fucose; green circle, mannose; blue square, N-acetyl glucosamine; purple diamond, sialic acid; yellow circle, galactose. The N-glycans are accompanied by their designated numbers as listed in Supplemental Table S1.

abundance of 1.5%, and six unique glycan structures constituting mainly α-2,3-linked complex sialyated isomers were observed in LIM2405 (aggressive) with 8% relative abundance of the total N-glycome (Figure 2).

(dHex)1, 911.3; (Hex)3 (HexNAc)2, 1057.2; (Hex)3 (HexNAc)2 (dHex)1) was observed across all three CRC cell lines with total relative abundance of 1.5%, 3.5%, and 2.7% in LIM1215, LIM1899, and LIM2405, respectively (Figure 3A). Sialylated and fucosylated structures were also found to be differentially presented on these cell lines. A higher proportion of sialylated and fucosylated N-glycans were observed in LIM2405 compared to LIM1215 and LIM1899 (Figure 3C). This observed difference was mainly due to an increase in sialylated isomers present in LIM2405. Diagnostic fragment ions (m/z 290.1 and 655.3) were used to determine terminal sialic acid residues linked to penultimate galactose-GlcNAc antennas.38 While α-2,6-linked sialic acid N-glycans were found present in all three CRC cell lines, α-2,3-linked sialic acid residues were exclusively present in LIM2405 although in lower proportions compared to α-2,6-linked sialic residues (Supplemental Figure S2). Figure 4 shows the EIC of m/z corresponding to sialylated N-glycans. Two isomers of sialyated N-glycans (α-2,6- and α-2,3-linked) were present in LIM2405, while only one α-2,6-linked sialic acid isomer was observed in LIM1215 and LIM1899. The retention times of the differently sialylated N-glycans of bovine fetuin were used to determine the PGC-LC retention profiles of α-2,3- and α-2,6-linked sialic acid-containing N-glycans.29

Differential Expression of N-Glycan Structures

Classification of the detected N-glycans into the main structural N-glycan types may help to identify major differences in the glycosylation patterns between the cell lines. The N-glycan structures were divided into four major types, namely, high mannose, hybrid, complex, and paucimannose (Figure 3A). High mannose structures (Man5‑9GlcNAc2) represented the most abundant type of N-glycans across the three CRC cell lines with LIM1215 containing the highest proportion of high mannose type N-glycans (88.5%), while LIM1899 and LIM2405 contain 82.3% and 72%, respectively. Figure 3B shows a summary of the different high mannose type N-glycans observed with Man9 representing the most abundant structure, i.e., 51%, 55%, and 45% relative abundance of high mannose type glycans for LIM1215, LIM1899, and LIM2405, respectively. Complex N-glycans represented the second most abundant N-glycan class, with the highest presence observed in LIM2405 with a relative abundance of 23% compared to 5.7% and 10% relative abundance in LIM1215 and LIM1899, respectively (Figure 3A). Low abundance of smaller N-glycans, such as paucimannose (m/z 895.4; (Hex) 2 (HexNAc)2 282



Article

Figure 3. Differential expression of membrane protein N-glycan structures and determinants carried by the LIM1215, LIM1899, and LIM2405 cell lines. (A) N-Glycan structures were divided into four major types, i.e., high mannose, hybrid, complex, and paucimannose. (B) Distribution of the different types of high-mannose (Man5-9) structures. Relative abundances were calculated based on area under curve analysis of each glycan normalized to 100%. (C) Distribution of terminal glycan determinants; fucosylated (core fucose and Lewis type structures) and sialylated (glycan structures carrying sialic acid epitopes) structures as well as a separate representation of percentage of structures carrying both fucose and sialic acid, determined using percentage relative abundance of complex and hybrid glycans structures. (D) Distribution of fucosylated structures including core fucose and Lewis type structures plus a separate representation of percentage of structures carrying both core and Lewis type epitopes, which were determined using percentage relative abundance of complex and hybrid glycans structures.

Lectin Fluorescence Assay and Gene Expression of CRC Cells

Differential expression of core and Lewis type fucosylated Nglycans between the three cell lines was another important observation (Figure 3D). Core fucosylation was higher than Lewis type fucosylation in the N-glycomes from all three cell lines (Figure 3D). Specific diagnostic ions were used to distinguish between core and terminal fucose-containing hybrid and complex N-glycans (e.g., m/z 350 and 553 for core fucose).34 Another interesting observation was the unique presence of a number of bisecting GlcNAc-containing N-glycans in LIM1215 cell line (metastatic) with relative abundance of 37.7% (out of the total amount of complex and hybrid type N-glycans, which have the potential to be modified with bisecting GlcNAc residues) when compared to LIM1899 where only two bisecting structures were observed with relatively low abundance (3%) and LIM2405 in which no bisecting structure was observed (Figure 5A). Representative masses of the observed bisecting GlcNAc-containing structures, which was based on the presence of the abundant diagnostic fragment ion D-221 (m/z 508 and 670)34 and their relative limited retention on PGC-LC, are shown in Figure 5D. As an example of a PGCLC−ESI-MS/MS based N-glycan characterization, a doubly charged fragment ion from the complex bisecting structure corresponding to m/z 986.9 (glycan#19) with retention time; 37.5 min is shown in Supplemental Figure S3 with assigned monosaccharide fragmentations.

Presence of unique bisecting N-glycans in the LIM1215 cell line was further confirmed by performing lectin fluorescence studies using biotinylated PHA-E lectin, which specifically recognizes bisecting GlcNAc residues. The results clearly showed that the levels of bisecting GlcNAc-containing structures were significantly higher in LIM1215 compared to LIM1899 and LIM2405 (Figure 5B). Mgat3 mRNA expression levels were also evaluated in all three cell lines using transcriptome sequencing (RNA-seq). We observed a significantly higher Mgat3 (coding for the gene product GlcNAc transferase III responsible for bisecting N-glycans) mRNA expression level in LIM1215 compared to LIM1899 and LIM2405 (Figure 5C).

■

DISCUSSION Alterations in N-linked oligosaccharide expression have been observed in various malignant diseases such as cancer, and certain glycan structures are well-known tumor markers. Many reports suggest that changes in cell surface glycosylation may play an important role in promoting tumor and may provide important new sources of markers for tumor progression. In this study we investigated the N-glycan profiles of membrane proteins and the gene expression of some key enzymes from the N-glycosylation machinery from three phenotypically different CRC cell lines. Comparative analysis of the three N283



Article

Figure 4. Variation in α-2,6- and α-2,3-linked sialylation of N-glycan isomers between the CRC cell lines. Extracted ion chromatogram for m/z corresponding to sialyated N-glycans. LIM2405 (aggressive CRC cell line) contains both α-2,6- and α-2,3-linked sialic acids, while α-2,6-sialylation is completely absent in LIM1215 and LIM1899. Peaks marked with “∗” are isomeric molecules that do not correspond to the respective N-glycan isomer shown.

mannose, consisting of ∼70−90% of the total N-glycan species, is the most abundant type of glycosylation, which may suggest incomplete maturation of the N-glycans in the glycosylation process.39 Moreover, all three membrane glycomes are rich in Man8 and Man9 glycans. These results contrast observations made in mammalian cells in which high mannose glycans are not common.40 However, elevation of high mannose glycans is consistent with previous reports in both cell lines and tumor sera. For example, de Leoz et al.13 observed an abundance of

glycomes based on distinctive grouping of the glycans, presence of unique glycan structures, and isomer variation within the three cell lines was performed. Differences and similarities in expression profiles of N-glycan determinants between the CRC cell lines, with no correlation to the protein secretion rate of the cell lines (Table 1), were established. A key observation was the dominance of high mannose type N-glycans, followed by complex-type glycans in the membrane proteins from all three CRC cell lines. The amount of high 284



Article

Figure 5. Bisecting structures observed in CRC cell lines. (A) Percentage relative abundance of bisecting GlcNAc N-glycans observed in each CRC cell line. (B) Lectin immunofluorescence microscopy using biotinylated PHA-E lectin that binds to bisecting GlcNAc residue (40 μg/mL of lectin; 40× magnification). For negative control, cells were incubated with PHA-E lectin that was preincubated with its inhibitor (0.05 M glycine). (C) RNA-Seq data (RPKM value) for Mgat3 in each CRC cell line. Highest Mgat3 expression is observed in LIM1215. (D) List of bisecting GlcNAc Nglycans observed in CRC cell lines and their percentage relative abundance. Each structure is accompanied by its designated number as listed in Supplemental Table S1.

high mannose type N-glycan structures, in particular Man9, during breast cancer progression. Similarly, a significant increase in the content of high mannose glycans was observed in sera of mice with implanted head and neck tumors compared to control samples of healthy mice.41 High mannose glycans are involved in initial steps in Nglycan processing and control of protein folding in the endoplasmic reticulum. The high abundance of high mannose structures, in particular Man9, suggests an incompletion of the

glycosylation process that normally trims back Man9 to produce complex and hybrid type oligosaccharides. This observation could be explained by expression levels of the key enzymes in the N-glycosylation pathway: (i) Man1a1, which encodes a class I mammalian Golgi 1,2-mannosidase, responsible for the conversion of Man9 to Man8 high mannose; (ii) Man1b1, which encodes a class I α-1,2-mannosidase, is responsible for trimming a single α-1,2-linked mannose residue from Man9 to produce Man8, but at high enzyme 285



Article

Table 2. RNA-Seq Data (RPKM) for Different Enzymes Involved in Glycosylation gene ID

name

MAN1A1

α-1,2-mannosidase IA

MAN1B1

α-mannosidase IB

MAN2A1 MGAT1

α-mannosidase 2 N-acetyl glucosaminyltransferase I α-1,6-fucosyltransferase α-1,3-fucosyltransferase β-galactoside α-2,6-sialyltransferase 1 CMP-N-acetylneuraminateβ-1,4-galactoside α-2,3sialyltransferase

FUT8 FUT6 ST6GAL1 ST3GAL3

function progressively trim α-1,2-linked mannose residues from Man(9)GlcNAc(2) to produce Man(5)GlcNAc(2) primarily trims Man(9)GlcNAc(2) to produce Man(8)GlcNAc(2), but at high enzyme concentrations, as found in the ER quality control compartment, it further trims the carbohydrates to Man(5−6)GlcNAc(2) catalyzes conversion of high mannose to complex N-glycans initiates complex N-linked carbohydrate formation; essential for the conversion of highmannose to hybrid and complex N-glycans catalyzes the addition of fucose in α-1,6 linkage to the first GlcNAc (core) residue catalyzes the transfer of fucose from GDP-β-fucose to α-2,3-sialylated substrates transfers sialic acid from the donor of substrate CMP-sialic acid to galactose containing acceptor substrates at α-2,6 catalyzes the formation of the NeuAc-α-2,3-Gal-β-1,4-GlcNAc-, NeuAc-α-2,3-Gal-β-1,3GlcNAc- or NeuAc-α-2,3-Gal-β-1,3-GalNAc- sequences found in terminal carbohydrate groups of glycoproteins and glycolipids

LIM1215 (RPKM)

LIM1899 (RPKM)

LIM2405 (RPKM)

2.56

0.89

3.67

2.51

3.48

5.47

3.04 1.58

2.89 4.00

2.37 3.17

3.03 1.55 4.56

5.60 0.24 0.51

1.40 0.51 1.22

0.03

0.16

0.04

CRC, due to elevations in α-1,6-fucosyltransferase (Fut8) level.49−51 Fucosylation is suggested as a promising target for cancer diagnosis and therapeutics and core fucosylation has been suggested as an important feature of tumor progression related to increased metastasis.52 Due to the multienzymatic biosynthetic machinery involved in glycosylation, it is intrinsically difficult to identify the precise mechanisms responsible for specific N-glycosylation alterations in an unambiguous manner. Fundamentally, N-glycosylation can be regulated by changes in glycosylation machinery (e.g., changes in glycosylation enzyme levels or location, levels of nucleotide sugars and the trafficking rate) or by alterations in the glycoproteome with no associated changes in the glycosylation machinery. The β-1,4 GlcNAc branched oligosaccharide structure, commonly referred to as bisecting GlcNAc, was observed at a high level in LIM1215. Bisecting GlcNAc is a glycan modification of hybrid or complex N-glycans where a β-1,4-linked GlcNAc unit is attached to the trimannosyl core structure catalyzed by β-1,4-N-acetylglucosaminyltransferase III (GnT-III), encoded by the Mgat3 gene. Introduction of a bisecting GlcNAc suppresses the formation of 1,6 GlcNAc branching catalyzed by GnT-V, which is strongly associated with cancer metastasis, since GnT-V cannot utilize the bisected oligosaccharide as an acceptor substrate.53 This exclusive observation of the unique bisecting structures in LIM1215 is somewhat surprising since these cells have been derived from omental metastasis.22 In summary, this study provides a comprehensive analysis of the membrane N-glycome in three CRC cell lines and identifies N-glycosylation differences that correlate with the histological and pathological features of the cell lines. We have successfully identified similar and unique N-glycosylation features in LIM1215, LIM1899, and LIM2405 and related the Nglycosylation characteristics to expression levels of glycosylation enzymes. The differences in the glycan signatures between the cell lines and the unique glycosylation phenotypes provide insights into glycan-specific changes associated with malignant progression in CRC and may therefore serve as a molecular feature to differentiate CRC disease stages.

concentration, as found in the ER quality control compartment, it further trims the carbohydrates to Man(5−6); and (iii) Mgat1, which encodes N-acetylglucosaminyltransferase I, a critical enzyme that catalyzes an essential first step in the conversion of high-mannose N-glycans to hybrid and complex N-glycans (Table 2). The elevation of high-mannose glycans, a common theme in different cancers, may be a more complex problem during the synthesis that prohibits the deletion and subsequent addition of carbohydrate residues. The high relative abundance of α-2,6-linked sialic acidcontaining N-glycans in all three cell lines was another notable observation. Increased expression of α-2,6-linked sialic acids on N-glycans often correlates with human cancer progression, metastatic spread, and poor prognosis. This can be explained by up-regulation of the ST6GALI gene encoding the enzyme ST6Gal-1, responsible for the addition of an α-2,6-linked sialic acid to galactose. ST6Gal-1 is expressed at different levels in human tissues with liver possessing higher levels of the enzyme,42 while colons of healthy individuals contain very low levels.43,44 ST6Gal-1 has been shown to be drastically elevated in colorectal carcinomas, leading to their high metastatic potential43,44 and has also been reported in other cancers including breast and cervix and hepatocellular carcinoma.45−47 Increasing the level of α-2,6-sialylation influences several factors that increase the metastatic ability of the tumor cells including decreased cell−cell interactions and increased invasiveness. The exclusive presence of α-2,3-linked sialic acid-containing N-glycan isomers (although in relative small proportions) in LIM2405, may represent an example of aberrant changes to glycomic profiles observed in different cancers, which suggest a correlation between sialic acid linkage and diseases such as cancer. Differential expression of α-2,6-linked and α-2,3-linked sialic acids may be linked to the expression of their corresponding enzymes. At the RNA level, a higher level of ST6Gal1 compared to ST3Gal3 was observed (Table 2). Core fucosylation (α-1,6 fucosylation) was significantly increased compared to nonreducing end (Lewis type) fucosylation across all three cell lines, which correlated well with the observed differences in the expression levels of their corresponding enzymes responsible FUT8 and FUT6, respectively (Table 2). Core fucosylation involving attachment of fucose to the innermost N-acetylglucosamine in N-glycans is synthesized by α-1,6 fucosyltransferase (Fut8),48 while Fut6 is involved in the synthesis of Lewis types of fucosylation. Increased core fucosylation has been shown in cancer, including

■

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org. 286



■

Article

(15) Liu, Y. C.; Yen, H. Y.; Chen, C. Y.; Chen, C. H.; Cheng, P. F.; Juan, Y. H.; Khoo, K. H.; Yu, C. J.; Yang, P. C.; Hsu, T. L.; Wong, C. H. Sialylation and fucosylation of epidermal growth factor receptor suppress its dimerization and activation in lung cancer cells. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (28), 11332−11337. (16) Saldova, R.; Fan, Y.; Fitzpatrick, J. M.; Watson, R. W.; Rudd, P. M. Core fucosylation and alpha2−3 sialylation in serum N-glycome is significantly increased in prostate cancer comparing to benign prostate hyperplasia. Glycobiology 2011, 21 (2), 195−205. (17) Cazet, A.; Julien, S.; Bobowski, M.; Krzewinski-Recchi, M. A.; Harduin-Lepers, A.; Groux-Degroote, S.; Delannoy, P. Consequences of the expression of sialylated antigens in breast cancer. Carbohydr. Res. 2010, 345 (10), 1377−1383. (18) Kudo, T.; Ikehara, Y.; Togayachi, A.; Morozumi, K.; Watanabe, M.; Nakamura, M.; Nishihara, S.; Narimatsu, H. Up-regulation of a set of glycosyltransferase genes in human colorectal cancer. Lab. Invest. 1998, 78 (7), 797−811. (19) Pan, S.; Chen, R.; Aebersold, R.; Brentnall, T. A. Mass spectrometry based glycoproteomics–from a proteomics perspective. Mol. Cell. Proteomics 2011, 10 (1), R110 003251. (20) Drake, P. M.; Cho, W.; Li, B.; Prakobphol, A.; Johansen, E.; Anderson, N. L.; Regnier, F. E.; Gibson, B. W.; Fisher, S. J. Sweetening the pot: adding glycosylation to the biomarker discovery equation. Clin. Chem. 2010, 56 (2), 223−236. (21) Fanayan, S.; Smith, J. T.; Lee, L. Y.; Yan, F.; Snyder, M.; Hancock, W. S.; Nice, E. Proteogenomic analysis of human colon carcinoma cell lines LIM1215, LIM1899, and LIM2405. J. Proteome Res. 2013, 12, 1732−1742. (22) Fanayan, S.; Smith, J. T.; Sethi, M. K.; Cantor, D.; Goode, R.; Simpson, R. J.; Baker, M. S.; Hancock, W. S.; Nice, E. Chromosome 7centric analysis of proteomics data from a panel of human colon carcinoma cell lines. J. Proteome Res. 2013, 12 (1), 89−96. (23) Whitehead, R. H.; Macrae, F. A., St; John, D. J.; Ma, J. A colon cancer cell line (LIM1215) derived from a patient with inherited nonpolyposis colorectal cancer. J. Natl. Cancer Inst. 1985, 74 (4), 759− 765. (24) Andrew, S. M.; the, J. G.; Johnstone, R. W.; Russell, S. M.; Whitehead, R. H.; McKenzie, I. F.; Pietersz, G. A. Tumor localization by combinations of monoclonal antibodies in a new human colon carcinoma cell line (LIM1899). Cancer Res. 1990, 50 (17), 5225− 5230. (25) Whitehead, R. H.; Zhang, H. H.; Hayward, I. P. Retention of tissue-specific phenotype in a panel of colon carcinoma cell lines: relationship to clinical correlates. Immunol. Cell Biol. 1992, 70 (Pt 4), 227−236. (26) Nagalakshmi, U.; Waern, K.; Snyder, M. RNA-Seq: a method for comprehensive transcriptome analysis. Curr. Protoc. Mol. Biol. 2010, Chapter 4, Unit 4.11.1−13. (27) Lee, A.; Kolarich, D.; Haynes, P. A.; Jensen, P. H.; Baker, M. S.; Packer, N. H. Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture. J. Proteome Res. 2009, 8 (2), 770−781. (28) Jensen, P. H.; Karlsson, N. G.; Kolarich, D.; Packer, N. H. Structural analysis of N- and O-glycans released from glycoproteins. Nat. Protoc. 2012, 7 (7), 1299−1310. (29) Pabst, M.; Bondili, J. S.; Stadlmann, J.; Mach, L.; Altmann, F. Mass + retention time = structure: a strategy for the analysis of Nglycans by carbon LC-ESI-MS and its application to fibrin N-glycans. Anal. Chem. 2007, 79 (13), 5051−5057. (30) Thaysen-Andersen, M.; Mysling, S.; Hojrup, P. Site-specific glycoprofiling of N-linked glycopeptides using MALDI-TOF MS: strong correlation between signal strength and glycoform quantities. Anal. Chem. 2009, 81 (10), 3933−3943. (31) Harmon, B. J.; Gu, X.; Wang, D. I. Rapid monitoring of sitespecific glycosylation microheterogeneity of recombinant human interferon-gamma. Anal. Chem. 1996, 68 (9), 1465−1473. (32) Gil, G. C.; Kim, Y. G.; Kim, B. G. A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted

AUTHOR INFORMATION

Corresponding Author

*Tel: 61-2-98508260. Fax: 61-2-98508313. E-mail: susan. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was facilitated by access to Australian Proteomics Analysis Facility (APAF). We thank Mr. Vignesh Venkatakrishnan for assistance with glycan data analysis and Mrs. Debra Birch (Macquarie University Microscopy Unit) for assistance with fluorescence microscopy.

■

ABBREVIATIONS CRC, colorectal cancer; CID, collision-induced fragmentation; HNPCC, hereditary nonpolyposis colon cancer; PNGase F, peptide-N-glycosidase F; GlcNAc, N-acetyl-glucosamine; EIC, extracted ion chromatogram; RT, retention time

■

REFERENCES

(1) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. Global cancer statistics. Ca-Cancer J. Clin. 2011, 61 (2), 69−90. (2) Tjalsma, H. Identification of biomarkers for colorectal cancer through proteomics-based approaches. Expert Rev. Proteomics 2010, 7 (6), 879−895. (3) Etzioni, R.; Urban, N.; Ramsey, S.; McIntosh, M.; Schwartz, S.; Reid, B.; Radich, J.; Anderson, G.; Hartwell, L. The case for early detection. Nat. Rev. Cancer 2003, 3 (4), 243−252. (4) Rennert, G. Prevention and early detection of colorectal cancer– new horizons. Recent Results Cancer Res. 2007, 174, 179−187. (5) Levin, T. R.; Zhao, W.; Conell, C.; Seeff, L. C.; Manninen, D. L.; Shapiro, J. A.; Schulman, J. Complications of colonoscopy in an integrated health care delivery system. Ann. Intern. Med. 2006, 145 (12), 880−886. (6) Morikawa, T.; Kato, J.; Yamaji, Y.; Wada, R.; Mitsushima, T.; Shiratori, Y. A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology 2005, 129 (2), 422−428. (7) Reis, C. A.; Osorio, H.; Silva, L.; Gomes, C.; David, L. Alterations in glycosylation as biomarkers for cancer detection. J. Clin. Pathol. 2010, 63 (4), 322−329. (8) Adamczyk, B.; Tharmalingam, T.; Rudd, P. M. Glycans as cancer biomarkers. Biochim. Biophys. Acta 2012, 1820 (9), 1347−1353. (9) Kim, E. H.; Misek, D. E. Glycoproteomics-based identification of cancer biomarkers. Int. J. Proteomics 2011, 2011, 601937. (10) Dennis, J. W.; Laferte, S. Oncodevelopmental expression of GlcNAc beta 1−6Man alpha 1−6Man beta 1-branched asparaginelinked oligosaccharides in murine tissues and human breast carcinomas. Cancer Res. 1989, 49 (4), 945−950. (11) Li, W. P.; Zuber, C.; Heitz, P. U.; Roth, J. Cytochemical staining for beta 1,6 branching of asparagine-linked oligosaccharides in variants of metastatic human colon carcinoma cells. Am. J. Pathol. 1994, 145 (2), 470−480. (12) Jin, X. L.; Liu, H. B.; Zhang, Y.; Wang, B. S.; Chen, H. L. Alteration in N -acetylglucosaminyltransferase activities and glycan structure in tissue and bile glycoproteins from extrahepatic bile duct carcinoma. Glycoconjugate J. 2004, 20 (6), 399−406. (13) de Leoz, M. L.; Young, L. J.; An, H. J.; Kronewitter, S. R.; Kim, J.; Miyamoto, S.; Borowsky, A. D.; Chew, H. K.; Lebrilla, C. B. Highmannose glycans are elevated during breast cancer progression. Mol. Cell. Proteomics 2011, 10 (1), M110 002717. (14) Muinelo-Romay, L.; Gil-Martin, E.; Fernandez-Briera, A. Alpha(1,2)fucosylation in human colorectal carcinoma. Oncol. Lett. 2010, 1 (2), 361−366. 287



Article

laser desorption/ionization time-of-flight mass spectrometry. Anal. Biochem. 2008, 379 (1), 45−59. (33) Harvey, D. J. Quantitative aspects of the matrix-assisted laser desorption mass spectrometry of complex oligosaccharides. Rapid Commun. Mass Spectrom. 1993, 7 (7), 614−619. (34) Harvey, D. J. Fragmentation of negative ions from carbohydrates: part 3. Fragmentation of hybrid and complex N-linked glycans. J. Am. Soc. Mass Spectrom. 2005, 16 (5), 647−659. (35) Harvey, D. J.; Royle, L.; Radcliffe, C. M.; Rudd, P. M.; Dwek, R. A. Structural and quantitative analysis of N-linked glycans by matrixassisted laser desorption ionization and negative ion nanospray mass spectrometry. Anal. Biochem. 2008, 376 (1), 44−60. (36) Harvey, D. J. Fragmentation of negative ions from carbohydrates: part 2. Fragmentation of high-mannose N-linked glycans. J. Am. Soc. Mass Spectrom. 2005, 16 (5), 631−646. (37) Harvey, D. J.; Jaeken, J.; Butler, M.; Armitage, A. J.; Rudd, P. M.; Dwek, R. A. Fragmentation of negative ions from N-linked carbohydrates, part 4. Fragmentation of complex glycans lacking substitution on the 6-antenna. J. Mass Spectrom. 2010, 45 (5), 528− 535. (38) Ito, H.; Yamada, K.; Deguchi, K.; Nakagawa, H.; Nishimura, S. Structural assignment of disialylated biantennary N-glycan isomers derivatized with 2-aminopyridine using negative-ion multistage tandem mass spectral matching. Rapid Commun. Mass Spectrom. 2007, 21 (2), 212−218. (39) Zhao, Y. Y.; Takahashi, M.; Gu, J. G.; Miyoshi, E.; Matsumoto, A.; Kitazume, S.; Taniguchi, N. Functional roles of N-glycans in cell signaling and cell adhesion in cancer. Cancer Sci. 2008, 99 (7), 1304− 1310. (40) Tao, S. C.; Li, Y.; Zhou, J.; Qian, J.; Schnaar, R. L.; Zhang, Y.; Goldstein, I. J.; Zhu, H.; Schneck, J. P. Lectin microarrays identify cellspecific and functionally significant cell surface glycan markers. Glycobiology 2008, 18 (10), 761−769. (41) Lattova, E.; Varma, S.; Bezabeh, T.; Petrus, L.; Perreault, H. Mass spectrometric profiling of N-linked oligosaccharides and uncommon glycoform in mouse serum with head and neck tumor. J. Am. Soc. Mass Spectrom. 2008, 19 (5), 671−685. (42) Paulson, J. C.; Weinstein, J.; Schauer, A. Tissue-specific expression of sialyltransferases. J. Biol. Chem. 1989, 264 (19), 10931−10934. (43) Dall’Olio, F.; Chiricolo, M.; Lau, J. T. Differential expression of the hepatic transcript of beta-galactoside alpha2,6-sialyltransferase in human colon cancer cell lines. Int. J. Cancer 1999, 81 (2), 243−247. (44) Gessner, P.; Riedl, S.; Quentmaier, A.; Kemmner, W. Enhanced activity of CMP-neuAc:Gal beta 1−4GlcNAc:alpha 2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients. Cancer Lett. 1993, 75 (3), 143−149. (45) Recchi, M. A.; Hebbar, M.; Hornez, L.; Harduin-Lepers, A.; Peyrat, J. P.; Delannoy, P. Multiplex reverse transcription polymerase chain reaction assessment of sialyltransferase expression in human breast cancer. Cancer Res. 1998, 58 (18), 4066−4070. (46) Wang, P. H.; Lee, W. L.; Lee, Y. R.; Juang, C. M.; Chen, Y. J.; Chao, H. T.; Tsai, Y. C.; Yuan, C. C. Enhanced expression of alpha 2,6-sialyltransferase ST6Gal I in cervical squamous cell carcinoma. Gynecol. Oncol. 2003, 89 (3), 395−401. (47) Pousset, D.; Piller, V.; Bureaud, N.; Monsigny, M.; Piller, F. Increased alpha2,6 sialylation of N-glycans in a transgenic mouse model of hepatocellular carcinoma. Cancer Res. 1997, 57 (19), 4249− 4256. (48) Uozumi, N.; Yanagidani, S.; Miyoshi, E.; Ihara, Y.; Sakuma, T.; Gao, C. X.; Teshima, T.; Fujii, S.; Shiba, T.; Taniguchi, N. Purification and cDNA cloning of porcine brain GDP-L-Fuc:N-acetyl-beta-Dglucosaminide alpha1→6fucosyltransferase. J. Biol. Chem. 1996, 271 (44), 27810−27817. (49) Peracaula, R.; Barrabes, S.; Sarrats, A.; Rudd, P. M.; de Llorens, R. Altered glycosylation in tumours focused to cancer diagnosis. Dis. Markers 2008, 25 (4−5), 207−218. (50) Noda, K.; Miyoshi, E.; Gu, J.; Gao, C. X.; Nakahara, S.; Kitada, T.; Honke, K.; Suzuki, K.; Yoshihara, H.; Yoshikawa, K.; Kawano, K.;

Tonetti, M.; Kasahara, A.; Hori, M.; Hayashi, N.; Taniguchi, N. Relationship between elevated FX expression and increased production of GDP-L-fucose, a common donor substrate for fucosylation in human hepatocellular carcinoma and hepatoma cell lines. Cancer Res. 2003, 63 (19), 6282−6289. (51) Muinelo-Romay, L.; Vazquez-Martin, C.; Villar-Portela, S.; Cuevas, E.; Gil-Martin, E.; Fernandez-Briera, A. Expression and enzyme activity of alpha(1,6)fucosyltransferase in human colorectal cancer. Int. J. Cancer 2008, 123 (3), 641−646. (52) Dennis, J. W.; Laferte, S.; Waghorne, C.; Breitman, M. L.; Kerbel, R. S. Beta-1−6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 1987, 236 (4801), 582− 585. (53) Gu, J.; Nishikawa, A.; Tsuruoka, N.; Ohno, M.; Yamaguchi, N.; Kangawa, K.; Taniguchi, N. Purification and characterization of UDPN-acetylglucosamine: alpha-6-D-mannoside beta 1−6 N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase V) from a human lung cancer cell line. J. Biochem. 1993, 113 (5), 614−619.

288


Comparative N-Glycan Profiling of Colorectal Cancer Cell Lines

Recommend Documents