Simultaneous analyses of N-linked and O-linked glycans of ovarian cancer cells using solid-phase chemoenzymatic method
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Glycans play critical roles in a number of biological activities. Two common types of glycans, N-linked and O-linked, have been extensively analyzed in the last decades. N-glycans are typically released from glycoproteins by enzymes, while O-glycans are released from glycoproteins by chemical methods. It is important to identify and quantify both N- and O-linked glycans of glycoproteins to determine the changes of glycans.
The effort has been dedicated to study glycans from ovarian cancer cells treated with O-linked glycosylation inhibitor qualitatively and quantitatively. We used a solid-phase chemoenzymatic approach to systematically identify and quantify N-glycans and O-glycans in the ovarian cancer cells. It consists of three steps: (1) immobilization of proteins from cells and derivatization of glycans to protect sialic acids; (2) release of N-glycans by PNGase F and quantification of N-glycans by isobaric tags; (3) release and quantification of O-glycans by β-elimination in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP).
We used ovarian cancer cell lines to study effect of O-linked glycosylation inhibitor on protein glycosylation. Results suggested that the inhibition of O-linked glycosylation reduced the levels of O-glycans. Interestingly, it appeared to increase N-glycan level in a lower dose of the O-linked glycosylation inhibitor. The sequential release and analyses of N-linked and O-linked glycans using chemoenzymatic approach are a platform for studying N-glycans and O-glycans in complex biological samples.
The solid-phase chemoenzymatic method was used to analyze both N-linked and O-linked glycans sequentially released from the ovarian cancer cells. The biological studies on O-linked glycosylation inhibition indicate the effects of O-glycosylation inhibition to glycan changes in both O-linked and N-linked glycan expression.
KeywordsChemoenzymatic Glycoprotein Glycomics Solid phase
glycoprotein immobilization for glycan extraction
high-performance liquid chromatography
matrix assisted laser desorption/ionization
Quaternary Amine Containing Isobaric Tag for Glycan
Glycosylation is one of the most abundant and diverse protein modifications. It plays essential roles in the biological and physiological functions of a living organism . Aberrant glycosylation is associated with different diseases, e.g. prostate cancer , ovarian cancer [3, 4], rheumatoid arthritis , diabetes , and cardiac diseases [7, 8]. Studies reveal that cancer cells often display their glycans at different levels of structures as compared to those observed on normal cells . Glycosylation can thus be harnessed for defining cancer malignancy and disease progression [10, 11]. The abnormal glycosylation may contribute to cancer metastasis [12, 13]. Therefore, it is important to characterize protein glycosylation in biological and clinical specimens.
The N-linked and O-linked glycans are two most commonly studied glycoforms in protein glycosylation. The N-glycan has common core structure (GlcNAc2Man3) that conjugates to the asparagine (Asn or N) residues in the consensus peptide motif of Asn-X-Ser/Thr [where X is any amino acid except proline (Pro)]; The O-glycan conjugates to serine (Ser) or threonine (Thr) without a consensus amino-acid motif. The structure of glycans is complex due to its non-template biosynthesis pathway. The complexity is predominantly due to its variable monosaccharides, branches, linkages, and isomers.
It is preferable to analyze both N-glycans and O-glycans from glycoproteins; technology development to achieve this goal has been the focus for glycomics [14, 15, 16, 17, 18, 19, 20]. Release of these glycans from glycoproteins can be fulfilled by enzymes or chemical reactions. PNGase F (peptide: N-glycosidase F) releases all N-glycans except for glycans with core-α(1,3)-fucose that are found only in slime molds, plants, insects, and parasites plant and insect , whereas PNGase A (peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase) releases these N-glycans from glycopeptides including core-α(1,3)-fucose and all N-glycans released by PNGase F . However, no universal O-glycosidase has been developed for the removal of all O-glycans except for core 1 (Gal-GalNAc) or core 3 (GlcNAc-GalNAc). The removal of O-glycans is usually performed through alkali treatment using β-elimination [23, 24] or hydrazinolysis [25, 26]. Chemical release is cost-effective and can be ubiquitously applied to release different types of glycans. Hydrazine hydrolysis releases both O-glycans (60 °C) and N-glycans (95 °C) [26, 27]. However, even at a relatively lower temperature for O-glycans release (60 °C), it can still result in N-glycan release. The recently reported oxidative strategy releases all types of glycans including N-glycans and O-glycans without specificity . It has been reported that O-glycans can be specifically released at a mild β-elimination such as ammonia ; however, others showed that ammonia (26–28%) alone could also release both N-glycans and O-glycans . Additional consideration with glycans released by the chemical methods is the sequential degradation of reducing-end monosaccharide units by consecutive β-elimination, also known as “peeling” [30, 31]. The peeling of the alditols on the reducing end is showed to be prevented by release of O-glycans in a mild medium in the presence of reagents for alditol capping . Several chemical compounds have been exploited for the capping of O-glycan alditol after β-elimination. Among them, pyrazolone derivatives have been used for capping the alditol and enhancing hydrophobicity of glycans for LC–ESI–MS [33, 34].
An integrated platform has been sought for the comprehensive profiling of glycans [16, 17, 18, 19, 20, 35]. Numerous N-glycan studies have shown that native sialic acid residues are fragile and may be easily lost during sample preparation and ionization in MALDI-MS [14, 36, 37, 38]. Stabilization by chemical methods such as amidation , methyl esterification , permethylation [18, 19, 40], and perbenzolylation  has been developed for analysis of sialylated glycans. For example, glycoproteins are systematically analyzed by immobilizing on polymer membranes for sequential release of N-glycans and O-glycans . Structural analysis can be achieved via sialidases or exoglycosidases in coupling with porous graphitized liquid chromatography-mass spectrometry . Mass spectrometric screening strategy is developed for characterizing glycan component of both glycosphingolipids and glycoproteins from a single sample . These methods have been widely used for analysis of glycans in biological specimens, such as ovarian cancers from serum and cell lines [4, 42, 43, 44].
It has been successfully demonstrated that sialic acid residues can be effectively stabilized using an in-solution amidation [37, 45]. Permethylation of the released glycans can protect sialic acids for both N- and O-glycans [46, 47]. Yet, the decomposition of O-acetyl groups may occur under the harsh conditions used for permethylation . Besides, the permethylated glycans may lose their reactivity on the reducing-ends, consequently preventing their further use for fluorophore, chromophore, or isobaric tag labeling . To this end, we recently developed a solid-phase chemoenzymatic platform termed as glycoprotein immobilization for glycan extraction (GIG) by conjugating glycoproteins on solid phase, protecting sialic acids, and sequentially releasing N- and O-linked glycans for MS analyses [14, 49, 50].
Glycoprofiling on ovarian cancer serum found that unique N-glycans were present in cancer patient . Profiling of N-glycans by a nanoLC mass spectrometric method observed up-regulation of the fucosylated N-glycans in healthy controls . Recent works discovered the glycosylation changes in ovarian cancers were influenced by aberrant regulation of gene expression. The characteristic glycan features that were unique to the ovarian cancer membrane proteins have been identified, including “bi-secting N-acetyl-glucosamine” and “N,N′-diacetyl-lactosamine” type N-glycans . These glycosylation changes in ovarian cancer may contribute to disease pathogenesis . Therefore, inhibition of protein glycosylation may be useful for ovarian cancer treatment. In this study, we applied the quantitative glycomics to the analyses of both N- and O-linked glycans in ovarian cancer cells in the presence and absence of inhibitor for O-linked glycosylation. The glycosylation changes on both N-glycans and O-glycans are described.
Reagents and sample preparation
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. Aminolink resin, spin columns (snap cap), and Zeba spin desalting columns were purchased from Life Technologies (Grand Island, NY). Alltech Extract-Clean Carbograph columns, analytical column [NanoViper, 75 μm (ID), 150 mm, 2 µm particle size], water, methanol, and acetonitrile (ACN) (HPLC grade) were purchased from Fisher Sci. (Waltham, MA). NaCl solution (5 M) was ordered from ChemCruz Biochemicals (Santa Cruz, CA). Chloroform was purchased from J.T. Baker (VWR, Radnor, PA). Cell lysis buffer consists of 1× PBS, 1% NP-40, 0.5% sodium deoxycholate (C24H39NaO4), 0.1% SDS, 2 mM EDTA, and 50 mM NaF. Micro-centrifuge tubes (1–2 mL) were purchased from Denville Scientific Inc. (Holliston, MA). Sep-Pak C18 1 cc Vac Cartridges (50 mg sorbent per cartridge, 55–105 μm particle size) were purchased from Waters Corporation. Peptide-N-glycosidase F (PNGase F), denaturing buffer (10×), and GlycoBuffer (G7; 10×) were from New England Biolabs (Ipswich, MA).
OVCAR-3 cell culture and treatment
OVCAR-3 cell line (ATCC® HTB-161TM) was purchased from ATCC (American Type Culture Collection). Cell culture was proceed according to ATCC protocol. The culture medium consists of RPMI-1640 (Thermo Fisher), 0.01 mg/mL bovine insulin (Sigma), and 20% fetal bovine serum (Sigma). OVCAR-3 cells were suspended in a 15-cm cell culture dish (Thermo Fisher). O-GalNAc inhibitor (Benzyl-α-GalNAc or BAG; Sigma) was dissolved in DMSO (Dimethyl sulfoxide; Sigma) (100 mM). A final concentration of BAG (0, 0.2, 1, 2 mM) was added to OVCAR-3 for 24-h treatment. Cells were washed by 1× PBS three times before harvest in 1.5 mL microcentrifuge tube, followed by cell lysis in 500 μL of 1× binding buffer. Protein concentration was determined by BCA assay (Thermo Fisher). One mg protein was used for glycan analysis.
Protein immobilization on solid phase and N-glycan release
Proteins were first extracted from cells using cell lysis buffer. Proteins (1 mg) were denatured at 100°C for 10 min in 100 μL solution consisting of 10 μL 10× denaturing buffer and 90 μL deionized (DI) water. After Aminolink resin was pre-conditioned by 1× binding buffer (500 μL; 3×) (pH 10; 100 mM sodium citrate and 50 mM sodium carbonate) , the denatured proteins were mixed with resin in a spin column by adding 350 μL DI water and 50 μL 10× binding buffer. The reaction proceeded up to 4 h with mixing at room temperature, followed by incubation for another 4 h after adding 25 μL of 1 M NaCNBH3. Next, resin was rinsed using 500 μL 1× PBS (3×) (Thermo Fisher). The conjugation continued for 4 h in 500 μL 1× PBS in the presence of 50 mM NaCNBH3. The active aldehyde sites on the resin were blocked using 500 μL 1× Tris–HCl (50 mM NaCNBH3). After washing the resin using 1 M NaCl and DI water (500 μL, 3×), the sialic acid residues were reacted with 1 M p-Toluidine (pT, Sigma) buffer via carbodiimide coupling (3 h). The pT buffer (465 μL) consisted of 400 μL of 1 M pT, 25 μL HCl (36–38%), and 40 μL EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide). To remove chemical compounds such as pT and EDC, the resin was extensively washed with four solutions (500 μL) in a sequential order of 10% formic acid (3×), 10% acetonitrile in 0.1% TFA (trifluoroacetic acid) (3×), 1 M NaCl (3×), and DI (3×). N-glycans were then enzymatically released by 2 μL PNGase F (1000 units; 360 μL DI, 40 μL 10× GlycoBuffer; 37 °C, 3 h). The released N-glycans were purified by Carbograph as described in our previous protocol .
Chemical release of O-glycans from solid phase
Mass spectrometry and data analysis
MALDI (matrix-assisted laser desorption/ionization) was performed using Shimadzu Resonance Maxima QIT-ToF. Laser energy was 140–160; 1 μL of DHB (2,5-dihydroxybenzoic acid)-DMA (dimethylaniline) was mixed with 1 μL of glycans. The modified glycans were cleaned by a C18-SPE trap column (Thermo Scientific; Dionex nanoViper Fingertight Fitting). Sample (12 μL) was injected to the trap column (C18) by the loading pump at a flow rate of 5 μL/min. The nano-flow pump (Thermo Scientific; Dinoex UltiMate 3000) was set at a flow rate of 0.25 μL/min; the LC gradient was set from 4% (acetonitrile, 0.1% TFA) to 50% within 70 min using an analytical column (Fisher Scientific; Thermo Scientific Acclaim PepMap 100 C18). The full scan MS1 mass range was from 400 to 1800 Da (m/z) using positive mode (Thermo Scientific; Orbitrap Velos; collision-induced dissociation: 30%). The MS2 parameters were as follows: collision energy 29%, isolation width 2.0, m/z, activation time 0.2 ms, and HCD (high-energy collision dissociation). Dynamic exclusion included repeat count 2, repeat duration 25 s, exclusion list size 500, and exclusion duration 5 s. Glycan spectra were analyzed using Thermo Xcalibur Qual Browser. Glycan composition was determined by (1) precursor matching and further confirmed by MS2 fragments (Additional file 1: Figure S1, 37 MS/MS); and (2) database matching using CFG (http://www.functionalglycomics.org), GlycomeDB (http://www.glycome-db.org) and Glycosciences (http://www.glycosciences.de/database/index.php) for those low abundance glycans. Glycans without MS/MS were given by their composition (N: HexNAc; H: Hexose; F: Fucose; S: NeuAc). The figures depicting the glycan structures were plotted using Glycoworkbench 2.1 software .
Results and discussion
GIG consists of three steps: (1) the denatured proteins are conjugated on a solid support (amine-reactive resin (aldehyde)) via reductive amination (Fig. 1a); the immobilized proteins are modified via carbodiimide coupling on the solid support for stabilization of the sialic acids (Fig. 1b); (2) N-glycans are released by PNGase F treatment (Fig. 1c) and labeled with isobaric tags (QUANTITY) for relative quantification  (Fig. 1d); (3) O-glycans are released from the solid support via β-elimination using ammonia in the presence of PMP (Fig. 1e). The labeled N-glycans and O-glycans are identified and quantified by LC–MS/MS.
Sequential release of N-glycans and O-glycans
Protection of sialylated O-glycans
The sialic acids are fragile and preferentially lost during sample preparation and ionization in MS. Sialic acid is negatively charged and hydrophilic, thus its identification is ineffective in the positive ionization mode for MS. The negative ionization mode is commonly used and has been well developed for the analysis of intact sialic acids [56, 57]. Modification of sialic acid provides several advantages: (1) stabilization of sialic acids, (2) neutralization of negative charge, and (3) enhanced hydrophobicity. Similar to modification on N-glycans , the sialic acid residues of O-glycans are simultaneously protected via carbodiimide coupling (Fig. 1b).
Analyses of N- and O-glycans from ovarian cancer cells treated with O-glycosylation inhibitor
We then applied the sequential release and analyses of N- and O-linked glycans from OVCAR-3 cells treated with Benzyl-α-GalNAc (BAG) to inhibit α-GalNAc biosynthesis. Different concentrations of BAG (0 mM (control), 0.2, 1, and 2 mM) were used to treat OVCAR-3 cells for 24 h. Cells were harvested and proteins were extracted. After protein (1 mg for each sample) immobilization, N-glycans were first released by PNGase F, followed by Carbograph cleanup . One tenth of the N-glycans was loaded onto MALDI-MS for comparing the glycan profile from OVCAR-3 cells treated with different concentrations of BAG. An internal standard (25 μM/1 μL DP7) was used to determine the abundance of N-glycans as indicated in the Additional file 2: Table S1 (MALDI-OV3-Nglycan). Several observations are evident from the MALDI-MS analysis of N-glycans: (1) Oligomannoses are highly abundant N-glycans in OVCAR-3 cells; (2) Among the abundant oligomannose glycans, Man6 is the most abundant compared to other oligomannose glycans; and (3) Most oligomannoses are upregulated in 0.2 mM BAG-treated cells. The MALDI-MS profile of BAG-treated cell lines indicated that oligomannoses are highly abundant N-linked glycans in OVCAR-3 cells and affected by treatments using different concentrations of BAG.
The detail mechanism of N-glycan upregulation in BAG treated ovarian cancer cells is unclear. It has been indicated that the glycosylation of proteins in Golgi and in-transit glycoproteins could be affected by BAG . Several polypeptide-N-acetyl-galactosaminyltransferases (ppGalNAcTs) are located throughout the Golgi, where N-glycans are synthesized. BAG inhibition could essentially affect many transcriptional factors that may regulate genes associated with N-glycan synthesis . Therefore, the inhibition of O-GalNAc glycans might indirectly affect N-glycan biosynthesis .
Mucin-type O-glycans are critically regulated in cancers. For example, when CA125, an ovarian cancer marker, purified from the spent media of OVCAR-3 cells, O-glycomic analysis revealed that the sialylated O-glycans were highly abundant, containing NS, NHS and N2H2S; three dominant non-sialylated O-glycans were N2H2, N3H2, and N3H3 . Our results indicate that the sialylated O-glycans in OVCAR-3 cells are effectively inhibited by BAG; however, non-sialylated O-glycans remain minimally regulated by inhibition of O-glycan biosynthesis. These observations are consistent with previous studies, indicating that BAG inhibition leads to a decrease of mucus secretion and a decreased intracellular amount of sialic acid [60, 63]. For example, BAG can impede the sialylation of O-glycosidic sugar chains on CD44, and the inhibition enhances experimental metastatic capacity in melanoma cells . Subsequent studies have explored the possibility that the change of sialic acids in cells might be a consequence of the metabolic processing of BAG into Gal-BAG, which is a potent competitive inhibitor of the Gal-GalNAc-α2,3-sialyltransferase [62, 66]. Further inhibition of O-GalNAc glycosylation can be achieved by increasing the concentration of BAG (4–8 mM) and extending the treatment up to 72 h [61, 64, 67].
A streamlined approach is used for the systematic identification and quantification of N-linked and O-linked glycans in the ovarian cancer cells. The performance of the platform is evaluated by the analysis of glycans in standard N- and O-linked glycoproteins. The stabilization of sialic acids by carbodiimide coupling to the solid support enhances the detection of sialylated glycans, which are not observed without sialic acid modification using in-solution β-elimination.
Inhibition of ovarian cancer cells by an O-GalNAc-targeted inhibitor appears to up-regulate N-glycans and down-regulate mucin-type O-glycans by two independent experiments using label-free glycomic analysis and isobaric labeled N-glycan analysis. To our knowledge, this is the first report to show the levels of N-glycans are regulated by O-linked glycosylation by O-GalNAc inhibitor. Even though the mechanism of this regulation is unclear, results indicate that a low concentration of O-GalNAc inhibitor might favor the biosynthesis of N-glycans in OVCAR-3 cells. The regulation of glycosylation biosynthesis by drugs should include considerations of their effects on both N-linked and O-linked glycans.
SY and HZ designed the method. SY drafted the manuscript and conducted the experiments. HZ revised the manuscripts. NH, YL, LC, and LZ helped on cell cultures and sample preparation. WY helps on the O-glycan identification. SL synthesized QUANTITY and helped on quantitation. All authors read and approved the final manuscript.
We thank Drs. Thomas Stefani and Punit Shah from Johns Hopkins for help on LC–MS.
The authors declare that they have no competing interests.
Availability of data and material
The Supporting Information is available free of charge via the Internet at https://clinicalproteomicsjournal.biomedcentral.com/.
This work was supported by the National Institutes of Health, National Cancer Institute, the Early Detection Research Network (EDRN, U01CA152813), the Clinical Proteomic Tumor Analysis Consortium (CPTAC, U24CA160036), National Heart Lung and Blood Institute, Programs of Excellence in Glycosciences (PEG, P01HL107153), and the National Institute of Allergy and Infectious Diseases (R21AI122382), by Maryland Innovation Initiative (MII), and by The Patrick C. Walsh Prostate Cancer Research Fund.
Consent for publication
This manuscript is solely submitted to Clinical Proteomics for consideration.
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