Differential expression of N-linked oligosaccharides in methotrexate-resistant primary central nervous system lymphoma cells

Background Oligosaccharides of glycoprotein, particularly negatively-charged sialylated N-glycans, on the surface of lymphomas play important roles in cell–cell interactions and bind immunoglobulin-like lectins, causing inflammatory responses and bioregulation. However, their characterizations have largely been unknown in central nervous system (CNS) lymphoma. Methods Here, we investigated expression patterns of N-linked oligosaccharides of glycoproteins in cells derived from CNS lymphomas and clinical specimens. Results We first generated methotrexate (MTX)-resistant cells derived from HKBML and TK as CNS lymphoma, and RAJI as non-CNS lymphoma and determined N-linked oligosaccharide structures in these cells and other non-CNS lymphoma-derived cells including A4/FUK, OYB, and HBL1. Major components of the total oligosaccharides were high-mannose type N-glycans, whose level increased in MTX-resistant HKBML and TK but decreased in MTX-resistant RAJI. We also detected sialylated biantennary galactosylated N-glycans with α1,6-fucosylation, A2G2F, and A2G2FB from HKBML, TK, and RAJI. Sialylated A4G4F was specifically isolated from RAJI. However, the ratios of these sialylated N-glycans slightly decreased against MTX-resistant compared to non-resistant cells. Interestingly, almost all complex-type oligosaccharides were α2,6-sialylated. Discussion This is the first study for the expression profile of N-oligosaccharides on MTX-resistant primary CNS lymphoma-derived cells HKBML and TK, and tumor tissues resected from patients with CNS lymphoma, Conclusion These results propose a possibility that the differential expression of high-mannose types and sialylated A2G2F, A2G2FB, and A4G4F on the surface of CNS lymphomas may provide a hint for targets for diagnoses and treatments of the oligosaccharide type-specific lymphomas.


Background
Oligosaccharides are saccharide polymers that have various functions such as cell-cell interactions and cell recognitions, including immune responses [1]. Glycans are normally formed by presenting sugar chains linked to lipids or amino acid side chains by Nor O-glycosidic bonds [2]. The O-linked oligosaccharide is attached to threonine (Thr) or serine (Ser) [3], while the N-linked oligosaccharide is generally pentasaccharides attached to asparagine (Asn) by β-linkage to amine nitrogen of side chains [4]. The N-oligosaccharides of glycoproteins show differential patterns of branching formations by N-acetylglucosaminyltransferase activities at N-acetylglucosamine (GlcNAc) residues in types of Pl-2, Pl-4, and B1 + 6 linkage to mannose (Man) residues of the core [5,6]. Previous studies have also demonstrated the mechanisms of intracellular trafficking of the cell surface of glycoproteins and their subsequent returns to the cell surface [7][8][9].
Several studies have clarified that the structures of Nlinked oligosaccharide chains on glycoproteins have been involved in tumor cell adhesion to the extracellular matrix (ECM), metastatic potentials, and cell proliferation and differentiation [8,[10][11][12][13][14][15]. Metastatic potentials of tumor cells have been shown to correlate with the expression of highly branched tri-and tetra-antennary β-1, 6-GlcNAc-bearing N-glycans [16,17]. The increased β1, 6-GlcNAc-bearing N-glycan expression is co-regulated by N-acetyl glucosaminyl transferases V (GnT-V) and the Ets-1 transcription factor, and the branching complex type N-glycans function in glioma invasivity [16]. N-acetyl glycan structures and physicochemical properties regulate cell proliferation and differentiation in leukemia [12]. A semi-automated systematic detection system for analyzing the N-linked oligosaccharides of glycoproteins has been developed [18,19]. N-linked oligosaccharides are relatively easily detectable from a small amount of acetone-precipitated sample (e.g., 1-2 mg) [20]. The oligosaccharides on the cell surface of gliomas are well-examined in T cell immune responses and sensitivity to killer lymphocytes [21][22][23]. Several studies clarify a strong correlation between the lectinbinding and the biological function in diffuse large B-cell lymphoma (DLBCL) [24][25][26][27][28]. However, the oligosaccharides on the primary central nervous system (CNS) lymphoma (PCNSL) surface have largely been unknown.
PCNSL is a rare subtype of DLBCL, which is an aggressive variant of extra-nodal non-Hodgkin's lymphoma (NHL) [29]. PCNSL only accounts for 3% of primary CNS tumors and 1% of NHLs in adults [30]. Methotrexate (MTX) is an antifolate that inhibits the dihydrofolate reductase activity in purine and thymidine syntheses and regulates the expression of glucocorticoid receptor α and β in human blood cells in vitro [31,32]. High-dose methotrexate (HD-MTX) is used as a first-line treatment in PCNSL [33]. Moreover, second-line treatments are also required for 10-35% of patients with refractory diseases and for another 35-60% or more who have relapse-acquired resistances [34]. Eventually, although treatment with HD-MTX is used as a standard treatment in PCNSL, most of the cases come to relapseacquired resistances to MTX [35].
Here, we generated MTX-resistant lymphoma cell lines derived from PCNSL and non-CNS lymphoma, which were applied to the semi-automated detection system for the N-linked oligosaccharides of glycoproteins on the cell surfaces by using N-pyridylamination fluorescent labels and reverse-and normal-phase high performance liquid chromatography (HPLC), in addition to CNS lymphoma specimens derived from the patients. Consequently, we obtained the results for differential expression patterns of N-oligosaccharides among PCNSL-derived cells, non-CNS lymphoma-derived cells, and clinical specimens of CNS lymphoma and for the slightly decreased expression in MTX-resistant lymphomas, including human brain malignant lymphomas HKBML and TK, and non-CNS lymphoma RAJI, which was seemed those in CNS lymphomas. This study is the first report for the expression patterns of N-oligosaccharides on tumor tissues resected from patients with CNS lymphomas and MTX-resistant PCNSLderived cells, including HKBML and TK. The results may be a hint for understanding the status and microenvironments of the surface glycans of lymphomas and may be useful for development of applied target therapies for cell recognitions.

Clinical specimens
Primary and secondary CNS lymphomas, which were pathologically diagnosed DLBCL tissue specimens, were obtained from Toyama Prefectural Central Hospital (Additional file 1: Table S1). All study protocols were approved by both of the Institutional Review Boards of Toyama Prefectural Central Hospital and Kyoto Prefectural University of Medicine (approval number 2011-1081), and experiments were performed in accordance with institutional guidelines. Written informed consents were obtained from all patients. Resected tumor tissues were immediately snap-frozen and fixed in 4% (v/v) paraformaldehyde (PFA) for 24 h, and then substituted with phosphate-buffered saline (PBS).

Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS)
Molecular masses of PA-sugar chains and their isobaric monosaccharide compositions were determined by MALDI/TOF-MS as described [44]. One microliter of matrix solution of 10 mg/mL 2,5-dihydroxybenzoic acid in 30% acetonitrile was spotted on the plate, 1 μL of sample solution was added and then dried by warm air. MALDI/TOF mass spectra were acquired using REFLEX mass spectrometer (Bruker-Franzen) in the positive and reflector mode at an acceleration voltage of 20 kV and delayed ion extraction. Standard PA-oligosaccharides were used to achieve a two-point external calibration for mass assignment of ions. The mass spectra shown were the sum of at least 30 laser shots.

Heatmap analysis
Heatmaps were constructed by color-coding standardized log or linear scales, indicating fold-differences of neutral and sialylated sugar chains in MTX-resistant cells compared to corresponding control cells, or by using the percent (%) area of fractionated peaks, designated by peak numbers, eluting at different retention times. Color configurations are shown in each panel. NA, not applicable.

Statistics
Statistics were performed using JMPv10 built-in-modules (SAS Institute Inc., Tokyo, Japan). Principal component analysis was performed to estimate Pearson correlation. P < 0.05 was considered statistically significant.

Generation of MTX-resistant lymphoma cells
The aim of this study is a comparison with N-glycan profiling between CNS lymphoma cells and their derived MTX-resistant cells and thereby an annotation clinical specimens of CNS lymphomas. First, to examine differential expression of oligosaccharides of glycoproteins on the surface of the cells, we generated MTX-resistant lymphoma cells (Fig. 1), including HKBML and TK as PCNSL, and RAJI as non-CNS lymphoma, in addition to HBL, OYB, and A4/FUK as other non-CNS lymphoma references (Additional file 1: Table S1, and Additional file 2: Figure S1A Fig. 1a-c), indicating that acquired resistances to MTX in PCNSL-derived TK and HKBML cells are weak than that in non-CNS lymphoma-derived RAJI cells. After collection of cells, samples were used to analyze the N-oligosaccharides of glycoproteins on the cell surface using pyridylamination fluolabeling and subsequent HPLC (Additional file 2: Figure S1B).

Differential expression patterns of N-linked oligosaccharides in MTX-resistant lymphoma cells
The ratios of peaks fractionating at appropriate retention time were calculated by converting to percent (%) area of the peaks in each cell (Fig. 2a-f and Additional file 6: Figure S5A, B), indicating almost expression ratios of distinct oligosaccharides of glycoproteins on surfaces of each cell. Expression ratios of each oligosaccharide were shown in a heat map as neutral sugar chains and neutral and sialylated sugar chains in nonresistant (control) and resistant cells for MTX ( Fig. 2g and Additional file 6: Figure S5C). M4B, M5A, and   Figure S4. g Summary for expression changes of neutral and sialyl sugar chains among HKBML and TK as PCNSL, and RAJI as non-CNS lymphoma. Color configuration indicates high (red) to low (green). S0: non-sialyl sugar chain, Neu: Neuraminidase-treated sugar chains. h Summary for expression changes of sialyl sugar chains among HKBML and TK as PCNSL, and RAJI as non-CNS lymphoma. Fold changes of sialyl sugar chains from MTX-resistant cells compared to the corresponding non-resistant cells, including HKBML, TK, and RAJI. Color configuration indicates high (red) to low (green). NA, not applicable. S0: non-sialyl sugar chain, Neu: Neuraminidase-treated sugar chains scale); M6B, M7A, M7B, M8A, and M9A were increased in MTX-resistant HKBML and TK (1.0-1.86fold in log scale); however, expression changes between neutral sugar chains and neutral and sialylated sugar chains were irregular (0.69-14.57-fold in log scale) (Fig.  2h). Ectopic fold-expression of A2G2F and A2G2FB were also observed, whereas slightly changes in MTXresistant HKBML, TK, and RAJI were observed compared to each corresponding control cell in neutral and sialylated sugar chains (0.74-1.09-fold in log scale) (Fig.  2h). A4G4F was only detected in MTX-resistant RAJI in neutral and sialylated sugar chains, but almost no changes were detected compared to the corresponding control cells (0.96-fold in log scale) (Fig. 2h). These results suggest that high-mannose types M5A and M6B may be possible targets for specific cells in PCNSL and non-CNS lymphoma. Besides, differential expression of sialylated A2G2F, A2G2FB, and A4G4F may be used to distinguish PCNSL and non-CNS lymphoma cells as a landmark in MTX-resistant lymphomas. However, the landmarks may function effectively, because there are almost no differences between the MTX-resistant and non-resistant cells in sialylated A2G2F, A2G2FB, and A4G4F.

Sialylated N-linked oligosaccharides of glycoproteins on the cell surface of lymphomas
Calculating ratios of the peaks of neutral and/or sialylated sugar chains successfully returned the ratios of sialylated sugar chains (Fig. 3a, b and Additional file 6: Figure S5D, E). Interestingly, as for sialylated oligosaccharides, expression of A2G2F and A2G2FB were low and high in HKBML and TK as PCNSL, and RAJI as non-CNS lymphoma, respectively, whereas almost no change between MTX-resistant and non-resistant cells was observed (Fig. 3b). Sialylated A4G4F was only detected in RAJI (Fig. 3b) and slightly decreased by 0.81fold in MTX-resistant RAJI compared to the nonresistant RAJI (Fig. 3c). However, fold-differences of complex type sialylated oligosaccharides detected in MTX-resistant HKBML, TK, and RAJI slightly decreased compared to the corresponding control cells (0.62-0.97fold) (Fig. 3c). In non-CNS lymphoma cells, including A4/FUK and OYB, M7A and M7B were expressed, and especially highly expressed in OYB (41.5%) (Additional file 6: Figure S5D, E). A2G2F, A2G2FB, and A4G4F were hard to detected in A4/FUK, HBL1, and OYB (Additional file 6: Figure S5D, E). Although expression of A2G2F, A2G2FB, and A4G4F almost did not change in neutral and sialylated sugar chains, as described above (Fig. 2h), the results in only sialylated sugar chains indicated that the expression slightly decreased in MTXresistant cells compared to the corresponding control cells, suggesting a careful attention for the strategies targeting sugar chains on the surface of MTX-resistant lymphomas. The ectopic expression of M7A and M7B was detected in OYB, which may be useful for marking distinct OYB-type non-CNS lymphoma from other types of lymphomas.
A2G2F was increased in glioblastoma tissues and glioma cell lines, while being less than 0.1% in normal brain tissue [51]. In the present study, A2G2F was detected in neutral and sialylated sugar chains as 2.19-5.0% but not in neutral sugar chains in the cell lines, which had a slight change in the MTX-resistant cells compared to the control cells as 0.92-1.08-fold (Fig. 2g,  h). In sialylated sugar chains, A2G2F was detected as 11.67-44.02% with decreased levels in the MTXresistant cells compared to the control cells as 0.76-0.81-fold (Fig. 3b, c). Besides, in the analysis for the CNS lymphoma tissues, average 6.59% (range: 2.77-14.54%) of A2G2F was detected in the four clinical specimens (Fig. 4a, b). Therefore, A2G2F in CNS lymphoma tissues and cell lines is much than normal brain tissue, whereas sialylated A2G2F in the MTX-resistant PCNSL-derived cells is slightly decreased compared to the control cells, which is consistent with glioma cell lines, glioblastoma tissues, PCNSL-derived cell lines, CNS lymphoma tissues, and non-CNS lymphoma cells. In glioblastoma and/or glioma, Lens culinaris agglutinin (LCA)-lectin binding to A2G2F inhibits cell proliferation of glioma through induction of apoptosis [51]. Therefore, A2G2F may also provide a useful marker candidate and a hint for diagnosis and development for target therapy in CNS lymphoma, as well as glioma/glioblastoma.
All of the four CNS lymphoma specimens and cell lines, including CNS lymphoma and non-CNS lymphoma, used in the study were human immunodeficiency virus (HIV)negative, except for HBL1 and OYB as no valid information for HIV (Additional file 1: Table S1). PCNSL-derived HKBML and non-CNS lymphoma-derived Raji are Epstein-Barr virus (EBV)-positive and PCNSL-derived TK is EBV-negative (Additional file 1: Table S1). However, the expression patterns of N-oligosaccharides in HKBML were similar to that in TK than Raji, in addition to differential expression with acquired MTX-resistance in neutral and sialylated sugar chains (Fig. 2g, h), and sialylated sugar chains (Fig. 3b, c). While, the EBV infection in OYB cells was unknown (Additional file 1: Table S1). Our previous study has clarified that EBV-positive PCNSLs are counted by 20% [52]. However, all of the four clinical specimens examined in the study were EBV-negative. Therefore, whether EBV-positive PCNSLs make a change to their oligopatterns should await future studies.

Conclusions
In this study, we demonstrated that the expression of specific sialylated N-linked oligosaccharides, including A2G2F and A2G2FB, in the MTX-resistant cells slightly decreased compared to the corresponding control cells. Similarly, the decreased expression of sialylated A2G2F and A2G2FB seemed to correlate with poor prognoses of the CNS lymphoma patients, despite the small sample number. Therefore, the differential expression and patterns of surface glycans on CNS lymphomas make it possible to escape the cell-cell recognition by immune cells, thereby, MTX-resistant malignant CNS lymphoma cells could re-grow. In conclusion, the above-mentioned oligosaccharides may be promising oligosaccharide marker candidates to recognize MTX-resistant cells, and primary and secondary CNS lymphomas, which may be useful for diagnosis marker development and/or applied molecular targeted therapies for CNS lymphomas.
Additional file 1: Table S1. Clinical information of cell lines and CNS lymphoma specimen.