Knockdown of N-Acetylglucosaminyltransferase-II Reduces Matrix Metalloproteinase 2 Activity and Suppresses Tumorigenicity in Neuroblastoma Cell Line

Abstract

Neuroblastoma (NB) development and progression are accompanied by changes in N-glycans attached to proteins. Here, we investigated the role of N-acetylglucosaminyltransferase-II (GnTII, MGAT2) protein substrates in neuroblastoma (NB) cells. MGAT2 was silenced in human BE(2)-C NB (HuNB) cells to generate a novel cell line, HuNB(-MGAT2), lacking complex type N-glycans, as in rat B35 NB cells. Changes in N-glycan types were confirmed by lectin binding assays in both cell lines, and the rescued cell line, HuNB(-/+MGAT2). Western blotting of cells heterologously expressing a voltage-gated K+ channel (Kv3.1b) showed that some hybrid N-glycans of Kv3.1b could be processed to complex type in HuNB(-/+MGAT2) cells. In comparing HuNB and HuNB(-MGAT2) cells, decreased complex N-glycans reduced anchorage-independent cell growth, cell proliferation, and cell invasiveness, while they enhanced cell-cell interactions. Cell proliferation, invasiveness and adhesion of the HuNB(-/+MGAT2) cells were more like the HuNB than HuNB(-MGAT2). Western blotting revealed lower protein levels of MMP-2, EGFR and Gab2 in glycosylation mutant cells relative to parental cells. Gelatin zymography demonstrated that decreased MMP-2 protein activity was related to lowered MMP-2 protein levels. Thus, our results support that decreased complex type N-glycans suppress cell proliferation and cell invasiveness in both NB cell lines via remodeling ECM.

Keywords: EGFR; Gab2; MMP-2; cell adhesion; cell invasiveness; cell proliferation; glycans; neuroblastoma.

Figure 1

Construction and characterization of an NB cell line with an introduced frameshift in MGAT2. The MGAT2 gene was silenced in the HuNB cell line using CRISPR/Cas9 technology. Coding sequences of the MGAT2 gene from 1 to 42 is shown for the parental and N-glycosylation mutant cell lines (A). The mutant cell line has a c inserted following the 22nd nucleotide, which is denoted in bold purple font. The purple font reveals the different codons in the sequence caused by the frameshift mutation. Typical flow cytometry plots of fluorescently labelled L-PHA (left panels), E-PHA (left middle panels), and GNL (right middle panels) bound to HuNB (top panels) and HuNB(-MGAT2) (bottom panels) cell lines are shown (B). Mean fluorescence intensity values of all three lectins bound to each of the cell lines were obtained from 4 experiments (C). * p < 0.01.

Figure 2

Identification of reduced levels of complex type N-glycans in NB cells with MGAT2 silenced. Lectin blots of whole cell lysates from HuNB and HuNB(-MGAT2) cell lines, along with the later cell line transiently transfected with MGAT2 to create the rescued cell line, called NB_1(-/+MGAT2) (A). Proteins separated on membranes were probed with L-PHA, E-PHA, and GNL, as indicated. Coomassie blue-stained SDS gels show that similar levels of protein were loaded per lane. Horizontal lines adjacent to blots and gel indicate molecular weight standards in kDa: 250, 150, 100, 75, 50, and 37, from top to bottom. Western blot of total membranes from HuNB, HuNB(-MGAT2), and HuNB(-/+MGAT2) cell lines transfected with glycosylated Kv3.1b (WT), along with HuNB transfected with unglycosylated Kv3.1b (DM) (B). (+) denotes the cell line studied. Black and gray arrows reflect complex and hybrid types of N-glycans respectively, attached to the Kv3.1b protein. The gray dotted line denotes oligomannose type glycans attached to Kv3.1b, and the black line denotes unglycosylated Kv3.1b. Western blots of WT Kv3.1b from total membranes of HuNB (C), and HuNB(-MGAT2) (D) cells treated with glycosidases, as indicated. Numerical values adjacent to western blots denote molecular weight markers.

Figure 3

Increased levels of hybrid and oligmannose types of N-glycans reduced HuNB cell growth. Typical DIC micrographs of cell colonies of HuNB (left panels) and HuNB(-MGAT2) (right panels) cell lines from the anchorage-independent cell growth assay (A). Bar graphs show the mean cell colony areas (B), and cell colony number per experiment (C) of each cell line from 3 experiments. * p < 0.02. The cell-attached proliferation assay was conducted three times in triplicate for the various NB cell lines (D). Mean differences were compared using one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc tests. A value of p < 0.05 was considered significant (*).

Figure 4

Higher levels of hybrid and oligomannose types of N-glycans in NB cells decrease disruption of cell–cell adhesion. Characteristic DIC images obtained from HuNB (left panels), HuNB(-MGAT2) (middle panels), and HuNB(-/+MGAT2) (right panels) cell lines (A). Scale bar is 50 µm. Cell clusters of greater than 5 cells, as encircled in white, were analyzed. The mean area of cell clusters from three cell dissociation experiments for each cell line were analyzed (B). * p < 0.01. Mean differences were compared using One-way ANOVA followed by Bonferroni’s post-hoc tests was used to compare differences in mean values. A value of p < 0.01 was considered significant (*).

Figure 5

Decreased complex type N-glycans in NB cells decreases cell–cell association. Selected micrographs acquired from HuNB (left panels), and HuNB(-MGAT2) cell lines from the hanging drop assay (A). Scale bar is 100 µm. Cell areas at or greater than 2000 pixels, encircled in yellow were analyzed. The mean area of cell clusters from three experiments were determined for each cell line (B). The unpaired Student’s t-test was used for comparing mean values at p < 0.00001 (*).

Figure 6

Higher levels of hybrid and oligomannose types of N-glycans diminished cell invasiveness. Selected images obtained from matrigel invasion assays of HuNB (left panels), HuNB(-MGAT2) (middle panels), and HuNB(-/+MGAT2) (right panels) (A). Bright purple invasive cells and pores in the membrane are visible. The mean number of invasive cells per well from the indicated cell lines were normalized to that of the HuNB cell line (B). Cell invasiveness was determined from three experiments performed in quadruplicate for each cell line. Micrographs of cell wound healing assays were acquired for the HuNB (upper panel) and the HuNB(-MGAT2) (lower panel) at 0 and 19 h (C). White lines at the edge of the monolayer are used to denote the width of the cell wound. AU represents the arbitrary unit. The bar graph indicates the percent of cell wound closure in 19 h from 11 experiments with at least 5 wounds per experiment of each cell line (D). Mean values were compared using the unpaired Student’s t-test at p < 0.05 (*).

Figure 7

Decreased complex type N-glycans lessens cell invasiveness of three-dimensional (3D) spheroids derived from HuNB cells. After 13 days, images of invading spheroids were acquired from HuNB and HuNB(-MGAT2) cell lines. Representative micrographs are shown of HuNB (left panel) and HuNB(-MGAT2) (right panel) (A). Scale bar is 100 µm. Cell invasion was determined by dividing invasive area (green line) by the sphere area (yellow dotted line) (B). The mean cell invasion area was calculated from three experiments of at least 172 spheroids (C). * p < 0.00001.

Figure 8

MMP-2 protein levels are lowered with increased hybrid and oligomannose types of N-glycans in NB cell lines. Conditioned medium was harvested from confluent plates. MMP-2 was detected by zymography (right panels), and western blots (middle panels) of the human and rat NB cell lines (A,B). Coomassie blue-stained gel (left panels) indicates that similar amounts of protein were present in the conditioned medium (A,B). Western blot of β-tubulin (right panels) and coomassie blue-stained gel (left panels) of whole cell lysates indicate that similar amounts of cells were used to generate MMP-2 in conditioned medium. (C,D). The reduction in activity and immunoband intensities between HuNB and HuNB(-MGAT2) were 29% ± 4% (n = 7) and 37% ± 11% (n = 4) respectively, and also, between ratNB and ratNB(-Mgat2), 40% ± 8% (n = 6) and 62% ±8% (n = 3), respectively. The n value denotes number of bands from three separate experiments for both human and rat. Horizontal lines adjacent to blots and gels indicate molecular weight standards in kDa: 250, 150, 100, 75, 50, 37, and 25, from top to bottom. Top marker was unobserved for western blot in panel C. Black and grey arrows point to MMP-2 and β-tubulin, respectively.

Figure 9

Lowered complex type N-glycans by NB cells altered EGFR and Gab2 protein levels. Western blots of EGFR (top panels) and Gab2 (middle panels) of total membranes from parental and mutant human (A) and rat (B) cell lines, as indicated. Coomassie blue stained gels (bottom panels) show that equal amounts of protein were loaded. Relative molecular weights (in KDa) of EGFR and Gab2 are next to western blots. Molecular weights (in KDa) of standards are adjacent to coomassie blue stained gel. Experiments were performed three times.