Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF-Stimulated Proliferation In Vitro
<p><b>Knockout of <span class="html-italic">MGAT1</span> blocks hybrid and complex N-glycan synthesis</b>. (<b>A</b>) There are three general types of N-glycans: oligomannose, hybrid, and complex. N-glycans attach to N-glycosylation sites (Asn-X-Ser/Thr) in glycoproteins (red lines). The conserved pentasaccharide is enclosed with a blue line. Oligomannose N-glycans have 0–6 mannose (Man) residues attached to the pentasaccharide. Hybrid N-glycans have Man residues attached to the Manα1–6 arm (right arm of the pentasaccharide), and at least one N-acetylglucosamine (GlcNAc) attached to the Manα1–3 arm (left arm of the pentasaccharide). Replacement of the Man residues with a GlcNAc residue on the right arm of hybrid N-glycans produces complex N-glycans. Complex N-glycans can have up to five branches initiated by GlcNAc and elongated with various monosaccharides. A fucose (Fuc) residue attached to the core is a common modification of hybrid and complex types of N-glycans. An N-acetylneuraminic acid (Neu5Ac) residue commonly caps hybrid and complex N-glycans. In some cases, a chain of Neu5Ac residues is attached to N-glycans, referred to as polysialylated N-glycans. (<b>B</b>) N-glycan processing, where oligomannose N-glycans are converted to hybrid N-glycans via GnT-I and then from hybrid to complex N-glycans via GnT-II. Green and red arrows show that the loss of GnT-I (<span class="html-italic">MGAT1</span>) results in the accumulation of oligomannose and reduced levels of hybrid and complex N-glycans, respectively. This is a simplified cartoon of the N-glycosylation pathway. (<b>C</b>) DNA sequences and chromatograms of the <span class="html-italic">MGAT1</span> gene showing the wild type (Wt) sequence in the top panel, and the three frameshift mutations in the lower panels. The frameshift mutations result from deletions of 4, 7, and 27 nucleotides within the <span class="html-italic">MGAT1</span> coding sequence.</p> "> Figure 2
<p><b>Altered lectin binding and oligomannosylated EGFR from BE(2)-C(MGAT1<sup>−/−</sup>) cells.</b> (<b>A</b>) GNL, L-PHA, and E-PHA lectin blots, along with Coomassie blue-stained gels, of whole cell lysates from BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>) cells. Black-filled ovals represent molecular weights markers of 150, 100, and 75 kDa for the lectin blots and the Coomassie blue gel; only 100 and 75 kDa markers are shown for the L-PHA blot. (<b>B</b>) A multiplexed EGFR/β-actin Western blot of both BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>) whole cell lysates. (<b>C</b>) EGFR Western blot of total membranes from BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>) (+) digested or (−) not digested with PNGase F or Endo H. The chevron signifies EGFR that is associated with complex N-glycans while the black arrowhead denotes oligomannosylated EGFR, and the black line represents EGFR with N-glycans removed.</p> "> Figure 3
<p><b>Loss of <span class="html-italic">MGAT1</span> impacted NB cell–cell adhesion, proliferation, and morphology.</b> (<b>A</b>) Representative DIC images of intact cell clusters and (<b>B</b>) the area of cell clusters from BE(2)-C (<span class="html-italic">n</span> = 926), BE(2)-C +GnT-I (<span class="html-italic">n</span> = 408), BE(2)-C(MGAT1<sup>−/−</sup>) (<span class="html-italic">n</span> = 739), and BE(2)-C(+/−<span class="html-italic">MGAT1</span>) (<span class="html-italic">n</span> = 898) following mechanical dissociation via pipetting. <span class="html-italic">n</span> denotes the number of cell clusters. (<b>C</b>) BrdU cell proliferation assay under 2D cell culturing conditions for BE(2)-C for Wt with (<span class="html-italic">n</span> = 4) and without (<span class="html-italic">n</span> = 8) GnT-I. (<b>D</b>) 3D cell spheroid BrdU proliferation assay (<span class="html-italic">n</span> = 8) normalized to BE(2)-C for Wt and mutant cell lines. (<b>E</b>) Representative DIC images of cell colonies from anchorage-independent growth of BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>) and (<b>F</b>) quantification of the area of clusters from BE(2)-C (<span class="html-italic">n</span> = 627) and BE(2)-C(MGAT1<sup>−/−</sup>) (<span class="html-italic">n</span> = 172) following 13 days of growth, where <span class="html-italic">n</span> is the number of cell clusters. (<b>G</b>) Examples of the cell types in BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>) cells, as indicated. (<b>H</b>) Percentage of cell type per field of Wt and MGAT1 mutant cells (<span class="html-italic">n</span> = 59), where n signifies the number of fields examined. In all cases, the data are normalized to BE(2)-C, except in panel (<b>B</b>). All quantifications are represented as the mean ± SEM. One-way ANOVA with a post hoc Holm–Bonferroni test was used when comparing more than 2 samples, where * <span class="html-italic">p</span>< 0.05 and ** <span class="html-italic">p</span>< 0.01. The Student’s t-test was used to compare Wt to MGAT1 mutant in panels (<b>D</b>,<b>F</b>,<b>H</b>), where * <span class="html-italic">p</span> < 0.04 and ** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 4
<p><b>Oligomannosylated EGFR heightened autophosphorylation following EGF stimulation.</b> (<b>A</b>) Multiplexed Western blots of pEGFR Y1173/β-actin (<b>Top</b>) and pan-EGFR/β-actin (<b>Bottom</b>) from BE(2)-C (<b>Left</b>) and BE(2)-C(MGAT1<sup>−/−</sup>) (<b>Right</b>) following EGF stimulation with EGF, as indicated. (<b>B</b>) Multiplexed pEGFR Y1173/β-actin Western blot (<span class="html-italic">n</span> ≥ 5) of BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>) following 0 ng/mL, 20 ng/mL, and 100 ng/mL EGF stimulation, and (<b>C</b>) quantification of the pEGFR immunoband with respect to β-actin. The Student’s <span class="html-italic">t</span>-test was used to compare Wt to the MGAT1 mutant, where ** <span class="html-italic">p</span> < 0.041 and * <span class="html-italic">p</span> < 0.063. All quantifications are represented as the mean ± SEM.</p> "> Figure 5
<p><b>Oligomannosylated EGFR sensitized NB to EGF-stimulated proliferation in 2D and 3D cell cultures.</b> (<b>A</b>) Multiplexed Western blot of EGFR/β-actin from either 2D or 3D whole cell lysates of BE(2)-C and BE(2)-C (<span class="html-italic">MGAT1<sup>−/−</sup></span>). (<b>B</b>) 2D (<span class="html-italic">n</span> ≥ 6) and 3D (<span class="html-italic">n</span> = 8) BrdU proliferation of BE(2)-C and BE(2)-C (<span class="html-italic">MGAT1<sup>−/−</sup></span>) following 20 ng/mL EGF stimulation. (<b>C</b>) 3D cell counts (<span class="html-italic">n</span> = 6) of BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>) following 20 ng/mL EGF stimulation. Two-way ANOVA used with correction; * <span class="html-italic">p</span> < 0.05. All quantifications are represented as the mean ± SEM.</p> "> Figure 6
<p><b>Loss of <span class="html-italic">MGAT1</span> decreased 2D invasion and migration.</b> (<b>A</b>) Representative images of either BE(2)-C or BE(2)-C(MGAT1<sup>−/−</sup>) cells from the bottom of membrane insert, indicating migration, following 4 h of incubation. (<b>B</b>) The average number of migratory cells (<span class="html-italic">n</span> = 16) that crossed the membrane insert. <span class="html-italic">n</span> represents the number of fields. (<b>C</b>) Images of invasive BE(2)-C or BE(2)-C(MGAT1<sup>−/−</sup>) cells following 22 h of incubation and their invasion through a Matrigel invasion chamber. (<b>D</b>) Average number (<span class="html-italic">n</span> = 5) of BE(2)-C cells and BE(2)-C(MGAT1<sup>−/−</sup>) cells that invaded through the Matrigel chamber. <span class="html-italic">n</span> signifies the number of wells. All quantifications are represented as the mean ± SEM. The Student’s <span class="html-italic">t</span>-test was used to compare the Wt to the MGAT1 mutant; ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 7
<p>Oligomannose N-glycans increased 3D spheroid invasiveness but EGF treatment impaired 3D invasion in both BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>). (<b>A</b>) Images of 3D spheroids from BE(2)-C and BE(2)-C(MGAT1<sup>−/−</sup>) following 22 h of invasion. (<b>B</b>) Quantification of the invasion area for BE(2)-C (<span class="html-italic">n</span> = 75) and BE(2)-C(MGAT1<sup>−/−</sup>) (<span class="html-italic">n</span> = 77). The Student’s t-test was used, where ** <span class="html-italic">p</span> < 0.0001. (<b>C</b>) DIC images of invading spheroids and (<b>D</b>) quantification of the invasion area of BE(2)-C without EGF (<span class="html-italic">n</span> = 79), BE(2)-C with 20 ng/mL EGF (<span class="html-italic">n</span> = 83), BE(2)-C(MGAT1<sup>−/−</sup>) without EGF (<span class="html-italic">n</span> = 66), and BE(2)-C(MGAT1<sup>−/−</sup>) with 20 ng/mL EGF (<span class="html-italic">n</span> = 35). <span class="html-italic">n</span> denotes the number of invading spheroids. Two-way ANOVA was used with correction; * <span class="html-italic">p</span> < 0.05. All quantifications are represented as the mean ± SEM.</p> "> Figure 8
<p><b>RhoA activation decreased 3D invasion in a glycan-independent manner.</b> (<b>A</b>) Micrographs of 3D invading spheroids at 22 h, as indicated. (<b>B</b>) Quantification of the invasion area of BE(2)-C without (<span class="html-italic">n</span> = 21), BE(2)-C with (<span class="html-italic">n</span> = 19), BE(2)-C(MGAT1<sup>−/−</sup>) without (<span class="html-italic">n</span> = 22), and BE(2)-C(MGAT1<sup>−/−</sup>) with 1 µg/mL RhoA activator (<span class="html-italic">n</span> = 15). <span class="html-italic">n</span> denotes the number of invading spheroids. Two-way ANOVA was used with correction; * <span class="html-italic">p</span> < 0.05. All quantifications are represented as the mean ± SEM.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines and Cell Culture
2.2. 3D Cell Spheroid Formation
2.3. Whole Cell Lysates
2.4. Total Membrane Isolation and Glycosidase Treatment
2.5. Western and Lectin Blotting
2.6. Cell Dissociation Assay
2.7. 2D and 3D BrdU Proliferation
2.8. Anchorage Independent Growth
2.9. Morphology
2.10. 2D Migration and Invasion
2.11. 3D Spheroid Invasion
2.12. EGF Treatment
2.13. RhoA Activation to Examine Invasion
2.14. Data Analysis
3. Results
3.1. Manipulation of the N-glycan Processing Pathway to Yield Oligomannosylated EGFR
3.2. Rescue of Altered Cell–Cell Adhesion, Proliferation, and Morphology in the MGAT1 Mutant NB Cell Line
3.3. Oligomannosylated EGFRs Have an Increased Response to EGF Treatment and Sensitize NB Cells to EGF-Stimulated Proliferation
3.4. Cell Migration Is Unaltered While Cell Invasiveness Is Suppressed by Nonfunctional GnT-I Using 2D Dispersed Cell Culture
3.5. Oligomannose N-glycans Promote Cell Spheroid Invasiveness, and Both EGF and RhoA Treatment of NB Cell Lines Markedly Suppress Cell Spheroid Invasiveness
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Burch, A.P.; Hall, M.K.; Wease, D.; Schwalbe, R.A. Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF-Stimulated Proliferation In Vitro. Int. J. Transl. Med. 2024, 4, 519-538. https://doi.org/10.3390/ijtm4030035
Burch AP, Hall MK, Wease D, Schwalbe RA. Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF-Stimulated Proliferation In Vitro. International Journal of Translational Medicine. 2024; 4(3):519-538. https://doi.org/10.3390/ijtm4030035
Chicago/Turabian StyleBurch, Adam P., M. Kristen Hall, Debra Wease, and Ruth A. Schwalbe. 2024. "Reduction of N-Acetylglucosaminyltransferase-I Activity Promotes Neuroblastoma Invasiveness and EGF-Stimulated Proliferation In Vitro" International Journal of Translational Medicine 4, no. 3: 519-538. https://doi.org/10.3390/ijtm4030035