Mutations in the N-glycan branching enzyme MGAT1 promote invasiveness and EGF-stimulated proliferation and adhesion in neuroblastoma cells.

Attachment and modification of N-glycans to proteins have been linked to the promotion of cancer, and specifically, more recent investigations have supported the involvement of less processed N-glycans. Herein, we investigated how reduced activity of N-acetylglucosaminyltransferase-I (GnT-I, MGAT1) influences aberrant cellular properties and epidermal growth factor receptor (EGFR) signaling in human NB cells. CRISPR/Cas9 technology was used to introduce mutations into MGAT1 in the parental BE(2)-M17 cell line, generating the MGAT1 mutant cell lines, Trunc and Δ45. Lectin binding studies showed increased oligomannose type N-glycans accompanied by decreased hybrid and complex types of N-glycans in both MGAT1 mutant cell lines relative to BE(2)-M17, and the level of oligomannose type N-glycans were reversed in the mutant cells by ectopic expression of GnT-I. Reduced GnT-I activity increased the percentage of S-type cells and decreased the percentage of N-type cells. Anchorage-independent cell growth was reduced in both MGAT1 mutant cell lines, and cell proliferation was slower at sub-confluency. Unexpectedly, cell proliferation rates were increased in confluent and over-confluent cell cultures, as they were in three-dimensional cell spheroids, in the MGAT1 mutant lines compared to the parental cell line. Trunc and Δ45 cells were less sensitive to mechanical dissociation and retained larger intact cell clusters, supporting that increased oligomannose N-glycans strengthen cell-cell adhesion. MGAT1 mutant cell lines were also more invasive than BE(2)-M17. Following EGF stimulation, Trunc and Δ45 cells had increased levels of P-EGFR (Y1068), along with enhanced EGF-stimulated cell proliferation and cell-cell adhesion, whereas the parental cell line was relatively unresponsive. Taken together, these results show that decreased GnT-I activity imparts NB cells to perpetually proliferate at high cell densities. Furthermore, increased oligomannose N-glycans strengthened cell-cell contacts, promoted cell invasiveness, and enhanced EGF-stimulated proliferation, supporting a novel role of oligomannose N-glycans in NB progression.