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.

Modifications in surface glycans attached to proteins via N-acetylglucosamine-β1-N-asparagine linkage have been linked to tumor development and progression. These modifications include complex N-glycans with high levels of branching, fucose and sialic acid residues. Previously, we silenced Mgat2 in neuroblastoma (NB) cells, which halted the conversion of hybrid type N-glycans to complex type, to generate a novel cell line, NB_1(-Mgat2). By comparing the aberrant cell properties of the NB_1(-Mgat2) cell line to the parental cell line (NB_1), we investigated the impact of eliminating complex type N-glycans on NB cell behavior. Further, the N-glycosylation pathway in the NB_1(-Mgat2) cell line was rescued by transiently transfecting cells with Mgat2, thus creating the NB_1(-/+Mgat2) cell line. Changes in the N-glycosylation pathway were verified by enhanced binding of E-PHA and L-PHA to proteins in the rescued cell line relative to those of the NB_1(-Mgat2) cell line. Also, western blotting of total membranes from the rescued cell line ectopically expressing a voltage-gated K+ channel (Kv3.1b) revealed that N-glycans of Kv3.1b were processed to complex type. By employment of various cell lines, we demonstrated that reduction of the complex type N-glycans diminished anchorage-independent cell growth, and enhanced cell-cell interactions. Two independent cell invasion assays showed that cell invasiveness was markedly lessened by lowering the levels of complex type N-glycans while cell mobility was only slightly modified. Neurites of NB cells were shortened by the absence of complex type N-glycans. Cell proliferation was reduced in NB cells with lowered levels of complex type N-glycans which resulted from hindered progression through G1+Go phases of the cell cycle. Overall, our results illustrate that reducing the ratio of complex to hybrid types of N-glycans diminishes aberrant NB cell behavior and thereby has a suppressive effect in cell proliferation, and cell dissociation and invasion phases of NB.

Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9) technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.

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