Abnormal modifications in N-glycosylation processing are commonly associated with neurological disorders, although the impact of specific N-glycans on neuronal excitability is unknown. By replacement of complex types of N-glycans with hybrid types in neuroblastoma cells, we provide the first study that addresses how distinct N-glycan types impact neuronal excitability. Using CRISPR/Cas9 technology, NB_1, a clonal cell line derived from rat neuroblastoma cells (NB), was modified to create an N-glycosylation mutant cell line, NB_1 (-Mgat2), which expresses predominantly hybrid type N-glycans. Western and lectin blotting, flow cytometry, TIRF and DIC microscopy, and patch clamp studies were conducted. Lectin binding revealed the predominant type of N-glycans expressed in NB_1 (-Mgat2) is hybrid while those of NB and NB_1 are complex. Kv3.1 b-expressing cells with complex N-glycans localized more glycosylated Kv3.1b to the neurites than cells with hybrid N-glycans. Further the absence of N-glycan attachment to Kv3.1b was critical for sub-plasma distribution of Kv3.1b to neurites in primary adult mammalian neurons, along with NB cells. Replacement of complex type N-glycans with hybrid type hindered the opening and closing rates of outward ionic currents of Kv3.1 b-expressing NB cells. The lacks of N-glycan attachment hindered the rates even more but were not significantly different between the NB cell lines. Taken together, our evidence supports N-glycosylation impacts the sub-plasma membrane localization and activity of Kv3.1 b-containing channels. We propose that N-glycosylation processing of Kv3.1 b-containing channels contributes to neuronal excitability, and abnormal modifications in N-glycosylation processing of Kv3.1b could contribute to neurological diseases.
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.
HighlightsCell surface N-glycans impact E-cadherin placement at the cell–cell border.E-cadherin retention at the cell–cell border is affected by cell surface N-glycans.Cell surface N-glycans influence E-cadherin-mediated cell–cell adhesion.N-glycans outside the cell contribute to plasma membrane structure.
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