Kv3-Expressing Cells Present More Elaborate N-Glycans with Changes in Cytoskeletal Proteins, Neurite Structure and Cell Migration.

The cues contained by N-glycans relay the quality, cellular destination, and interactions of proteins, thereby, impacting cellular physiology. Voltage-gated K⁺ (Kv3) channels have two conserved N-glycosylation sites which are vital for Kv3 channel activity, and primary motor neuron development. Our previous studies showed that the parental (NB_1) and N-glycan mutant (NB_1(-Mgat1), NB_1(-Mgat2), and NB_1(-Mgat3)) Neuroblastoma (NB) cell lines have compromised N-acetylglucosaminyltransferase activities: GnT-I, GnT-II, or GnT-III. Herein, we stably expressed Kv3.1b in the parental and engineered N-glycan mutant Neuroblastoma (NB) cell lines to examine how changes in N-glycans alter the cytoskeleton, and subsequently cellular properties. MALDI-TOF MS verified that the parental and mutant cell lines had different N-glycan profiles. When Kv3.1b was expressed in NB cells with an intact Mgat1, the N-glycan population had more complex N-glycans with increased branching. Further NB cells with an intact Mgat2 had higher and lower levels of hybrid and oligomannose N-glycans, respectively. N-Glycan populations changed cytoskeletal protein abundancies and cell morphology. Moreover, all Kv3.1b-expressing cells, except NB_1(-Mgat2), had changed levels of F-actin, neurofilament and vimentin, along with modified neurite formation. In all cases, migratory rates were enhanced when cells expressed Wt Kv3.1b. Glycan populations and glycans attached to Kv3.1b altered spatial arrangement in membranes and both ER folding and transport proteins were not increased by expression of unglycosylated Kv3.1b. Kv3.1b expression in NB cells alters N-glycan populations and mediates adjustments in cytoskeletal proteins, and thereby cell morphology and cell migration, supporting roles in neuronal development and maintenance.