Impaired N-Glycosylation Processing Impacts Neuronal Function
Modifications of membrane proteins are carried out primarily using N-linked glycosylation. N-glycosylation has the potential to co-translationally and post-translationally modify protein function by the addition and processing of oligosaccharides. These oligosaccharides are known as glycans and are broken up into three types: oligomannose, hybrid and complex. Modification of oligosaccharides is performed by enzymes known as N-acetylglucosaminyltransferases (GnTs). Encoding of GnT proteins is performed by genes known as Mgats. Kv3.1b is an alpha subunit of a voltage-gated potassium channel known as Kv3 channels. Kv3 channels have the ability to open and close much more rapidly than other K channels, thus allowing neurons to create trains of action potentials. Kv3.1b is N-glycosylated at two sites, the N220 site and N229. The first aim of this study was to examine caudal primary (CaP) motor neuron development and the motor activity of WT AB larval zebrafish when the N220 site of a Kv3.1b alpha subunit is abolished. The zebrafish expressing N220Q Kv3.1b protein in CaP motor neuron displayed maldeveloped CaP neurons and a significant reduction in motor activity throughout larval development compared to those expressing Wt Kv3.1b protein or EGFP in CaP neurons. The second aim was to determine the effect of atypical N-glycosylation processing in developing zebrafish. Herein, CRISPR/Cas9 was implemented to create the Mgat1b -/-and Mgat1a -/- fish strains for current and future experiments. Mgat1 genes are responsible for the encoding of GnT enzymes. The Mgat1b -/- strain has increased oligomannose type glycans, as demonstrated by the Galanthus Nivalis Lectin (GNL) lectin binding assay. Up until now, the Mgat1b strain has been established, while the Mgat1a strain is still in progress. The locomotor studies indicated that the Mgat1b -/- strain is less active than the WT AB strain. The confocal images suggest cell morphology of CaP neurons are lacking branches. The third and final aim of this study examined the fully glycosylated (Wt) or the un-glycosylated (N220/229Q) Kv3.1b proteins in two different neuroblastoma (NB) cell lines. The NB_1 cell line (control), along with the N-glycosylation mutant (NB_1( -Mgat1) cell line were utilized for confocal microscopy experiments. The earlier and later cell lines express primarily complex and solely oligomannose types of N-glycans, respectively. Both cell lines were designed to stably express either Wt or N220/229Q Kv3.1b proteins. To examine whether occupancy influenced the distribution of Kv3.1b to the neurites (outgrowth) and soma (cell body), cell lines expressing Wt or N220/229Q Kv3.1b proteins was compared. To determine the effect due to the type of N-glycan, then Wt Kv3.1b protein expressed in each of the cell lines were compared. It was found that the percent of Kv3.1b protein present in outgrowths was higher when the Kv3.1b was N-glycosylated. Further attachment of complex type N-glycan to the Kv3.1b protein had more Kv3 channels, containing Kv3.1b protein, in outgrowth than when oligomannose was associated with the Kv3.1b glycoprotein. My studies along with laboratory's research, conclude that N-glycosylation processing of Kv3.1b in zebrafish is critical in CaP neuron development and function, and thereby swimming locomotor activity. Further N-glycosylation processing of Kv3.31b has a vital role in distribution of Kv3.1b protein to the subdomains of NB cells.
glycans, motor neurons, Kv3 channels, neuronal development, motor activity, excitability, cell surface localization, neurodegenerative disorders, spinal cord, GnTI, Mgat1b
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