Compromised N-Glycosylation Processing of Kv3.1b Correlates with Perturbed Motor Neuron Structure and Locomotor Activity

Neurological difficulties commonly accompany individuals suffering from congenital disorders of glycosylation, resulting from defects in the N-glycosylation pathway. Vacant N-glycosylation sites (N220 and N229) of Kv3, voltage-gated K+ channels of high-firing neurons, deeply perturb channel activity in neuroblastoma (NB) cells. Here we examined neuron development, localization, and activity of Kv3 channels in wildtype AB zebrafish and CRISPR/Cas9 engineered NB cells, due to perturbations in N-glycosylation processing of Kv3.1b. We showed that caudal primary (CaP) motor neurons of zebrafish spinal cord transiently expressing fully glycosylated (WT) Kv3.1b have stereotypical morphology, while CaP neurons expressing partially glycosylated (N220Q) Kv3.1b showed severe maldevelopment with incomplete axonal branching and extension around the ventral musculature. Consequently, larvae expressing N220Q in CaP neurons had impaired swimming locomotor activity. We showed that replacement of complex N-glycans with oligomannose attached to Kv3.1b and at cell surface lessened Kv3.1b dispersal to outgrowths by altering the number, size, and density of Kv3.1b-containing particles in membranes of rat neuroblastoma cells. Opening and closing rates were slowed in Kv3 channels containing Kv3.1b with oligomannose, instead of complex N-glycans, which suggested a reduction in the intrinsic dynamics of the Kv3.1b α-subunit. Thus, N-glycosylation processing of Kv3.1b regulates neuronal development and excitability, thereby controlling motor activity

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

Reduction in N-Acetylglucosaminyltransferase-I Activity Decreases Survivability and Delays Development of Zebrafish

A lack of complex and hybrid types of N-glycans in mice is embryonically lethal due to neural tube maldevelopment. N-acetylglucosaminyltransferase-I (GnT-I; Mgat1) catalyzes a required step for converting oligomannose N-glycans into hybrid and complex N-glycans. Unlike mice, zebrafish have two Mgat1a/b genes. Herein, CRISPR/Cas9 technology was used to knockdown GnT-Ib activity in zebrafish, referred to as Mgat1b−/−, to examine the impact of a decrease in complex types of N-glycans on survival and development, and sensory and motor functions. Genotyping verified the occurrence of edited Mgat1b, and LC-ESI-MS and lectin blotting identified higher levels of oligomannose and lower levels of complex N-glycans in Mgat1b−/− relative to Wt AB. The microscopic visualization of developmental stages and locomotor studies using an automated tracking unit and manual touch assays revealed reduced survivability, and delayed motor and sensory functions in Mgat1b−/−. Moreover, embryonic staging linked reduced survivability of Mgat1b−/− to disruption in brain anlagen formation. Birefringence measurements supported delayed skeletal muscle development, which corresponded with motor and sensory function impediments in Mgat1b−/−. Furthermore, GnT-Ib knockdown hindered cardiac activity onset. Collectively, Mgat1b−/− displayed incomplete penetrance and variable expressivity, such that some died in early embryonic development, while others survived to adulthood, albeit, with developmental delays. Thus, the results reveal that reducing the amount of complex-type N-glycans is unfavorable for zebrafish survival and development. Moreover, our results support a better understanding of human congenital disorders of glycosylation

Limited N-Glycan Processing Impacts Chaperone Expression Patterns, Cell Growth and Cell Invasiveness in Neuroblastoma

Enhanced N-glycan branching is associated with cancer, but recent investigations supported the involvement of less processed N-glycans. Herein, we investigated how changes in N-glycosylation influence cellular properties in neuroblastoma (NB) using rat N-glycan mutant cell lines, NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3), as well as the parental cell line NB_1. The two earlier mutant cells have compromised N-acetylglucosaminyltransferase-I (GnT-I) and GnT-II activities. Lectin blotting showed that NB_1(-Mgat3) cells had decreased activity of GnT-III compared to NB_1. ESI-MS profiles identified N-glycan structures in NB cells, supporting genetic edits. NB_1(-Mgat1) had the most oligomannose N-glycans and the greatest cell invasiveness, while NB_1(-Mgat2) had the fewest and least cell invasiveness. The proliferation rate of NB_1 was slightly slower than NB_1(-Mgat3), but faster than NB_1(-Mgat1) and NB_1(-Mgat2). Faster proliferation rates were due to the faster progression of those cells through the G1 phase of the cell cycle. Further higher levels of oligomannose with 6–9 Man residues indicated faster proliferating cells. Human NB cells with higher oligomannose N-glycans were more invasive and had slower proliferation rates. Both rat and human NB cells revealed modified levels of ER chaperones. Thus, our results support a role of oligomannose N-glycans in NB progression; furthermore, perturbations in the N-glycosylation pathway can impact chaperone systems

The formation of low-temperature sedimentary pyrite and its relationship with biologically-induced processes

This contribution is an updated review on sedimentary pyrite and on its role in well-consolidated research topics, such as the biogeochemical cycles and the studies on sediment-hosted ore deposit studies, as well as new frontiers of research, such as astrobiology. Textural and compositional information preserved in sedimentary pyrite from sediment-hosted ore deposits has contributed to elucidate their environment of forzmation. In particular, the content of redox-sensitive elements such as Ni, Co, Mo, and V has implications for defining the syn- and post-sedimentary conditions. In addition, the stable isotope compositions are useful indicators of the pathways of both biogenic and abiogenic pyrite formation. Despite the longstanding research on pyrite and the mechanism of its formation, there are still significant gaps in our knowledge. In this nonexhaustive review, we briefly touch on different current aspects of research on sedimentary pyrite, exemplifying how sedimentary pyrite remains relevant to geoscientists, and becomes more and more relevant in understanding some basic aspects of knowledge, such as the origin of life and the search for extraterrestrial life, as well as aspect of classical applied science, such as the implications for ore deposition