The Role of Complex Type N-Glycans in Neuronal Development

Abstract

Oligosaccharides are attached to up to 70% of human proteins. N-linked glycosylation is one of the predominant forms of co- and post-translational modifications in vertebrates. Discrepancies in glycan processing result in congenital disorders of glycosylation (CDGs), a group of rare genetic disorders characterized by impaired glycan synthesis or processing, leading to a wide range of clinical manifestations. CDGs are divided into two classes: type 1 and type 2. Regarding N-glycosylation, CDG type 1 disorders result from improper glycan synthesis or transfer of the precursor oligosaccharide, while CDG type 2 disorders result from disrupted processing of the precursor oligosaccharide linked to protein. Many identified CDG disorders are lethal within the first year of life, and those that survive often have neurological complications. Those who survive are impacted by epilepsy, locomotor skill deficiency, and slowed or stunted growth. N-linked oligosaccharides are processed by many enzymes, such as N-acetylglucosaminyltransferases (GnTs) and transporters. The three common types of N-glycans are oligomannose, hybrid, and complex. The first GnT to act is GnT-I, which is responsible for the processing of oligomannose to hybrid N-glycans via addition of a N-acetylglucosamine (GlcNaC) residue to the conserved core of N-glycans. The importance of glycan processing by GnT-I has been implicated in several studies using knockdown organismal and cell models. Global GnT-I knockout in mice was found to be lethal at the embryonic age of 8.5-10.5 days. This timeframe coincides with neurogenesis and the neural tube formation from the neural plate, which involves cell migration and proliferation. GnT-I was inactivated in developing neural tissue of mice, which perturbed neuron development. Further, these mice suffered from a shortened life span and reduced body size. These findings highlight the critical role of GnT-I in neurogenesis, growth, and longevity. This study used zebrafish as an organismal model since embryonic and larval development stages are well-characterized, and the number of individual fish studied can be relatively large. Zebrafish, unlike mice, possess two gene copies of GnT-I referred to as mgat1a and mgat1b, resulting from multiple whole genome duplications. Inactivation of one of the two mgat1 genes, mgat1b, reduced the level of glycan processing from oligomannose to hybrid and complex N-glycans. Glycomics profiling supported this knockout with an increase in the Glc2Man5 structure acted upon by GnT-I. The GnT-Ib knockout fish had reduced complex N-glycans. Survivability was greatly reduced in the mutant fish line which began to drop around 10hpf. Developmental milestones were delayed when mgat1b was knocked out such as the presence of a heartbeat and swim bladder inflation. Sensory motor function was reduced in mutant line fish as indicated by reduced motor activity and slow touch or vibrational startle response as well as motor coordination and stamina. Muscle structure development of mgat1b knockout fish was delayed until up to 72 hpf via birefringence microscopy. Spinal cord caudal primary (CaP) primary motor neurons transiently expressing electric green fluorescent protein (EGFP) were examined using fluorescent microscopy with the mutant line having reduced collateral branching. These results indicate the importance of complex N-glycosylation on zebrafish development and motor function.