Abstract
Protein glycosylation is one of the most common and most important post-translational modifications. Despite the growing knowledge on N-glycosylation, the research on O-glycosylation is lagging behind. This study investigates the importance of O-glycosylation in the post-embryonic development of insects using the red flour beetle, Tribolium castaneum, as a model. We identified 28 O-glycosylation-related genes (OGRGs) in the genome of the red flour beetle. 14 OGRGs were selected for functional analysis based on their involvement in the initial attachment of the carbohydrate in the different O-glycosylation pathways or the further elongation of the most abundant O-glycans and, in addition, showing severe RNAi-induced phenotypes in Drosophila melanogaster. The expression profile of these OGRGs was mapped throughout the developmental stages of the insect and in the different tissues of the pupa and adult. Subsequently, these genes were silenced using RNA interference (RNAi) to analyze their role in development. A broad spectrum of phenotypes was observed: from subtle effects and disrupted wing formation when silencing the genes involved in O-mannosylation, to blockage of pupation and high mortality after silencing of the genes involved in O-GalNAc and core 1 O-glycan (O-GalNAc-Gal) synthesis. RNAi experiments were also performed to assess the effects of blocking multiple pathways of O-glycosylation. However, the observed phenotypes induced by multiple RNAi were similar to those of the single gene RNAi experiments. The silencing of OGRGs often resulted in high mortality and wing phenotypes, indicating the importance of O-glycosylation for the survival of the insect and the formation of wings during the post-embryonic development of T. castaneum.
Highlights
The decoration of proteins with carbohydrates is one of the most important post-translational modifications
We discovered that most O-glycan transferases reached a peak of gene expression in the pupal stage, and a decrease in O-glycosylation-related genes (OGRGs) transcription levels resulted in mortality as well as abnormal wings and antennae
OGRGs in the genome of T. castaneum were identified through BLAST searches
Summary
The decoration of proteins with carbohydrates is one of the most important post-translational modifications. In contrast to N-glycosylation, where an oligosaccharide (glycan precursor) is transferred to the nascent protein in the endoplasmic reticulum (ER), O-glycans are synthesized by the stepwise addition of monosaccharides in the ER, Golgi or cytosol (Spiro, 2002; De Pourcq et al, 2010; Dell et al, 2010; Figure 1). O-glycans consist mainly of eight monosaccharides –N-acetylgalactosamine (GalNAc), galactose (Gal), N-acetylglucosamine (GlcNAc), fucose (Fuc), mannose (Man), glucose (Glc), glucuronic acid (GlcA) and xylose (Xyl)– which can be assembled into oligosaccharides or polysaccharides. The attached residue in O-glycosylation can be GalNAc, GlcNAc, Fuc, Man, Glc, Xyl (Haltom and Jafar-Nejad, 2015; Walski et al, 2017), while the attached monosaccharide residue in
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