RNAi-Mediated Silencing of Pgants Shows Core 1 O-Glycans Are Required for Pupation in Tribolium castaneum

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.

Developmental O‐glycan profile analysis shows pentasaccharide mucin‐type O‐glycans are linked with pupation of Tribolium castaneum

“…Throughout the development of T. castaneum, GalNAc and the core 1 O-glycans are present at relatively high levels, suggesting that these O-glycans play important roles in development and are probably required in each stage. This is in agreement with the lethal phenotype reported after disruption of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (PGANT) activity, an enzyme required for the attachment of GalNAc to the peptide (Li et al, 2021;Tian & Ten Hagen, 2007;Tran et al, 2012;Yamamoto-Hino et al, 2015). The relative abundance of the O-GalNAcGal, O-GalNAcGal 2, and O-GalNAcGalGlcA glycans is significantly lower at the pupa stage compared with the larval stage.…”

“…This could be attributed to the higher abundance of more complex mucin-type O-glycans, such as O-GalNAc(GalNAc)GlcA, O-GalNAcGalGlcAGlcNAc, and O-GalNAc(GlcNAcGlcA)GalGlcA, at this stage requiring the smaller mucin O-glycan structures for their synthesis. While the enzymes required for the synthesis of the more complex mucin-type O-glycans have not yet been identified (Li et al, 2020), PGANT activity was shown to be involved in the metamorphosis of T. castaneum (Dönitz et al, 2015;Li et al, 2021). For instance, downregulation of C1GalTA, the galactosyltransferase required for the elongation of O-GalNAc with a Gal residue, was shown to cause pupal lethality (Li et al, 2021;Lin et al, 2008).…”

“…While the enzymes required for the synthesis of the more complex mucin-type O-glycans have not yet been identified (Li et al, 2020), PGANT activity was shown to be involved in the metamorphosis of T. castaneum (Dönitz et al, 2015;Li et al, 2021). For instance, downregulation of C1GalTA, the galactosyltransferase required for the elongation of O-GalNAc with a Gal residue, was shown to cause pupal lethality (Li et al, 2021;Lin et al, 2008). These observations together with our current analysis of the O-glycan profile, revealing a shift to more complex O-glycan structures at pupal stage, yield strong indications that the more complex mucin-type O-glycans are required for pupal development or metamorphosis in T. castaneum.…”

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“…Both O-and N-glycosylation are important for insect physiology and development, as shown by RNAi experiments. Indeed, silencing of different enzymes important for the glycosylation process in insects caused failure of postembryonic development and wing formation [109,113].…”