O -GlcNAc transferase regulates centriole behavior and intraflagellar transport to promote ciliogenesis
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Protein & Cell
LETTER
Dear Editor, O-GlcNAcylation is a nutrient sensor that is particularly sensitive to environmental glucose (Hardiville and Hart, 2014). Glucose can be converted to UDP-GlcNAc through the hexosamine biosynthetic pathway, providing a substrate for O-GlcNAcylation. Two enzymes participate in this reversible modification, O-GlcNAc transferase (OGT), which adds a single GlcNAc residue to the serine/threonine sites of proteins, and O-GlcNAcase (OGA), which removes the residue (Yang and Qian, 2017). OGT is a highly conserved, single gene-encoded protein that is ubiquitously expressed in higher eukaryotes, and human OGT shares more than 65% sequence identity with its Caenorhabditis elegans and Drosophila melanogaster orthologs (Jinek et al., 2004). OGlcNAcylation can influence protein conformation, activity, interaction, half-life, and subcellular localization. Almost all functional proteins are present among the pool of O-GlcNAcylated proteins, including enzymes, structural proteins, and transcription factors. Accordingly, O-GlcNAcylation can regulate complex processes, such as the cell cycle and embryonic development (Yang and Qian, 2017). Dysregulation of O-GlcNAcylation has been implicated in a wide range of pathologies, including cancer, neurodegeneration, cardiovascular diseases, and diabetes. The level of OGlcNAcylation is greatly dysregulated by the abnormal glucose metabolism in diabetic mice and patients (Brownlee, 2001). In addition, we have demonstrated that dysregulation of O-GlcNAcylation is related to diabetic complications due to defects in cilia (Yu et al., 2019), which are hairlike protrusions present on the surface of most mammalian cells. However, the molecular details regarding the role of OGT in cilium assembly are still unclear. To investigate the function of OGT in cilium formation, we generated OGT haploinsufficient mice. Because Ogt is an X-linked gene and complete knockout of OGT is lethal in mice, we first obtained female Ogtfl/+Cre+ and Ogtfl/+Cre− mice by crossing Ogtfl/fl mice with Ubc-Cre-ERT2 mice (Figs. 1A and S1A). We then obtained Ogt+/− and Ogt+/+ mice through intraperitoneal injection of tamoxifen (Fig. 1B). Two months after tamoxifen-induced knockdown of OGT, we © The Author(s) 2020
found that the levels of OGT and protein O-GlcNAcylation were significantly reduced in Ogt+/− mice (Fig. S1B). We then examined the morphology of cilia in these OGT haploinsufficient mice. Immunostaining of primary cilia and motile cilia in different tissues revealed a number of defects in Ogt+/− mice. For example, retinal photoreceptor cilia, which are modified primary cilia, were fewer and shorter in Ogt+/− mouse eyes than in Ogt+/+ mouse eyes; ciliary length reduced from ∼1.5 μm to ∼1.0 μm (Fig. 1C–E). In addition, we found fewer and shorter motile cilia in Ogt+/− mouse trachea (Fig. 1F–I). Line profiles along the arrow-indicated regions revealed that OGT knockdown caused a significant decrease in the fluorescence intensity of tracheal epithelial cilia (Fig. 1F and 1G), and quantification revea
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