Victória Trindade Maller Carvalho1, Ana Luiza Lopes1, Adriane Regina Todeschini1, Wagner Barborsa Dias1
The post-translational modification known as O-GlcNAcylation consists of the dynamic and reversible addition of a N-acetylglucosamine (GlcNAc) moiety to serine/threonine residues of intracellular proteins. This process is catalyzed by the enzyme O-GlcNAc transferase (OGT). Its donor sugar, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), reaches high intracellular concentrations and functions as a metabolic sensor and influencing the activity of glycosyltransferases and transporters involved in other glycosylation pathways, such as those of mannose, hyaluronic acid, and UDP-GalNAc. Additionally, O-GlcNAcylation directly influences the regulation of cellular processes through competition for shared substrates. The intestinal epithelium is composed of diverse specialized cell types that play essential roles in maintaining intestinal homeostasis, which encompasses digestion, nutrient absorption, and interactions with the gut microbiota. As a nutrient and hormone sensor, O-GlcNAcylation has been implicated in the regulation of metabolism and tissue integrity; however, its mechanisms of action within the intestinal context remain poorly understood. This study aims to investigate the effects of epithelial-specific deletion of the OGT enzyme in the colon on metabolism and intestinal homeostasis. To this end, C57BL/6 mice with tissue-specific deletion of Ogt (Cdx2-Cre/Ogt^flox) will be used, generated by crossing OGT F/F (female) and OGT F/Y (male) mice with Cdx2-Cre transgenic mice. Deletion will be validated by PCR targeting Cre and Ogt genes in colonic epithelial cells and confirmed by immunofluorescence. Intestinal epithelial cells will be isolated through chemical dissociation followed by flow cytometry sorting using epithelial markers, enabling cell-specific analyses of molecular alterations. To assess the influence of O-GlcNAcylation on gut microbial composition, 16S rRNA gene sequencing-based metagenomics will be performed, including microbial diversity analyses and correlation of these data with the glycosylated surface proteome. In addition, fecal metabolomics will be conducted to quantify short-chain fatty acids and infer the metabolic consequences of OGT deletion. MS-Imaging will be used to trace UDP-GlcNAc levels within tissues, with a focus on the availability of this activated sugar and its impact on competing glycosylation pathways. Complementary analyses will include targeted proteomics and metabolomics aimed at identifying alterations in N- and O-linked glycosylation pathways, both in colonic tissue and in isolated epithelial cells. Expression of genes involved in other glycosylation pathways will be assessed via RT-qPCR and tissue immunofluorescence. To investigate epithelial barrier integrity, in vivo intestinal permeability assays will be performed, along with analyses of junctional proteins expression. This project is currently in its early stages, and it is expected that by employing a murine model with tissue-specific OGT deletion and integrating multi-omics approaches, it will be possible to identify epithelial biomarkers associated with OGT deficiency in the colon. Moreover, this study aims to elucidate how key O-GlcNAcylation-regulated targets influence UDP-GlcNAc metabolic flux, potentially redirecting this substrate toward other glycosylation pathways and thereby broadly impacting intestinal physiology.
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