O-glycan initiation directs distinct biological pathways and controls epithelial differentiation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE141387 | GEO | 2020/01/10
REPOSITORIES: GEO
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Project description:Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, thus understanding molecular changes associated with EOC progression could lead to the identification of essential therapeutic targets. Glycosylation is a post-translational modification (PTM) that participates in major pathophysiology events, including tumorigenesis. Aberrant mucin-type or O-glycosylation in cancer is frequently attributed to altered expression of different N-acetylgalactosaminyltransferases (GalNAc-Ts). We have previously identified the N-acetylgalactosaminyltransferase 3 (GALNT3) gene as a potential EOC oncogene, possibly implicated in aberrant O-glycosylation in EOC cells. Literature reports are suggestive for genetic and functional redundancy between different GalNAc-Ts, and our previous data were indicative for an induction of the GALNT6 expression upon GALNT3 suppression in EOC cells. Here we show that GALNT3 gene knockout (KO) in the A2780s EOC cells leads to strong GALNT6 upregulation. Moreover, A2780s cell migration, invasion, proliferation and cell cycle analysis were significantly reduced in double GALNT3/T6 gene KO cells compared to single GALNT3 gene KO. Furthermore, MUC1 expression was downregulated in both GALNT3 KO and GALNT3/6 KO, with a more prominent decrease observed in the double KO clone. In vivo studies supported the observed functional redundancy imposed by GALNT6, as animals injected with double GALNT3/T6 KO EOC cell clones displayed a significant increase in survival rates compared to those injected with the control and the GALNT3 KO clones. Collectively our data strongly suggest for possible functional redundancy of the two GalNAc-Ts (GALNT3 and T6) in EOC, with the perspective to use both of these genes as EOC markers and/or therapeutic targets.
2019-05-07 | GSE104861 | GEO
Project description:Previously, we have identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that GALNT3 is strongly overexpressed in both LMP and HG serous EOC tumors, thus suggesting that epigenetic mechanisms might be implicated in GALNT3 overexpression in serous epithelial ovarian cancer (EOC). Moreover, GALNT3 expression significantly correlated with shorter progression-free survival (PFS) periods in serous EOC patients with advanced disease. Knockdown of the GALNT3 expression in EOC cells led to sharp decrease of cell proliferation and induced S-phase cell cycle arrest. Additionally, GALNT3 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon GALNT3 suppression, while some tumor suppressor genes were induced. Moreover, GALNT3 downregulation was associated with reduced MUC1 protein expression in EOC cells, probably related to destabilization of the MUC1 protein due to lack of GALNT3 glycosylation activity.Taken together, our data are indicative for a strong oncogenic potential of the GALNT3 gene in advanced EOC and identify this transferase as a novel EOC biomarker and putative EOC therapeutic target. Our findings also suggest that GALNT3 overexpression might contribute to ovarian etiology through aberrant mucin O-glycosylation. To better understand the molecular mechanisms of GALNT3 gene action in ovarian cancer cells, we employed the Agilent Whole Human Genome microarrays, containing ~ 44,000 genes to identify global gene expression changes upon GALNT3 suppression in A2780s cells. We compared the gene expression of the previously selected clone shRNA- GALNT3-knockdown clones 1 & 2 (sh-cl1 & sh-cl2) against the corresponding control (ctrl) clone. The microarray experiments were performed in duplicates, as four hybridizations were carried out for the GALNT3-suppressing cell clones against the corresponding control, using a fluorescent dye reversal (dye-swap) technique.
2014-05-22 | E-GEOD-52602 | biostudies-arrayexpress
Project description:Previously, we have identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that GALNT3 is strongly overexpressed in both LMP and HG serous EOC tumors, thus suggesting that epigenetic mechanisms might be implicated in GALNT3 overexpression in serous epithelial ovarian cancer (EOC). Moreover, GALNT3 expression significantly correlated with shorter progression-free survival (PFS) periods in serous EOC patients with advanced disease. Knockdown of the GALNT3 expression in EOC cells led to sharp decrease of cell proliferation and induced S-phase cell cycle arrest. Additionally, GALNT3 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon GALNT3 suppression, while some tumor suppressor genes were induced. Moreover, GALNT3 downregulation was associated with reduced MUC1 protein expression in EOC cells, probably related to destabilization of the MUC1 protein due to lack of GALNT3 glycosylation activity.Taken together, our data are indicative for a strong oncogenic potential of the GALNT3 gene in advanced EOC and identify this transferase as a novel EOC biomarker and putative EOC therapeutic target. Our findings also suggest that GALNT3 overexpression might contribute to ovarian etiology through aberrant mucin O-glycosylation.
2014-05-22 | GSE52602 | GEO
Project description:Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. The majority of glycans are classified as N-linked chains, where the carbohydrate moiety is attached to asparagine residues, or O-linked chains, most commonly linked to a serine or threonine. N-linked glycosylation is initiated by the oligosaccharyltransferase complex with only two paralogs of the catalytic subunit, whereas O-glycan initiation is more complex. There are several types of O-linked glycosylation, but among the most diverse is the mucin or GalNAc type (hereafter referred to as O-glycosylation). O-glycosylation is initiated by 20 evolutionarily conserved polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the first step in the O-glycosylation of proteins by adding GalNAc residues to threonine, serine, and tyrosine amino acids (Fig 1A). Each of the GalNAc-Ts are differentially expressed in various tissues and have both distinct and overlapping peptide substrate specificities [2-12]. Thus, the repertoire of GalNAc-Ts expressed in a given cell determines the subset and O-glycosite pattern of glycosylated proteins [13]. Substantial efforts have been made to characterize and predict the substrate specificities of GalNAc-Ts in vitro, but understanding of the in vivo specificities of the individual GalNAc-Ts or their biological functions is limited [13-15]. This lack of insight prevents an understanding of how site-specific O-linked glycosylation affects diseases, such as metabolic disorders, cardiovascular disease, and various malignancies, that have been associated with GalNAc-Ts through genome-wide association studies and other linkage studies [16-26]. Therefore, it is imperative that we establish how O-glycosylation at specific sites in proteins affects protein function. A major task in achieving this goal is to identify the non-redundant biological functions of site-specific O-glycosylation. We and others recently developed new strategies for identifying specific sites on proteins that undergo O-glycosylation in different cell types and tissues [27-31]. Characterization of the O-glycoproteomic landscape in isolated human cells and multiple human cell lines suggests that more than 80 % of all proteins that traffic through the secretory pathway are O-glycoproteins [28, 30]. Probing the non-redundant contributions of individual GalNAc-Ts in cells with and without specific GalNAc-Ts [32-34] has revealed broad substrate specificities for some of the individual isoforms, whereas others seem to have very restricted substrate specificities [33-35]. Assessing all of the mapped O-glycosylation sites to identify associations between O-glycosites and protein annotations, we recently found that O-glycans are over-represented close to tandem repeat regions, protease cleavage sites, within propeptides, and on a select group of protein domains [28, 30, 36]. Although such general associations between the location of O-glycans and protein functions may direct future investigations, the strategy does not define the function of site-specific glycosylation. Further progress in discovering and defining novel functions of site-specific glycosylation events requires direct quantitative analysis of potential biological responses induced by the loss of distinct GalNAc-T isoforms, and such biological responses are not easily observed in single cell culture systems. Instead, more complex model systems can be used to examine and dissect the molecular mechanisms underlying the important biological functions of site-specific glycosylation. We previously used an organotypic tissue model equipped with genetically engineered cells to decipher the function of elongated O-glycans [29]. In the present study, we use the model combined with quantitative O-glycoproteomics and phosphoproteomics to perform open-ended discovery of the biological functions of site-specific glycosylation governed by GalNAc-Ts (Fig 1B). With this combinatorial strategy, we demonstrate that loss of individual GalNAc-T isoforms has distinct phenotypic consequences through their effect on distinct biological pathways, suggesting specific roles during epithelial formation.
2020-05-22 | PXD016618 | Pride
Project description:Post-translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O-glycosylation is among the most abundant and diverse PTMs. Initiation of O-GalNAc glycosylation is regulated by 20 distinct GalNAc-transferases (GalNAc-Ts), and deficiencies in individual GalNAc-Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc-Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc-Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc-T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc-T1 targets are associated with components of the endomembrane system, GalNAc-T2 targets with cell-ECM adhesion, and GalNAc-T3 targets with epithelial differentiation. Thus, GalNAc-T isoforms serve specific roles during human epithelial tissue formation.
| S-EPMC7271655 | biostudies-literature
Project description:We used RNA_Seq to quantify cellular transcripts in human immortalized N/TERT-1 keratinocytes upon knock out of genes delineating key subtypes of glycosylation pathways in humans.
2020-05-15 | GSE148284 | GEO
Project description:The intestinal anaerobic bacterium Akkermansia muciniphila is specialized in the degradation of mucins, which are heavily O-glycosylated proteins that constitute the major components of the mucus lining the intestine. Despite that adhesion to mucins is considered critical for the persistence of A. muciniphila in the human intestinal tract, our knowledge of how this intestinal symbiont recognizes and binds to mucins is still limited. Here, we first show that the mucin-binding properties of A. muciniphila are independent of environmental oxygen concentrations and not abolished by pasteurization. We then dissected the mucin-binding properties of pasteurized A. muciniphila by use of a recently developed cell-based mucin array that enables display of the tandem repeats of human mucins with distinct O-glycan patterns and structures. We found that A. muciniphila recognizes the unsialylated LacNAc (Galβ1-4GlcNAc1-R) disaccharide selectively on core2 and core3 O-glycans. This disaccharide epitope is abundantly found on human colonic mucins capped by sialic acids, and we demonstrated that endogenous A. muciniphila neuraminidase activity can uncover the epitope and promote binding. In summary, our study provides insights into the mucin-binding properties important for colonization of a key mucin-foraging bacterium.
2024-05-30 | PXD051738 | Pride
Project description:Dr. Clausen's laboratory is interested in the structure, biosynthesis and genetic regulation of glycoconjugates, with a major focus on mucins. This laboratory is studying the glycosylation and secretion process of mucins and biological functions involving mucins. This lab is also interested in receptor modulation mediated by glycosylation or through glycosphingolipids. We have a particular interest in understanding how mucin type O-glycosylation is regulated. Mucin type O-glycosylation is controlled by the ppGalNAc-transferase multigene family. An estimated 23 iso-forms belong to this family, so the specific involvement of each individual iso-form in any given organ seems complex to understand. An initial attempt to comprehend how this multigene family regulates mucin type O-glycosylation, is to get insight into which iso-forms are expressed in different organs and cell types. For this reason we have generated monoclonal antibodies towards 7 iso-forms, and the list of ppGalNAc-T iso-form specific monoclonals is currently growing. We have done some immuno histochemistry on lung using the available monoclonals, but are keen on assessing the number of ppGalNAc-T's expressed in lung as determined this Glyco-array. We have focused on lung because a number of ppGalNAc-T acceptor polypeptides, belonging to the mucin gene family, are expressed in this organ. An RNA sample pooled from two independent adult human healthy lung tissue samples was analyzed
2010-11-30 | E-GEOD-25712 | biostudies-arrayexpress
Project description:Dr. Clausen's laboratory is interested in the structure, biosynthesis and genetic regulation of glycoconjugates, with a major focus on mucins. This laboratory is studying the glycosylation and secretion process of mucins and biological functions involving mucins. This lab is also interested in receptor modulation mediated by glycosylation or through glycosphingolipids. We have a particular interest in understanding how mucin type O-glycosylation is regulated. Mucin type O-glycosylation is controlled by the ppGalNAc-transferase multigene family. An estimated 23 iso-forms belong to this family, so the specific involvement of each individual iso-form in any given organ seems complex to understand. An initial attempt to comprehend how this multigene family regulates mucin type O-glycosylation, is to get insight into which iso-forms are expressed in different organs and cell types. For this reason we have generated monoclonal antibodies towards 7 iso-forms, and the list of ppGalNAc-T iso-form specific monoclonals is currently growing. We have done some immuno histochemistry on lung using the available monoclonals, but are keen on assessing the number of ppGalNAc-T's expressed in lung as determined this Glyco-array. We have focused on lung because a number of ppGalNAc-T acceptor polypeptides, belonging to the mucin gene family, are expressed in this organ.
2010-11-30 | GSE25712 | GEO
Project description:Aberrant glycosylation is a characteristic of tumor cells. The expression of certain glycan structures has been associated with poor prognosis. In cervical carcinoma has been reported changes in the gene expression level of some glycogenes that has been associated with lymph invasion. Human papilloma virus (HPV) infection is one of the most important factors to develop cervical cancer. HPV oncoproteins E6 and E7 have been implicated in cervical carcinogenesis. The role of these oncoproteins in glycosylation changes has not been reported. To know the effect of these oncoproteins on glycogene expression we realized a partial silencing of oncogenes E6 and E7 in HeLa cells, we made a microarray expression assay and we identified glycogenes that modified its expression. The analysis showed alteration in some glycosylation pathways, like glycosphingolipid, O-glycan mucin-type, and O-glycan non mucin-type glycosylation. Our results suggest that E6 and E7 oncoproteins could modified glycosylation of structures implicated in proliferation, adhesion, and apoptosis.
2016-12-07 | GSE90930 | GEO