Mechanism of lacritin-dependent production of the important dry eye mucin 'MUC16'
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE31930 | GEO | 2011/09/06
SECONDARY ACCESSION(S): PRJNA145025
REPOSITORIES: GEO
Similar Datasets
Project description:Lacritin targets its coreceptor 'syndecan-1' in lacritin-dependent production of the important dry eye mucin 'MUC16'. Real-time PCR analysis suggests that this effect is post-transcriptional. In contrast, serum stimulation of MUC16 by these cells is transcriptionally dependent. Our goal is to determine whether this regulation is global to other heavily O-glycosylated proteins, and if so, if it is dependent on transcription of a Golgi glycosyltransferases. If not, the mechanism might be microRNA dependent. Among other glycogenes, expression of heparanases, sulfotransferases and epimerase are also of interest. Heparanase is required for lacritin binding to its co-receptor syndecan-1 and this binding site may require a sulfated iduronic acid. In request 1705, we recently asked core D whether they could generate this sulfate iduronic acid-containing glycan (iduronic acid (2-O-sulfated)-N-acetylglucosamine-glucuronic acid-N-acetylglucosamine-[glucuronic acid-N-acetylglucosamine]5-glucuronic acid-galactose-galactose-SERINE) to test as a competitive inhibitor in binding studies. Our experiment is to compare Glyco-gene expression by human corneal epithelial cells in response to cell communication glycoprotein 'lacritin' versus negative control deletion construct 'C-25 lacritin' and versus positive control serum.
2011-09-05 | E-GEOD-31930 | biostudies-arrayexpress
Project description:Purpose. How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. We sought to identify vitamin A responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial cell line (HCjE) grown with all-trans-retinoic acid (RA). The analysis showed that the membrane-associated mucin MUC16 was induced by RA and that secretory phospholipase A2 Group IIA (sPLA2-IIA), the gene most upregulated by RA, was induced earlier. Since eicosanoids, metabolites of arachidonic acid, which is produced by sPLA2 catalysis of membrane phospholipids, have been demonstrated to affect mucin production, we sought to determine if the sPLA2 induction in HCjE cells was associated with RA induction of MUC16. Methods. HCjE cells were cultured with or without RA for 3, 6, 24 and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips (HG-U133A; Affymetrix) and analyzed using Rosetta Resolver software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA2 is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad spectrum PLA2 inhibitor, aristolochic acid (ArA) or the specific sPLA2-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis. Results. After RA addition, 28 transcripts were upregulated and 6 downregulated by over 2.0-fold (p < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA2, significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA2 upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA2-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (p < 0.01). Conclusion. The retinoic acid-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA2-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface. Experiment Overall Design: Time course of retinoic acid treatment of human conjunctival epithelial cells: 0 (control), 3, 6, 12, 24, and 48 hours. 2 samples per time point.
2007-12-12 | E-GEOD-2835 | biostudies-arrayexpress
Project description:Purpose. How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. We sought to identify vitamin A responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial cell line (HCjE) grown with all-trans-retinoic acid (RA). The analysis showed that the membrane-associated mucin MUC16 was induced by RA and that secretory phospholipase A2 Group IIA (sPLA2-IIA), the gene most upregulated by RA, was induced earlier. Since eicosanoids, metabolites of arachidonic acid, which is produced by sPLA2 catalysis of membrane phospholipids, have been demonstrated to affect mucin production, we sought to determine if the sPLA2 induction in HCjE cells was associated with RA induction of MUC16. Methods. HCjE cells were cultured with or without RA for 3, 6, 24 and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips (HG-U133A; Affymetrix) and analyzed using Rosetta Resolver software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA2 is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad spectrum PLA2 inhibitor, aristolochic acid (ArA) or the specific sPLA2-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis. Results. After RA addition, 28 transcripts were upregulated and 6 downregulated by over 2.0-fold (p < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA2, significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA2 upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA2-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (p < 0.01). Conclusion. The retinoic acid-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA2-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface. Keywords: time course
2005-12-20 | GSE2835 | GEO
Project description:While growing in the human intestine, C. jejuni grows within the mucus layer. The largest constituents of this layer are the large mucin glycoproteins. A transcriptomic profile of C. jejuni NCTC11168 growing in a mucin-containing minimal medium seeks to describe the effect of the presence of mucin proteins on the transcriptome of C. jejuni.
2011-04-01 | GSE28295 | GEO
Project description:While growing in the human intestine, C. jejuni grows within the mucus layer. The largest constituents of this layer are the large mucin glycoproteins. A transcriptomic profile of C. jejuni NCTC11168 growing in a mucin-containing minimal medium seeks to describe the effect of the presence of mucin proteins on the transcriptome of C. jejuni. Microarray data was collected from three independent biological replicates and 9 technical replicates for each biological replicate.
2011-04-01 | E-GEOD-28295 | biostudies-arrayexpress
Project description:Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, we recently introduced a new mucinase (SmE) and demonstrated it has complementary recognition patterns to StcE. Here, we optimized our previous enrichment method to increase throughput and lower sample input amounts while maintaining mucin selectivity and enhancing glycopeptide signal. We also compare the ability of different mucinases, lectins, and mucin binding domains to enrich mucins from various cell lines and human serum. Overall, we demonstrate that our improved method StcE effectively isolates more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, which will allow for enhanced analysis of the mucinome.
2024-04-10 | PXD046534 | Pride
Project description:Mechanism of lacritin-dependent production of the important dry eye mucin 'MUC16'
| PRJNA145025 | ENA
Project description:Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N-glycoconjugates in human lysosomal storage disorders, is that proteolysis and glycan degradation occur largely independently. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O-glycosylated mucin domains, without prior deglycosylation. Using activity screening coupled with mass spectrometry we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis resulted in accumulation of mucins in liver-resident macrophages. Our findings suggest that mucin-degrading activity is not limited to the bacterial kingdom and is a component of glycoprotein catabolism in mammalian tissues.
2022-09-03 | PXD027616 | Pride
Project description:Our experiment is to compare Glyco-gene expression by interferon-gamma/TNF-treated human corneal epithelial cells also treated either with cell communication glycoprotein 'lacritin' or negative control deletion construct 'C-25 lacritin'. In a newly developing story, lacritin but not C-25 protects human corneal epithelial cells from INFG/TNF-induced cell death. This expression array will help work out the mechanism of protection to complement phosphoblotting and shRNA approaches currently underway. Lacritin targets its coreceptor 'syndecan-1' in lacritin-dependent cell proliferation and secretion. Lacritin appears to be important in human 'dry eye' and in promoting corneal epithelial cell survival in the presence of inflammatory cytokines.
2011-09-05 | E-GEOD-31931 | biostudies-arrayexpress
Project description:How novel gene functions evolve is a fundamental question in biology. Mucin proteins, a functionally but not evolutionarily defined group of proteins, allow to study convergent evolution of gene function. By analyzing the genomic variation of mucins across a wide range of mammalian genomes, we propose that exonic repeats and their copy number variation contribute substantially to the de novo evolution of new gene functions. By integrating bioinformatic, phylogenetic, proteomic, and immunohistochemical approaches, we identified 15 undescribed instances of evolutionary convergence, where novel mucins originated by gaining densely O-glycosylated exonic repeat domains. Our results suggest that secreted proteins rich in proline are natural precursors for acquiring mucin function. Our findings have broad implications for understanding the role of exonic repeats in the parallel evolution of new gene functions, especially those involving protein glycosylation.
2022-10-15 | PXD033197 | Pride