| Status | 已發表Published |
| Identifying a role of N-glycosylation in C. elegans epithelial polarity by an RNAi-based biosynthetic-pathway-screen approach | |
Zhang, H. ; Abraham, N.; Khan, L.; Zhang, N.; Gobel, V.
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| 2015-06-24 | |
| Source Publication | 20th International C. elegans Conference Full Abstracts
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| Abstract | The contributions of non-proteinaceous molecules (e.g. lipids and sugars) to polarity and tubulogenesis are poorly understood. In an unbiased tubulogenesis screen, we previously identified a requirement of lipid-biosynthetic enzymes for C. elegans intestinal polarity. A follow-on tiered lipid-biosynthetic-pathway-screen approach* allowed us to identify glycosphingolipids (GSLs), presumed membrane raft components, as the synthesized compounds mediating the enzyme’s function. The analysis procured in vivo evidence for GSLs’ role in apical sorting and supported aspects of the controversial lipid-raft theory on polarity. Similarly controversial is a proposed requirement for sugars, specifically N-glycans, for polarity via apical sorting. To systematically investigate an in vivo requirement for sugars in polarity, we here use this biosynthetic-pathway-screen approach to query carbohydrate biosynthesis. In an initial broad tier-I screen we examined apical marker displacement subsequent to RNAi with each of 209 molecules predicted to affect synthesis, transfer, modification and transport of nucleotide-sugars, N-glycans, O-glycans, glycosaminoglycans and GPI-anchors. Only two enzyme knockdowns affected polarity: F52B11.2, a phosphomannomutase homolog, catalyzing activated nucleotide-sugar synthesis; and BUS-8, an alpha-1,3-mannosyltransferase homolog, needed for lipid-linked oligosaccharide N-, but not O-glycosylation. The follow-on tightened tier-II RNAi screen was thus targeted towards N-glycosylation and detected phenocopy when depleting 7 more genes: F09E5.2, a BUS-8 paralog; four oligosaccharyltransferase-complex components and a dolichol-phosphate-mannosyl-transferase-complex- and GDP-mannose-pyrophosphorylase subunit each. These screens thus identify an in vivo requirement for N-glycan biosynthesis in apical polarity. To examine whether N-glycan diversification was required for function, we next knocked down 104 N-glycan-derivative-specific lectins. This tier III screen, combined with triple mutant analyses of critical enzymes, failed to detect any such requirement. In contrast, an RNAi screen of N-glycosylated proteins revealed that this class of molecules is enriched for proteins required for apical marker placement and intestinal lumen morphogenesis. This class of molecules contains lumen-associated mucins as well as most secreted ECM components, raising the possibility that these molecules, via their sugar-containing portions, affect trafficking pathways for apical polarity. |
| Keyword | N-glycosylation epithelial polarity RNAi biosynthetic-pathway-screen |
| URL | View the original |
| Language | 英語English |
| The Source to Article | PB_Publication |
| PUB ID | 14674 |
| Document Type | Conference paper |
| Collection | DEPARTMENT OF BIOMEDICAL SCIENCES Faculty of Health Sciences |
| Recommended Citation GB/T 7714 | Zhang, H.,Abraham, N.,Khan, L.,et al. Identifying a role of N-glycosylation in C. elegans epithelial polarity by an RNAi-based biosynthetic-pathway-screen approach[C], 2015. |
| APA | Zhang, H.., Abraham, N.., Khan, L.., Zhang, N.., & Gobel, V. (2015). Identifying a role of N-glycosylation in C. elegans epithelial polarity by an RNAi-based biosynthetic-pathway-screen approach. 20th International C. elegans Conference Full Abstracts. |
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