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- Chen, Yen Hsi, et al.
(författare)
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The GAGOme : a cell-based library of displayed glycosaminoglycans
- 2018
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 15:11, s. 881-888
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Tidskriftsartikel (refereegranskat)abstract
- Glycosaminoglycans (GAGs) are essential polysaccharides in normal physiology and disease. However, understanding of the contribution of specific GAG structures to specific biological functions is limited, largely because of the great structural heterogeneity among GAGs themselves, as well as technical limitations in the structural characterization and chemical synthesis of GAGs. Here we describe a cell-based method to produce and display distinct GAGs with a broad repertoire of modifications, a library we refer to as the GAGOme. By using precise gene editing, we engineered a large panel of Chinese hamster ovary cells with knockout or knock-in of the genes encoding most of the enzymes involved in GAG biosynthesis, to generate a library of isogenic cell lines that differentially display distinct GAG features. We show that this library can be used for cell-based binding assays, recombinant expression of proteoglycans with distinct GAG structures, and production of distinct GAG chains on metabolic primers that may be used for the assembly of GAG glycan microarrays.
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| 2. |
- Sun, Lingbo, et al.
(författare)
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Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins
- 2021
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Ingår i: Journal of Biological Chemistry. - : Elsevier. - 0021-9258 .- 1083-351X. ; 298:2
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Tidskriftsartikel (refereegranskat)abstract
- The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as Selectins, Galectins, and Siglecs are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human HEK293 cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knock-in of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1-3GalNAcα1-O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3O-sulfotransferase 2 (GAL3ST2) in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3O-sulfotransferase 4 (GAL3ST4) resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step towards expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.
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