Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins
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Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins. / Sun, Lingbo; Konstantinidi, Andriana; Ye, Zilu; Nason, Rebecca; Zhang, Yuecheng; Büll, Christian; Kahl-Knutson, Barbro; Hansen, Lars; Leffler, Hakon; Vakhrushev, Sergey Y; Yang, Zhang; Clausen, Henrik; Narimatsu, Yoshiki.
In: Journal of Biological Chemistry, Vol. 298, No. 2, 101382, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins
AU - Sun, Lingbo
AU - Konstantinidi, Andriana
AU - Ye, Zilu
AU - Nason, Rebecca
AU - Zhang, Yuecheng
AU - Büll, Christian
AU - Kahl-Knutson, Barbro
AU - Hansen, Lars
AU - Leffler, Hakon
AU - Vakhrushev, Sergey Y
AU - Yang, Zhang
AU - Clausen, Henrik
AU - Narimatsu, Yoshiki
N1 - Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
U2 - 10.1016/j.jbc.2021.101382
DO - 10.1016/j.jbc.2021.101382
M3 - Journal article
C2 - 34954141
VL - 298
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 2
M1 - 101382
ER -
ID: 290599197