TY - JOUR
T1 - Human UDP-galactose 4′-epimerase (GALE) is required for cell-surface glycome structure and function
AU - Broussard, Alex
AU - Florwick, Alyssa
AU - Desbiens, Chelsea
AU - Nischan, Nicole
AU - Robertson, Corrina
AU - Guan, Ziqiang
AU - Kohler, X. Jennifer J.
AU - Wells, Lance
AU - Boyce, Michael
N1 - Funding Information:
This work was supported by a scholar award from the Rita Allen Foundation (to A. B. and M. B.); NIGMS, National Institutes of Health Grant GM069338 and NEI, National Institutes of Health Grant EY023666 (to Z. G.), Welch Foundation Grant I-1686 (to J. J. K.), and a postdoctoral fellowship from the German Academic Exchange Service (to N. N.). The authors declare that they have no conflicts of interest with the contents of this article. The con-tent is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020
Y1 - 2020
N2 - Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulateNSlevels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cellsurface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE-/- cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.
AB - Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulateNSlevels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cellsurface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE-/- cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.
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U2 - 10.1074/jbc.RA119.009271
DO - 10.1074/jbc.RA119.009271
M3 - Article
C2 - 31819007
AN - SCOPUS:85078870818
VL - 295
SP - 1225
EP - 1239
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 5
ER -