Abstract
Glycosylation, a common modification of cellular proteins and lipids, is often altered in diseases and pathophysiological states such as hypoxia, yet the underlying molecular causes remain poorly understood. By utilizing lectin microarray glycan profiling, Golgi pH and redox screens, we show here that hypoxia inhibits terminal sialylation of N- and O-linked glycans in a HIF- independent manner by lowering Golgi oxidative potential. This redox state change was accompanied by loss of two surface-exposed disulfide bonds in the catalytic domain of the α-2,6-sialyltransferase (ST6Gal-I) and its ability to functionally interact with B4GalT-I, an enzyme adding the preceding galactose to complex N-glycans. Mutagenesis of selected cysteine residues in ST6Gal-I mimicked these effects, and also rendered the enzyme inactive. Cells expressing the inactive mutant, but not those expressing the wild type ST6Gal-I, were able to proliferate and migrate normally, supporting the view that inactivation of the ST6Gal-I help cells to adapt to hypoxic environment. Structure comparisons revealed similar disulfide bonds also in ST3Gal-I, suggesting that this O-glycan and glycolipid modifying sialyltransferase is also sensitive to hypoxia and thereby contribute to attenuated sialylation of O-linked glycans in hypoxic cells. Collectively, these findings unveil a previously unknown redox switch in the Golgi apparatus that is responsible for the catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I.
Highlights
Glycosylation is a ubiquitous modification of cell surface proteins and lipids, and important for a multitude of recognition events between cells, cells and extracellular matrix components, signaling molecules and invading pathogens [1]
We found that hypoxia exposure of cells induced marked changes in both N- and O-linked glycans (Fig. 1a), increasing mainly the level of terminal galactose residues
The data described above provides a direct mechanistic link between hypoxia, attenuated sialylation of N- and O-glycans, lowered Golgi oxidative potential, reduced disulfide bond formation and the loss of catalytic activity of a relevant ST6Gal-I N-sialyltransferase and its ability to interact with its binding partner B4GalT-I
Summary
Glycosylation is a ubiquitous modification of cell surface proteins and lipids, and important for a multitude of recognition events between cells, cells and extracellular matrix components, signaling molecules and invading pathogens [1] They are dynamic and altered in certain environmental or pathophysiological conditions including cancers, inflammation, and diabetes [2,3,4,5,6], giving rise give to different cellular phenotypes or cell behaviors. This is evident in cancers, in which the more branched and over-sialylated N-glycans as well as truncated O-glycans are common and implicated as being important for tumorigenesis [3,7,8,9,10], as they typically increase cancer cell invasion and metastatic spread into adjacent tissues. Besides enzyme level changes, other defects must exist and need be identified
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