A Golgi-associated redox switch regulates catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I

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.

Golgi inCOGnito: From vesicle tethering to human disease

“…Also, the luminal environment within the Golgi governs glycosylation and Golgi homeostasis. Each Golgi cisternae has a unique pH and ionic environment, which contributes to correct enzyme localization [ 20 ] and also regulates enzyme activity [ 21 , 22 ]. The contribution of these factors to Golgi homeostasis has been reviewed by Kellokumpu [ 136 ] ].…”

Golgi pH, Ion and Redox Homeostasis: How Much Do They Really Matter?

“…Accordingly, hypoxia has been shown to alter expression levels of both glycosyltransferase and nucleotide sugar transporter genes, and to inhibit membrane trafficking between the ER and the Golgi (Koike et al, 2004; Shirato et al, 2011; Belo et al, 2015; Bensellam et al, 2016; Taniguchi et al, 2016). We recently showed that hypoxia modulates the Golgi redox state and glycosylation without markedly affecting Golgi pH homeostasis (Hassinen et al, 2019; see also below). Oxygen levels not only affect the redox state of the Golgi lumen, but also the production of NO levels by modulating the activity and expression of various NO synthase isoforms, including neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) (Jeffrey Man et al, 2014).…”

“…Intriguingly, the same enzymes (except B4GalT-I) were also found to mislocalize in chloroquine treated cells (unpublished observations), suggesting that heteromer formation may contribute to their retention or retrieval in the Golgi membranes. However, in other cases such as B4GalT-I and ST6Gal-I, heteromer formation was not affected by an increase in Golgi luminal pH (Hassinen et al, 2011), but rather by an altered Golgi redox state (Hassinen et al, 2019; see also Section “Golgi Redox Homeostasis and Altered Glycosylation” last paragraph). These observations point to fundamental differences in the way enzyme heteromers form in the Golgi lumen, and perhaps reflecting the high specificity of the interactions needed to prevent irrational interactions that could otherwise lead to the synthesis of mixed or irrelevant glycan structures (Kellokumpu et al, 2016).…”

“…We have recently shown that hypoxia also modulates glycosylation in a HIF-independent manner by reducing Golgi redox state (Hassinen et al, 2019). Specifically, we demonstrated that even moderate hypoxia (5% O 2 ) lowers the Golgi oxidizing potential to the level found in the ER of normoxic cells, consistent with possible problems in disulfide bond formation.…”

Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains

“…The ER and Golgi microenvironments have primary roles in governing the type of assembly made, in addition to contributing to the preservation of the Golgi structure and ER-Golgi trafficking (10,11). Accordingly, the redox state difference between these two compartments regulates the interaction between B4GALT1 and ST6GAL1, whereas the pH gradient (pH 7 versus pH 6.3) affects B4GALT1 and ST3GAL3 interactions as well as interactions between glycosyltransferases that act on O-glycans (6,12). Changes in Golgi redox potential in hypoxic cells correlates with loss of ␣-2,6 sialylation and inactivation of ST6GAL1 and its inability to bind B4GALT1.…”