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Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylation.

ABSTRACT: Glycosylation, the most common posttranslational modification of proteins, is a stepwise process that relies on tight regulation of subcellular glycosyltransferase location to control the addition of each monosaccharide. Glycosyltransferases primarily reside and function in the endoplasmic reticulum (ER) and the Golgi apparatus; whether and how they traffic beyond the Golgi, how this trafficking is controlled, and how it impacts glycosylation remain unclear. Our previous work identified a connection between N-glycosylation and Rab11, a key player in the post-Golgi transport that connects recycling endosomes and other compartments. To learn more about the specific role of Rab11, we knocked down Rab11 in HeLa cells. Our findings indicate that Rab11 knockdown results in a dramatic enhancement in the sialylation of N-glycans. Structural analyses of glycans using lectins and LC-MS revealed that α2,3-sialylation is selectively enhanced, suggesting that an α2,3-sialyltransferase that catalyzes the sialyation of glycoproteins is activated or upregulated as the result of Rab11 knockdown. ST3GAL4 is the major α2,3-sialyltransferase that acts on N-glycans; we demonstrated that the localization of ST3GAL4, but not the levels of its mRNA, protein, or donor substrate, was altered by Rab11 depletion. In knockdown cells, ST3GAL4 is densely distributed in the trans-Golgi network, compared with the wider distribution in the Golgi and in other peripheral puncta in control cells, whereas the α2,6-sialyltransferase ST6GAL1 is predominantly localized to the Golgi regardless of Rab11 knockdown. This indicates that Rab11 may negatively regulate α2,3-sialylation by transporting ST3GAL4 to post-Golgi compartments (PGCs), which is a novel mechanism of glycosyltransferase regulation.

SUBMITTER: Kitano M

PROVIDER: S-EPMC7949161 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIt is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation.MethodsHepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM EtOH for 72 hours. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I), and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by three-dimensional structured illumination microscopy (3D SIM).ResultsFirst, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor; however, the high-mannose-type N-glycans are increased. Further analysis by 3D SIM revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of ADP-ribosylation factor 1 (Arf1) GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (i) EtOH specifically blocks activation of Arf1, and (ii) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin sensitive.ConclusionsThus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans.

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Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylation.

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