Study of Ethanol‐Induced Golgi Disorganization Reveals the Potential Mechanism of Alcohol‐Impaired N‐Glycosylation

Abstract

Background It 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. Methods HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM ethanol for 72 h. 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 3D Structured Illumination Microscopy (SIM). Results First, 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 microscopy 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 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 (a) EtOH specifically blocks activation of Arf1, and (b) 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. Conclusions Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans.

The Role of Golgi Morphology in Post-Alcohol Recovery of Hepatocytes: Observations in Cellular and Animal Models

“…To examine the precise role of giantin in post-alcohol recovery, we monitored the Golgi morphology in VA-13 cells after EtOH withdrawal in presence of giantin siRNAs. As shown in Figure 3A and in agreement with our previous observation of these cells [19], Golgi morphology (stained by GM130) in EtOH-treated cells (35 mM EtOH for 72 h) looks predominantly disorganized, which returns to the classical perinuclear position when cells recovered after EtOH under normal growing conditions for another 48 h. As we showed previously, giantin KD has no significant impact on Golgi morphology [25]. However, in cells lacking giantin, post-alcohol Golgi failed to recollect membranes into the organized structure (Figure 3A and B).…”

“…Some of these Golgi resident enzymes exhibit altered re-localization due to EtOH-induced impairment of COPI vesicles, which normally deliver these enzymes to appropriate sites within the Golgi [25,26]. Also, recently we and others found that giantin represents a Golgi docking site for different Golgi resident proteins [18,[27][28][29][30], and EtOH-induced alteration of giantin dimerization results in impaired Golgi targeting of mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), the key enzyme of N-glycosylation [25]. This, in turn, causes abnormal glycosylation of ASGP-R and could be a potential reason for the release of carbohydrate-deficient transferrin, the widely available test for determining recent alcohol consumption [31].…”

Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

“…Our model suggests that giantin dimerization is a necessity for successful Golgi biogenesis. Since the formation of giantin-dimer requires functional COPII [51] and, broadly speaking, the dynamic of Golgi membranes is linked to the integrity of ER exit site [36,135], we believe that any substantial disturbance of ER function would inevitably result in Golgi dysfunction and its subsequent disorganization.…”

“…Accumulation of the Golgi resident proteins in the nascent Golgi stacks was suggested to be sequential, starting from the trans-proteins and followed by their cis-medial counterparts [80,81]. We recently found that the cis-Golgi protein, α-1,2-mannosidase (Man-I) may use GM130-GRASP65 as the Golgi docking site, whereas the next to Man-I enzyme in the N-glycosylation pathway, medial-Golgi glycosyltransferase MGAT1, appeared giantin-responsive [51]. Given that GM130 and giantin reside predominantly in the cisand medial-Golgi, respectively [43,82,83], we analyzed in BFA-treated HeLa cells the dynamics of colocalization of Man-I and MGAT1 with GM130 and giantin, accordingly.…”

Golgi Renaissance: the pivotal role of the largest Golgi protein giantin

“…Recently, we have reveled that ethanol (EtOH) treatment blocks activation of SAR1A GTPase, thus preventing COPII vesicle-mediated Golgi targeting of protein disulfide isomerase A3 (PDIA3), the chaperone that catalyzes dimerization of giantin (Petrosyan et al, 2015b, Petrosyan, Holzapfel et al, 2014. In EtOH-treated cells, Golgi is consequently disorganized and Golgi targeting of mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), the key enzyme of N-glycosylation, is altered (Casey, Bhat et al, 2016). In addition, we previously showed that giantin determines Golgi localization for core 2 O-glycosylation enzymes (both Nacetylglucosaminyltransferase 2/M and 1/L, C2GnT-M and C2GnT-L) (Petrosyan et al, 2012a.…”