Profiling impaired hepatic endoplasmic reticulum glycosylation as a consequence of ethanol ingestion

Abstract

Alcoholic liver disease (ALD) is a prominent cause of morbidity and mortality in the United States. Alterations in protein folding occur in numerous disease states, including ALD. The endoplasmic reticulum (ER) is the primary site of post-translational modifications (PTM) within the cell. Glycosylation, the most abundant PTM, affects protein stability, structure, localization, and activity. Decreases in hepatic glycosylation machinery have been observed in rodent models of ALD, but specific protein targets have not been identified. Utilizing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, glycoproteins were identified in hepatic microsomal fractions from control and ethanol-fed mice. This study reports for the first time a global decrease in ER glycosylation. Additionally, the identification of 30 glycoproteins within this fraction elucidates pathway-specific alterations in ALD impaired glycosylation. Among the identified proteins, triacylglycerol hydrolase (TGH) is positively affected by glycosylation, showing increased activity following the addition of sugar moieties. Impaired TGH activity is associated with increased cellular storage of lipids and provides a potential mechanism for the observed pathologies associated with ALD.

Figure 1

Chronic ethanol ingestion results in hepatic lipid accumulation and marked oxidative stress. (A.) H&E stained liver (400x) displays significant lipid accumulation following ethanol ingestion. Areas of significant lipid accumulation are designated by arrows; (B.) Immunohistochemical staining for 4-HNE shows increased staining in the ethanol-fed mice. (C.) Quantification of 4-HNE immunopositive staining (n = 6 pairs; p < 0.05). PT, portal triad; CV, central vein.

Figure 2

Chronic ethanol ingestion results in an apparent progressive decrease in glycosylation of microsomal proteins. 2-dimensional gel electrophoresis using a pI 3-10 non-linear IPG strip and 10% acrylamide gel was performed using microsomal fractions isolated from livers of both control and ethanol-fed mice. Gels were then stained with the Pro-Q Emerald 300 Glycoprotein Gel Stain Kit and visualized on an Ettan DIGE Imager.

Figure 3

2-dimensional gel electrophoresis was performed using a pI 3-10 non-linear IPG strip and a 17cm, 8% polyacrylamide gel. (A.) Microsomal fractions isolated from livers of control and ethanol-fed mice were stained for glycosylation. (B.) Paired gels were Silver Stained and spots were picked from the control gel, as staining was more intense. Gel spots (indicated) were then subjected to tryptic digest and analyzed using LC-MS/MS for protein identification.

Figure 4

Immunoblotting reveals no change in protein expression of identified targets of glycosylation. (A.) Glycoproteins analyzed via western blotting in (B.) are indicated for reference on 2-dimensional gels. (B.) Western blotting was performed using microsomal fractions from control and ethanol-fed mice for protein expression of Glucose Regulated Protein 94 (Grp94), Grp78, Protein Disulfide Isomerase (PDI), Calreticulin (CRT) and Carboxylesterase 3 (TGH). No change in expression was observed in these proteins.

Figure 5

Depiction of identified glycoproteins in murine microsomal fractions from liver extracts. “Reported Identification” designates all proteins identified that have been reported to be glycosylated in the UniProt database. “Novel Identification” designates all proteins identified that are predicted to be glycosylated that have not been previously reported in the literature. “Unreported” designates all proteins that are unreported and not predicted to be glycosylated.

Figure 6

Functional annotation clustering analysis using DAVID. The identified proteins were clustered based on molecular functions. Enrichment scores ≥ 2.0 were reported and are depicted by the open bars on the left y-axis. Total percentage of proteins identified in each category is represented by the closed bars in the right y-axis.