title: N-glycosylation is essential for ileal ASBT function and protection against proteases.
abstract: The bile acid transporter ASBT is a glycoprotein responsible for active absorption of bile acids. Inhibiting ASBT function and bile acid absorption is an attractive approach to lower plasma cholesterol and improve glucose imbalance in diabetic patients. Deglycosylation of ASBT was shown to decrease its function. However, the exact roles of N-glycosylation of ASBT, and how it affects its function, is not known. Current studies investigated the roles of N-glycosylation in ASBT protein stability and protection against proteases utilizing HEK-293 cells stably transfected with ASBT-V5 fusion protein. ASBT-V5 protein was detected as two bands with molecular mass of ~41 and ~35 kDa. Inhibition of glycosylation by tunicamycin significantly decreased ASBT activity and shifted ASBT bands to ~30 kDa, representing a deglycosylated protein. Treatment of total cellular lysates with PNGase F or Endo H glycosidases showed that the upper 41-kDa band represents a fully mature N-acetylglucosamine-rich glycoprotein and the lower 35-kDa band represents a mannose-rich core glycoprotein. Studies with the glycosylation deficient ASBT mutant (N10Q) showed that the N-glycosylation is not essential for ASBT targeting to plasma membrane. However, mature glycosylation significantly increased the half-life and protected ASBT protein from digestion with trypsin. Incubating the cells with high glucose (25 mM) for 48 h increased mature glycosylated ASBT along with an increase in its function. These results unravel novel roles for N-glycosylation of ASBT and suggest that high levels of glucose alter the composition of the glycan and may contribute to the increase in ASBT function in diabetes mellitus.
authors: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors: Saminathan
authors: S
authors: Muthusamy
authors: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors: Pooja
authors: P
authors: Malhotra
authors: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors: Mobashir
authors: M
authors: Hosameddin
authors: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors: Amish K
authors: AK
authors: Dudeja
authors: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors: Sujata
authors: S
authors: Borthakur
authors: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors: Seema
authors: S
authors: Saksena
authors: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors: Ravinder K
authors: RK
authors: Gill
authors: Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; and Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors: Pradeep K
authors: PK
authors: Dudeja
authors: Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; and Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois walrefai@uic.edu.
authors: Waddah A
authors: WA
authors: Alrefai
mesh_terms: D001647:Bile Acids and Salts / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006207:Half-Life; D006801:Humans; D007082:Ileum / Q000201:enzymology*; D008970:Molecular Weight; D009154:Mutation; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D021381:Protein Transport; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D001647:Bile Acids and Salts; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter; D010447:Peptide Hydrolases; D005947:Glucose
keywords: N-glycans; bile acid absorption; bile acids; intestinal transport proteins