N-glycosylation is essential for ileal ASBT function and protection against proteases.

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.

Common food additive carrageenan stimulates Wnt/ ß-catenin signaling in colonic epithelium by inhibition of nucleoredoxin reduction.

Exposure to the common food additive carrageenan was previously associated with increased Wnt9A expression and increased cytoplasmic ß-catenin in human colonic epithelial cells. In this report, exposure of human colonic epithelial cells in culture and of mouse colonic epithelium in vivo to low concentrations of carrageenan is shown to activate the Wnt/ß-catenin signaling pathway, leading to increases in nuclear ß-catenin, T-cell factor/lymphoid enhancer factor activation, and cyclin D1 expression and decline in bone morphogenetic protein-4. These effects are mediated through carrageenan-induced reactive oxygen species (ROS), and inhibited by the ROS scavenger Tempol. Carrageenan exposure and ROS production inhibited thioredoxin reductase activity and increased oxidation of nucleoredoxin, a member of the thioredoxin family of redox proteins. When oxidized, nucleoredoxin co-immunoprecipitation with dishevelled (DVL) declined, enabling DVL to interact with and inhibit the cytoplasmic ß-catenin destruction complex, and facilitating nuclear translocation of ß-catenin. Both nucleoredoxin silencing and carrageenan exposure produced similar declines in thioredoxin reductase activity. In addition to activation of Wnt signaling, carrageenan exposure also increased Wnt9A mRNA expression in the mouse colonic epithelium and the human colonic epithelial cells, thereby potentially permitting ongoing stimulation of the Wnt/ß-catenin pathway. These findings suggest how a common dietary ingredient can contribute to colon carcinogenesis by effects on Wnt signaling and Wnt expression.