Title : Two N-linked glycans are required to maintain the transport activity of the
bile salt export pump (
ABCB11 ) in MDCK II cells
Abstract :
- The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [ Bsep ( ATP-binding cassette B11)]
- Rat Bsep was fused with yellow fluorescent protein , and the following mutants, in which Asn residues of putative glycosylation sites ( Asn(109), Asn(116), Asn(122), and Asn(125) ) were sequentially replaced with Gln , were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q
- Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated
- Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells
- This decrease resulted from rapid decay of the mutant Bsep protein ; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively
- Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular
- MG-132 but not bafilomycin A(1) extended the half-life, suggesting a role for the proteasome in Bsep degradation
- To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans
- In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane
Output (sent_index, trigger,
protein,
sugar,
site):
- 1. glycosylation, , bile salt export pump, -, -
- 1. glycosylation, , protein, -, -
- 2. glycosylation, , -, -, Asn(109), Asn(116), Asn(122), and Asn(125)
- 2. glycosylation, , -, -, sites
- 2. sites, , -, -, Asn(109), Asn(116), Asn(122), and Asn(125)
- 2. sites, , -, -, sites
- 3. glycosylated, , -, -, site
- 8. Bsep, , Bsep, one glycan, -
- 8. glycosylation, , -, glycans, site
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 2. ABCB11, -, Asn(109), Asn(116), Asn(122), and Asn(125)