TwoN-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 (Asn109, Asn116, Asn122, and Asn125) 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 A1 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.

No Contribution of the ABCB11 p.444A Polymorphism in Japanese Patients with Drug-Induced Cholestasis

“…TC transport activity was evaluated, as described previously (Mochizuki et al, 2007). In brief, MDCK II cells were replated on Transwell membrane inserts (no.…”

The Role of the Sodium-Taurocholate Cotransporting Polypeptide (NTCP) and of the Bile Salt Export Pump (BSEP) in Physiology and Pathophysiology of Bile Formation

“…BSEP is a glycoprotein with four putative N-linked glycosylation sites and with a molecular weight of 140 to 170 kDa [34, 94,100,217,240]. Rat Bsep is glycosylated at all four putative glycosylation sites [225]. In addition to glycosylation, phosphorylation of Bsep [114,238] and ubiquitination occur as additional posttranslational modifications.…”

Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

“…Mutagenic removal of one or two of the four N-glycosylation sites from the apical bile salt export pump (ABCB11) did not significantly affect its stability, trafficking, or function (56). However, removal of three or all four N-glycosylation sites dramatically increased the rate of proteasome-dependent degradation and resulted in significant intracellular retention of the protein.…”

“…However, removal of three or all four N-glycosylation sites dramatically increased the rate of proteasome-dependent degradation and resulted in significant intracellular retention of the protein. Comparison of mutants of ABCB11 with various combinations of mutated glycosylation sites showed that presence of at least two N-glycans is required for normal folding and trafficking of the protein (56).…”

“…Removal of N-glycans from many apical proteins has been shown to decrease their abundance in the apical membrane. For example, mutagenic removal of N-glycosylation sites in the gastric H-K-ATPase ␤-subunit (90), bile salt export pump (56), and glycine transporter 2 (51, 94) significantly decreased their apical content and increased their intracellular accumulation (Table 3). Similarly, prevention of N-glycosylation by tunicamycin resulted in complete intracellular retention of the normally apically located membrane-bound enteropeptidase (95) ( Table 3).…”