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1 Department of Gastroenterology, Tokai University School of Medicine, Isehara, Kanagawa, Japan
2 Physiology, Tufts Universtiy School of Medicine, Boston, Massachusetts, United States
3 Laboratories for Structure and Function Research, Tokai University School of Medicine, Isehara, Kanagawa, Japan
4 NICHD and Unit for Cellular Polarity, National Institutes of Health, Bethesda, Maryland, United States
* To whom correspondence should be addressed. E-mail: kagawa{at}is.icc.u-tokai.ac.jp.
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 bile salt export pump (Bsep, ABCB11). Rat Bsep was fused with a 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 determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76 h, 3.65 h, 3.24 h, 1.35 h and 0.52 h in wild-type, single, double, triple and quadruple mutant, respectively. Wild type, single and double mutants were distributed exclusively along the apical membranes, whereas triple and quadruple mutants remained intracellular. MG132 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.
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