Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells.
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.
Mochizuki
Kaori
K
Department of Internal Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
Kagawa
Tatehiro
T
Numari
Asano
A
Harris
Matthew J
MJ
Itoh
Johbu
J
Watanabe
Norihito
N
Mine
Tetsuya
T
Arias
Irwin M
IM
D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000502:physiology*; D019943:Amino Acid Substitution; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000502:physiology*; D061988:Proteasome Inhibitors; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D013656:Taurocholic Acid / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490548:Abcb11 protein, rat; D001647:Bile Acids and Salts; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008164:Luminescent Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D011134:Polysaccharides; D061988:Proteasome Inhibitors; D011993:Recombinant Fusion Proteins; D027981:Symporters; D014415:Tunicamycin; C071908:sodium-bile acid cotransporter; D013656:Taurocholic Acid; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde