Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells
Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells
复制标题
DOI：
10.1152/ajpgi.00415.2006
复制
发表时间：
2007-03-01
影响因子：
4.5
通讯作者：
Arias, Irwin M.
中科院分区：
文献类型：
--
作者：
Mochizuki, Kaori;Kagawa, Tatehiro;Arias, Irwin M.
本研究的目的是确定 N 连接糖基化在蛋白质稳定性、细胞内运输和胆汁盐输出泵 [Bsep（ATP 结合盒 B11）] 的胆汁酸转运活性中的作用。将大鼠Bsep与黄色荧光蛋白融合，通过定点诱变构建了假定糖基化位点（Asn（109）、Asn（116）、Asn（122）和Asn（125））的Asn残基依次被Gln取代的突变体：单N109Q、双N109Q+N116Q、三重N109Q+ N116Q + N122Q，以及四联 N109Q + N116Q + N122Q + N125Q。免疫印迹和糖苷酶裂解分析表明每个位点都被糖基化。根据极化 MDCK II 细胞的测定，去除聚糖会降低牛磺胆酸盐转运活性。这种下降是由于突变 Bsep 蛋白的快速衰减所致；野生型、单突变体、双突变体、三突变体和四突变体细胞中的生化半衰期分别为3.76、3.65、3.24、1.35和0.52 h。野生型以及单突变和双突变蛋白仅沿着顶膜分布，而三突变和四突变蛋白则保留在细胞内。 MG-132（而非巴弗洛霉素 A(1)）延长了半衰期，表明蛋白酶体在 Bsep 降解中发挥作用。为了确定特定糖基化位点或聚糖数量是否对蛋白质稳定性至关重要，我们研究了 N-聚糖缺陷突变体组合的蛋白质表达，并观察到具有一个聚糖的 Bsep 与含有两个或多个聚糖的 Bsep 相比相当不稳定。总之，Bsep 蛋白稳定性、细胞内运输和顶膜功能需要至少两个 N 连接聚糖。
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.