Mechanisms of intrahepatic cholestasis and biochemical analysis of bile salt export pump(ABCB 11)

(1)在MDCK II细胞中，维持胆盐输出泵(ABCB11)的转运活性需要两个N-连接的糖链。本研究的目的是阐明N-连接的糖基化在胆盐输出泵(BSEP，ABCB-11)的蛋白质稳定性、细胞内转运和胆汁酸转运活性中的作用。将大鼠BSEP与一个黄色荧光蛋白在C端进行融合，并通过定点突变构建了以GIN取代ASN的糖基化位点(Asn^&lt；109&gt；109&gt；，Asn^&lt；16&gt；，Asn1^&lt；22&gt；和Asn^&lt；125&gt；)的突变体：单突变体N109Q；双突变体N109Q N116Q；三突变体N109Q N116Q N122Q和四突变体N109Q N116Q N125Q。免疫印迹和糖苷酶裂解分析表明，MDCK II细胞裂解产物的每个位点都携带一个碳水化合物，BSEP上不存在其他位点。极化的MDCK II细胞的牛磺胆酸盐转运活性为…更多的是由于多糖的去除而减少的。野生型、单突变体、双突变体、三重突变体和四重突变体的生化半衰期分别为3.76h、3.65h、3.24h、1.35h和0.52h。野生型、单突变体和双突变体仅分布在顶膜上，而三重和四重突变体则保留在细胞内。MG132延长了半衰期，但不能延长半衰期，提示蛋白酶体在降解中起作用。单突变体与双突变体、三突变体与四突变体之间的降解速率有显著差异。为了阐明特定的糖基化位点或糖链数目是否对蛋白质的稳定性起关键作用，我们研究了N-糖链缺失的Bsep突变体在不同组合下的蛋白质表达，发现与含有两个或更多糖链的Bsep相比，含有一个糖链的Bsep蛋白非常不稳定。总而言之，BSEP顶膜的正确折叠、蛋白质稳定性以及随后的细胞内转运和功能至少需要四个N-连接多糖中的两个。(2)PFIC2和BRIC2的表型差异与突变BSEP的蛋白质稳定性和MDCK II细胞牛磺胆酸分泌受损有关。进行性家族性胆汁淤积症(PFIC)2和良性复发性肝内胆汁淤积症(ERIC)2是由胆盐输出泵(BSEP，ABCB11)基因突变引起的，然而，它们的预后不同。PFIC2进展为肝硬变，需要肝移植，而BRIC2在临床上是良性的。为了确定导致表型差异的分子机制(S)，将8个PFIC2和2个BRIC2突变引入大鼠BSEP，并将其导入MDCK II细胞。在极化的MDCK II单层膜上研究了这些突变蛋白的牛磺胆酸转运活性、蛋白表达和亚细胞分布。牛磺胆酸转运活性在BRIC2突变体(A570T和R1050C)中约为野生型(WT)的一半，在两个PFIC2突变体(D482G和E297G)中显著低于野生型(WT)，在其他六个突变体(K461Em G982R、R1153C、R1268Q、3767-3768insC和E1057X)中几乎被取消。除R1057X外，BSEP蛋白表达水平与转运活性密切相关。D482G突变体的半衰期比WT突变体短(1.35h比3.49h)。BRIC2突变体和3个pFIC突变体(D482G、E297G、R1057X)主要分布在顶膜。其他的pFIC2突变体保持在细胞内。R1057X突变体蛋白稳定表达并运输到顶膜，表明C末端的尾巴是运输活动所必需的，但不是正确靶向所必需的。综上所述，牛磺胆酸转运功能受阻于BSEP蛋白的快速聚集，其排列顺序依次为：A570T和R1050C&GT；D482G&GT；E297G&GT；K461E；G982R；R1153C；R1268Q；3767-3768insC和R1057X。这些结果可能解释了BRIC2和PFIC2的表型差异。较少

(1) TWO N-LINKED GLYCANS ARE REQUIRED TO MAINTAIN TRANSPORT ACTIVITY OF THE BILE SALT EXPORT PUMP (ABCB11) IN MDCK II CELLSThe aim of this study was to elucidate the role of N-linked glycosylation in protein stability, intracellular trafficking and bile acid transport activity of the bile salt export pump (Bsep, ABCB 11). Rat Bsep was fused with a yellow fluorescent protein at the C-terminus and mutants in which Asn residues of putative glycosylation sites (Asn^<109>, Asn^<16>, Asn1^<22> and Asn^<125>) were sequentially replaced with GIn were constructed by site-directed mutagenesis: single mutant N109Q; double mutant N109Q + N116Q; triple mutant N109Q + N116Q + N122Q, and quadruple mutant N109Q + N116Q + N122Q + N125Q. Analysis by immunoblot and cleavage with glycosidases of cell lysates from transfected MDCK II cells demonstrated that each site carried a carbohydrate and no other sites were present on Bsep. The taurocholate transport activity determined in polarized MDCK II cells was … More diminished by the removal of glycans. This decrease was caused by the rapid decay of the mutant Bsep protein; the biochemical half-lives were 3.76 h, 3.65 h, 3.24 h, 1.35 h and 0.52 h in the wild-type, single, double, triple and quadruple mutant, respectively. The wild type, single and double mutants were distributed exclusively along the apical membranes, whereas triple and quadruple mutants remained intracellular. MG132 but not bafilomycin Al extended the half-life, suggesting the role of the proteasome in the degradation. There was a significant difference in the rate of degradation between single and double, and triple and quadruple mutants. To elucidate whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of N-glycan deficient Bsep mutants with various combinations and found that Bsep with one glycan was extremely unstable compared with Bsep harboring two and more glycans. In conclusion, at least two of four N-linked glycans were required for proper folding, protein stability, and subsequent intracellular trafficking and function in the apical membrane of Bsep.(2) Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cellsProgressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis(ERIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene, however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in, rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461Em G982R, R1153C, R1268Q, 3767-3768insC, and E1057X). Bsep protein expression levels correlated closely with transport activty, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35h vs. 3.49h in the form). BRIC2 mutants and three PFIC mutants (D482G, E297G, R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the C-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in propotion to rapid dagradation of Bsep protein in the mutants which ewre aligned in the following order, A570T and R1050C>D482G>E297G>K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2. Less