Baeckström D, Karlsson N, Hansson G C
Department of Medical Biochemistry, University of Göteborg, Gothenburg, Sweden.
J Biol Chem. 1994 May 20;269(20):14430-7.
Purification of sialyl-Le(a)-carrying mucins from primary human bile by trichloroacetic acid precipitation, delipidation, and gel filtration in guanidinium chloride gave three separable fractions, one of which was further purified by affinity chromatography. These fractions, named SBG1 (for soluble bile glycoprotein), SBG2, and SBG3 had molecular masses of > 1100, 800-950, and 100-250 kDa, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their mucin characteristics were indicated by a high carbohydrate content, ranging from 74 to 95%. The carbohydrate compositions indicated the presence of very long fucosylated polylactosamine chains. Amino acid analyses showed high abundance of serine and threonine in all three fractions (19-36%), confirming their mucin-like nature. Immunochemical analyses of deglycosylated samples detected the MUC1 mucin apoprotein in SBG2 and the MUC3 protein in SBG1. To our knowledge, this is the first report of a MUC3 mucin being purified. This mucin showed no significant reduction in size upon trypsin treatment or disulfide bond reduction and alkylation. Gel filtration of three samples of secondary bile showed that the size distribution of sialyl-Le(a)-carrying glycoproteins was similar to that found in primary bile, and immunochemical analysis showed that the MUC1 protein was present in all three samples. In one sample an additional fraction was isolated, which was insoluble in 6 M guanidinium chloride, but was solubilized upon reduction and alkylation. mRNAs from gallbladder epithelia were analyzed in Northern blot hybridizations showing that the MUC1 and MUC3 but not the MUC2 mucin apoprotein genes were expressed.
通过三氯乙酸沉淀、脱脂以及在氯化胍中进行凝胶过滤，从原发性人胆汁中纯化携带唾液酸化-Le(a)的黏蛋白，得到了三个可分离的组分，其中一个组分通过亲和层析进一步纯化。这些组分分别命名为SBG1（可溶性胆汁糖蛋白）、SBG2和SBG3，根据十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，它们的分子量分别大于1100 kDa、800 - 950 kDa和100 - 250 kDa。它们的黏蛋白特性表现为碳水化合物含量高，范围在74%至95%之间。碳水化合物组成表明存在非常长的岩藻糖基化多乳糖胺链。氨基酸分析显示，所有三个组分中丝氨酸和苏氨酸含量都很高（19% - 36%），证实了它们类似黏蛋白的性质。对去糖基化样品的免疫化学分析在SBG2中检测到MUC1黏蛋白载脂蛋白，在SBG1中检测到MUC3蛋白。据我们所知，这是关于MUC3黏蛋白被纯化的首次报道。该黏蛋白经胰蛋白酶处理或二硫键还原及烷基化后，大小没有明显减小。对三个继发性胆汁样品进行凝胶过滤显示，携带唾液酸化-Le(a)的糖蛋白的大小分布与原发性胆汁中的相似，免疫化学分析表明所有三个样品中都存在MUC1蛋白。在一个样品中分离出了一个额外的组分，它不溶于6 M氯化胍，但在还原和烷基化后可溶解。在Northern印迹杂交中分析了胆囊上皮细胞的mRNA，结果显示MUC1和MUC3黏蛋白载脂蛋白基因表达，但MUC2基因不表达。
