Purification and characterization of sialyl-Le(a)-carrying mucins of human bile; evidence for the presence of MUC1 and MUC3 apoproteins.
Purification and characterization of sialyl-Le(a)-carrying mucins of human bile; evidence for the presence of MUC1 and MUC3 apoproteins.
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人胆汁中携带唾液酸化 Le(a) 的粘蛋白的纯化和表征；
DOI：
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发表时间：
1994
影响因子：
4.8
通讯作者：
G. Hansson
中科院分区：
文献类型：
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作者：
D. Baeckström;N. Karlsson;G. Hansson
通过三氯乙酸沉淀、脱脂以及在氯化胍中进行凝胶过滤，从人原发性胆汁中纯化携带唾液酸化Le(a)的黏蛋白，得到三个可分离的组分，其中一个组分通过亲和色谱进一步纯化。这些组分分别命名为SBG1（可溶性胆汁糖蛋白）、SBG2和SBG3，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计，它们的分子量分别大于1100、800 - 950和100 - 250 kDa。它们的黏蛋白特性表现为碳水化合物含量高，范围从74%到95%。碳水化合物组成表明存在非常长的岩藻糖基化多聚乳糖胺链。氨基酸分析显示所有三个组分中丝氨酸和苏氨酸含量丰富（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黏蛋白脱辅基蛋白，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。这种黏蛋白在胰蛋白酶处理或二硫键还原和烷基化后大小没有显著减小。对三份继发性胆汁样本进行凝胶过滤表明，携带唾液酸化Le(a)的糖蛋白的大小分布与原发性胆汁中发现的相似，并且免疫化学分析显示MUC1蛋白在所有三个样本中都存在。在一个样本中分离出一个额外的组分，它不溶于6 M氯化胍，但在还原和烷基化后可溶解。通过Northern杂交分析胆囊上皮的mRNA，结果显示MUC1和MUC3黏蛋白脱辅基蛋白基因有表达，但MUC2黏蛋白脱辅基蛋白基因没有表达。
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.