O-glycosylated MUC2 monomer and dimer from LS 174T cells are water-soluble, whereas larger MUC2 species formed early during biosynthesis are insoluble and contain nonreducible intermolecular bonds
O-glycosylated MUC2 monomer and dimer from LS 174T cells are water-soluble, whereas larger MUC2 species formed early during biosynthesis are insoluble and contain nonreducible intermolecular bonds
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DOI：
10.1074/jbc.273.30.18864
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发表时间：
1998-07-24
影响因子：
4.8
通讯作者：
Hansson, GC
中科院分区：
文献类型：
--
作者：
Axelsson, MAB;Asker, N;Hansson, GC
MUC 2粘蛋白是小肠和大肠中主要的凝胶形成粘蛋白。由于其与血管性血友病因子的序列相似性，已建议在内质网中二聚化并在高尔基体网络中二聚化。使用O-糖基化敏感性MUGS抗血清，已经显示在LS 174 T细胞的内质网中发生二聚化（Asker，N.，Axelsson，M. A. B.，Olofsson，S.- O.，和Hansson，G. C.（1998）J.Biol.Chem.278，18857-18863）。使用抗血清免疫沉淀O-糖基化MUGS粘蛋白，单体和二聚体仅显示以可溶形式存在于LS 174 T细胞的裂解物中。O-糖基化二聚体的量很小，即使经过长时间的追踪，也没有发现更大的物质。然而，大多数标记的MUGS粘蛋白在细胞裂解物的沉淀碎片中发现。这种不溶性MUC 2粘蛋白在二硫键还原后通过免疫沉淀回收。琼脂糖凝胶电泳分析显示两条带，其中较小的作为O-糖基化单体迁移，较大的作为细胞裂解上清液的O-糖基化二聚体迁移。不溶于6 M氯化胍的粘蛋白也可以从LS 174 T细胞中获得。这样的粘蛋白早先已在小肠中发现（Carlstedt，I.，Herrmann，A.，Karlsson，H.，希恩，J.，弗兰森湖，一、和Hansson，G. C.（1993）J.Biol.Chem.268，18771-18781）。还原粘蛋白，然后通过等密度密度梯度超离心纯化，并通过琼脂糖凝胶电泳分析，发现两条带与去糖基化后的抗MUC 2串联重复抗体反应。这些条带的迁移与代谢标记所显示的条带相同，并且它们也可以通过速率带超离心分离。这些结果表明，MUC 2粘蛋白在生物合成早期形成不可还原的分子间键，但在初始O-糖基化之后。
The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUGS antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 278, 18857-18863). Using an antiserum immunoprecipitating O-glycosylated MUGS mucin, monomers and dimers mere shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUGS mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L.-A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.