The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment
The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment
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DOI：
10.1074/jbc.m208483200
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发表时间：
2002-12-06
影响因子：
4.8
通讯作者：
Hansson, GC
中科院分区：
文献类型：
--
作者：
Godl, K;Johansson, MEV;Hansson, GC
人MUC2粘蛋白的N末端(氨基酸1-1397)已在中国仓鼠卵巢细胞中以重组标记蛋白的形式表达。胞内形式是内切糖苷酶H敏感的单体，而分泌形式是低聚物，在二硫键还原时产生单体。分泌的MUC2 N端含有一个抗胰酶的核心片段。Edman测序和质谱分析将该核心片段定位于重组蛋白的C末端。这个核心保持了它的低聚性质，其表观质量类似于240 kDa。还原后，发现了与85 kDa相似的多肽，表明N末端形成三聚体。这一解释也得到了完整MUC2 N末端的凝胶电泳和凝胶过滤的支持。电子显微镜显示了三个球状结构域，每个结构域都通过一个延伸而灵活的区域以三叶草的方式连接到中心部分。金标抗体免疫染色定位于三个球状结构的N末端，针对Myc和附着在C末端的绿色荧光蛋白标记的抗体定位于中央三叶的茎侧。因此，MUC2粘蛋白的N端被组装成含有蛋白水解性稳定部分的三聚体，这表明MUC2只能被肠道蛋白酶部分降解，从而能够维持保护肠道的粘蛋白网络。
The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of similar to240 kDa. Upon reduction, peptides of similar to85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.