An autocatalytic cleavage in the c terminus of the human MUC2 mucin occurs at the low pH of the late secretory pathway
An autocatalytic cleavage in the c terminus of the human MUC2 mucin occurs at the low pH of the late secretory pathway
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DOI：
10.1074/jbc.m210069200
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发表时间：
2003-04-18
影响因子：
4.8
通讯作者：
Hansson, GC
中科院分区：
文献类型：
--
作者：
Lidell, ME;Johansson, MEV;Hansson, GC
在CHO-K1细胞中表达的重组MUC2 C末端的纯化过程中，用pH为6.0的缓冲液可部分切割该蛋白。当使用pH值较高的缓冲液时，裂解较少。二硫键结合在一起生成的两个片段，因为这些片段只有在还原后才能观察到。对C-末端110-kDa片段的Edman测序显示，裂解发生在Asp-Pro键上，该位置在前面描述的位置，在二硫键还原后产生所谓的“连接肽”。体外对卵裂条件的研究表明，在pH低于6.0时，卵裂发生的方式与时间有关。此外，该反应不是由酶介导的，因为它发生在MUC2 C末端的纯制剂中，并且不被蛋白酶抑制剂抑制。在粘蛋白产生细胞系LS 174T中表达时，C末端的切割程度高于CHO-K1细胞。用N-H4Cl或巴菲罗星A1中和分泌途径可抑制这种切割。综上所述，我们的结果表明，切割是一个自动催化反应，发生在晚期分泌途径的酸性环境中。此外，裂解产生了一个新的活性C末端，它有可能将粘蛋白附着到自己或其他分子上。由于在晚期分泌途径和粘膜表面可以达到pH低于6的水平，因此这种切割和可能的交联会具有生物学意义。
During purification of a recombinant MUC2 C terminus expressed in CHO-K1 cells, the protein was partly cleaved when buffers with a pH of 6.0 were used. When buffers with higher pH values were used, less cleavage was found. Disulfide bonds held the two fragments generated together as these were only observed after reduction. Edman sequencing of the C-terminal 110-kDa fragment revealed that the cleavage had occurred at an Asp-Pro bond, a site described previously to generate the so-called "link peptide" after disulfide bond reduction. In vitro studies on the conditions for cleavage showed that it occurred in a time-dependent manner at a pH below 6.0. Furthermore, the reaction was not enzyme-mediated as it occurred in pure preparations of the MUC2 C terminus and was not inhibited by protease inhibitors. When expressed in the mucin producing cell line LS 174T, the C terminus was cleaved to a higher extent compared with the CHO-K1 cells. Neutralizing the secretory pathway with either N-H4Cl or bafilomycin A1 inhibited this cleavage. Altogether, our results suggest that the cleavage is an autocatalytic reaction that occurs in the acidic environment of the late secretory pathway. Furthermore, the cleavage produced a new, reactive C terminus that has the potential to attach the mucin to itself or other molecules. Because a pH below 6 can be reached in the late secretory pathway and on mucosal surfaces, the cleavage and possible cross-linking are likely to be of biological importance.