The MUC1 SEA module is a self-cleaving domain
The MUC1 SEA module is a self-cleaving domain
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DOI：
10.1074/jbc.m506047200
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发表时间：
2005-09-30
影响因子：
4.8
通讯作者：
Wreschner, DH
中科院分区：
文献类型：
--
作者：
Levitin, F;Stern, O;Wreschner, DH
MUC 1是一种由多种人类腺癌过度表达的糖蛋白，是一种I型跨膜蛋白（MUC 1/TM），在其合成后不久在其胞外结构域中经历蛋白水解切割。这种切割产生两个亚基，α和β，它们特异性地相互识别并以强的非共价相互作用结合在一起。蛋白水解发生在SEA模块内，SEA模块是一个120个氨基酸的结构域，在许多高度糖基化的粘蛋白样蛋白中高度保守。SEA模块的翻译后切割发生在糖蛋白IgHepta和MUC 3中与MUC 1相似的位点。然而，在含有切割的SEA模块的其他蛋白质的情况下，MUC 1蛋白水解的机制尚未阐明。选择性剪接产生两种跨膜MUC 1同种型，命名为MUC 1/Y和MUC 1/X。我们在这里证明，MUC 1/X，其细胞外结构域是唯一的SEA模块除了30 MUC 1 N-末端氨基酸组成，在相同的位点进行蛋白水解切割的MUC 1/TM蛋白。相反，MUC 1/Y同种型，由N-末端截短的SEA模块组成，不被切割。MUC 1/X丝氨酸残基（Ser-63）的半胱氨酸或苏氨酸突变紧邻切割位点的C-末端产生切割的蛋白质，而MUC 1/X的Ser-63残基突变为17个氨基酸中的任何其它氨基酸不导致切割。在体外孵育的高度纯化的前体MUC 1/X蛋白导致自切割。此外，加入羟胺，一个强的亲核试剂，显着增强裂解。这两个特征都是自切割蛋白的特征，我们得出结论，MUC 1经历了由切割位点处的N-> O-酰基重排介导的自蛋白水解，然后是不稳定酯的水解拆分和伴随的切割。很可能所有裂解的含SEA模块的蛋白遵循类似的途径。
MUC1, a glycoprotein overexpressed by a variety of human adenocarcinomas, is a type I transmembrane protein (MUC1/TM) that soon after its synthesis undergoes proteolytic cleavage in its extracellular domain. This cleavage generates two subunits, alpha and beta, that specifically recognize each other and bind together in a strong noncovalent interaction. Proteolysis occurs within the SEA module, a 120-amino acid domain that is highly conserved in a number of heavily glycosylated mucin-like proteins. Post-translational cleavage of the SEA module occurs at a site similar to that in MUC1 in the glycoproteins IgHepta and MUC3. However, as in the case of other proteins containing the cleaved SEA module, the mechanism of MUC1 proteolysis has not been elucidated. Alternative splicing generates two transmembrane MUC1 isoforms, designated MUC1/Y and MUC1/X. We demonstrated here that MUC1/X, whose extracellular domain is comprised solely of the SEA module in addition to 30 MUC1N-terminal amino acids, undergoes proteolytic cleavage at the same site as the MUC1/TM protein. In contrast, the MUC1/Y isoform, composed of an N-terminally truncated SEA module, is not cleaved. Cysteine or threonine mutations of the MUC1/X serine residue (Ser-63) immediately C-terminal to the cleavage site generated cleaved proteins, whereas mutation of the Ser-63 residue of MUC1/X to any other of 17 amino acids did not result in cleavage. In vitro incubation of highly purified precursor MUC1/X protein resulted in self-cleavage. Furthermore, addition of hydroxylamine, a strong nucleophile, markedly enhanced cleavage. Both these features are signature characteristics of self-cleaving proteins, and we concluded that MUC1 undergoes autoproteolysis mediated by an N-->O-acyl rearrangement at the cleavage site followed by hydrolytic resolution of the unstable ester and concomitant cleavage. It is likely that all cleaved SEA module-containing proteins follow a similar route.