Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains - Roles of the CGLCG motif in the D1- and D3-domains
Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains - Roles of the CGLCG motif in the D1- and D3-domains
复制标题
DOI：
10.1074/jbc.273.51.34527
复制
发表时间：
1998-12-18
影响因子：
4.8
通讯作者：
Hill, RL
中科院分区：
文献类型：
--
作者：
Perez-Vilar, J;Hill, RL
通过定点突变对编码猪颌下粘蛋白氨基端的质粒进行修饰，以评估单个半胱氨酸残基在二硫键连接的粘蛋白多聚体组装中的作用，含有C1199 A的质粒的COS-7细胞主要表达单体，表明D3结构域中的半胱氨酸1199参与形成粘蛋白多聚体，该残基位于序列C（1199）SWRYEPCG中，其在其他分泌型粘蛋白和人前原冯维勒布兰德因子的D3结构域中高度保守。与此相反，细胞与含有C1276 A的质粒表达的三聚体一样：与未突变的质粒，表明半胱氨酸1276是不参与形成二硫键结合的多聚体，在CGLCG基序中的半胱氨酸在二硫键结合的多聚体的粘蛋白的组装的作用也进行了评估。具有质粒的细胞中，粘蛋白D1或D3结构域基序中的两个半胱氨酸都被分泌不良的丙氨酸表达蛋白质取代，这表明这些突变损害了表达蛋白质的正常折叠。具有突变D1结构域基序的质粒表达单体，而具有突变D3结构域基序的质粒表达单体和三聚体，然而，由后一种质粒表达的三聚体在非酸性区室中组装，这通过在莫能菌素存在下的表达研究来判断，莫能菌素抑制未突变质粒而不是突变质粒的三聚体形成。这些结果表明，CGLCG基序在HI结构域中的多聚化所需的trans-Golgi复合物。然而，在D3结构域中的CGLCG基序似乎阻止在细胞的非酸性区室中形成粘蛋白多聚体，编码D1和D2结构域、D1和D3结构域或仅D3结构域的质粒在莫能菌素存在下也表达寡聚体，这表明三个D结构域必须是连续的以避免在非酸性区室中的多聚化。粘蛋白中的这些基序可能在粘蛋白的二硫键结合的多聚体组装期间参与硫醇-二硫键交换反应。
Plasmids encoding the amino-terminal region of porcine submaxillary mucin were modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of mucin, COS-7 cells with the plasmid containing C1199A expressed primarily monomers, suggesting that half-cystine 1199 in the D3-domain is involved in forming mucin multimers, This residue is in the sequence C(1199)SWRYEPCG, which is highly conserved in the D3-domain of other secreted mucins and human prepro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those: with unmutated plasmid, suggesting that half-cystine 1276 is not involved in formation of disulfide-bonded multimers, The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmidis in which both half-cystines in the motif in the D1- or D3-domain of mucin are replaced by alanine expressed proteins that were poorly secreted, suggesting that these mutations impair normal folding of the expressed proteins, A plasmid with a mutant D1-domain motif expressed monomers, whereas one with a mutant D3-domain motif expressed monomers and trimers, However, the trimers expressed by the latter plasmid were assembled in nonacidic compartments, as judged by expression studies in the presence of monensin, which inhibits trimer formation by unmutated plasmid, but not by the mutant plasmid. These results suggest that the CGLCG motif in the HI-domain is required for multimerization in the trans-Golgi complex. However, the CGLCG motif in the D3-domain appears to prevent formation of mucin multimers in non-acidic compartments of the-cell, Plasmids encoding the D1- and D2-domains, the D1- and D3-domains, or only the D3-domain also expressed oligomers in the presence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that these motifs in mucins are engaged in the thiol-disulfide interchange reactions during the assembly of disulfide-bonded multimers of mucin.