PG0026 Is the C-terminal Signal Peptidase of a Novel Secretion System of Porphyromonas gingivalis
PG0026 Is the C-terminal Signal Peptidase of a Novel Secretion System of Porphyromonas gingivalis
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DOI：
10.1074/jbc.m112.369223
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发表时间：
2012-07-13
影响因子：
4.8
通讯作者：
Reynolds, Eric C.
中科院分区：
文献类型：
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作者：
Glew, Michelle D.;Veith, Paul D.;Reynolds, Eric C.
牙龈卟啉单胞菌（Porphyromonas gingivalis）的新型分泌系统的蛋白质底物含有保守的C-末端结构域（CTD），其类似于70-80个氨基酸残基，对于它们的分泌和附着到细胞表面是必需的。CTD本身尚未在成熟底物中检测到，这表明它可能被一种新的信号肽酶去除。超过10种蛋白质已被证明是分泌系统的正常功能所必需的，其中之一，PG 0026，是一种预测的半胱氨酸蛋白酶，也含有CTD，这表明它可能是分泌系统的分泌成分和CTD信号肽酶的候选者。将PG 0026缺失突变体与编码改变的催化Cys残基的PG 0026 C690 A靶向突变体一起沿着构建。通过SDS-PAGE和质谱法分析澄清的培养液级分显示，CTD在野生型菌株中完整地释放到周围培养基中，但在PG 0026突变菌株中没有。蛋白质印迹实验表明，模型底物的成熟停滞在CTD去除步骤中，特别是在PG 0026突变体中，全细胞ELISA实验证明底物部分分泌到细胞表面。CTD还显示在PG 0026突变体中在细胞表面可接近，表明CTD被分泌但不能被切割。数据表明，PG 0026负责在底物分泌穿过OM后切割CTD信号。
Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of similar to 70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.