Glew Michelle D, Veith Paul D, Chen Dina, Seers Christine A, Chen Yu-Yen, Reynolds Eric C
Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Victoria 3010, Australia.
Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Victoria 3010, Australia.
J Proteomics. 2014 Oct 14;110:72-92. doi: 10.1016/j.jprot.2014.07.033. Epub 2014 Aug 9.
Membrane complexes of Porphyromonas gingivalis were analyzed using two dimensional-blue native-PAGE. The molecular mass of the gingipain complexes, RgpA and Kgp, ranged from 450 kDa to greater than 1200 kDa, and did not change in single rgpA and kgp mutants indicating that the proteolytically processed polyproteins were independently capable of forming complexes. The outer membrane protein, LptO, which is essential for gingipain secretion, was found in up to seven different complex sizes. PG0026, also important for secretion, was observed to interact with the largest LptO complex [VII] at 480 kDa, supporting a cooperative role in secretion. Two pro-form RgpB intermediates formed a complex before cleavage of their C-terminal secretion signal domains (CTDs) such that complex formation may occur during secretion and processing. This may also be the case for other CTD-proteins as not only modified, mature RgpB, but also CPG70 was found to exist as multi-subunit complexes. RagA and RagB were observed in three different complex sizes. Elimination of the abundant gingipains enabled the identification of many inner and outer membrane protein complexes: TonB:ExbB:ExbD, Omp85, P51:PG2168, PorK:PorN, PG0056, PG0241, PG1430 and five proposed respiratory chain complexes (Mmd, Nqr, Rnf, Frd/Sdh and Atp).
Porphyromonas gingivalis is a major oral pathogen associated with chronic periodontitis in humans. Secreted gingipains are considered major virulence factors of this pathogen and are secreted by a newly described type IX secretion system. This work has used 2D-BN-PAGE and MS to demonstrate that mature gingipains can independently form complexes and that substrate intermediates and mature secreted proteins of the type IX secretion system form multi-subunit complexes. Based on this work we propose that the substrates of this secretion system are secreted as large multi-subunit protein complexes. Two known important components of the secretion machinery, PG0026 and the integral outer membrane protein, LptO, were found to interact which would anchor PG0026 to the outer membrane and perhaps aid in the function of PG0026 to cleave the CTD from secreted substrates. The work has also identified more than 100 membrane proteins forming multi-subunit complexes.
使用二维蓝色天然聚丙烯酰胺凝胶电泳分析牙龈卟啉单胞菌的膜复合物。牙龈蛋白酶复合物RgpA和Kgp的分子量范围为450 kDa至大于1200 kDa，在单rgpA和kgp突变体中未发生变化，表明经蛋白水解加工的多聚蛋白能够独立形成复合物。对牙龈蛋白酶分泌至关重要的外膜蛋白LptO，被发现有多达七种不同的复合物大小。对分泌也很重要的PG0026，被观察到与480 kDa的最大LptO复合物[VII]相互作用，支持其在分泌中的协同作用。两种前体形式的RgpB中间体在其C末端分泌信号域（CTD）切割之前形成复合物，使得复合物形成可能发生在分泌和加工过程中。其他CTD蛋白可能也是如此，因为不仅修饰后的成熟RgpB，而且CPG70也被发现以多亚基复合物的形式存在。观察到RagA和RagB有三种不同的复合物大小。去除丰富的牙龈蛋白酶能够鉴定出许多内膜和外膜蛋白复合物：TonB:ExbB:ExbD、Omp85、P51:PG2168、PorK:PorN、PG0056、PG0241、PG1430以及五个推测的呼吸链复合物（Mmd、Nqr、Rnf、Frd/Sdh和Atp）。
牙龈卟啉单胞菌是与人类慢性牙周炎相关的主要口腔病原体。分泌的牙龈蛋白酶被认为是该病原体的主要毒力因子，并由一种新描述的IX型分泌系统分泌。这项工作使用二维蓝色天然聚丙烯酰胺凝胶电泳和质谱证明，成熟的牙龈蛋白酶能够独立形成复合物，并且IX型分泌系统的底物中间体和成熟分泌蛋白形成多亚基复合物。基于这项工作，我们提出该分泌系统的底物以大型多亚基蛋白复合物的形式分泌。发现分泌机制的两个已知重要成分PG0026和整合外膜蛋白LptO相互作用，这将把PG0026锚定在外膜上，并可能有助于PG0026从分泌的底物上切割CTD的功能。这项工作还鉴定出100多种形成多亚基复合物的膜蛋白。
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