Seers Christine A, Slakeski Nada, Veith Paul D, Nikolof Todd, Chen Yu-Yen, Dashper Stuart G, Reynolds Eric C
Cooperative Research Centre for Oral Health Science, School of Dental Science, University of Melbourne, 720 Swanston Street, Victoria, Australia.
J Bacteriol. 2006 Sep;188(17):6376-86. doi: 10.1128/JB.00731-06.
Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the P. gingivalis outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain P. gingivalis mutant (YH522AB) and an Arg- and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.
牙龈卟啉单胞菌产生附着于外膜的蛋白质，其中包括与毒力相关的蛋白酶RgpA和RgpB（精氨酸牙龈蛋白酶）以及Kgp（赖氨酸牙龈蛋白酶）。我们分析了牙龈卟啉单胞菌的外膜蛋白质组，并鉴定出许多蛋白质，其C末端结构域在序列上与RgpB、RgpA和Kgp的相似，这表明这些结构域可能具有共同功能。以RgpB为模型研究C末端结构域的作用，我们在精氨酸牙龈蛋白酶缺陷型牙龈卟啉单胞菌突变株（YH522AB）和精氨酸及赖氨酸牙龈蛋白酶双缺陷型突变株（YH522KAB）中表达了全长酶原形式的RgpB（重组RgpB [rRgpB]）、具有催化性Cys244Ala突变的rRgpB [rRgpB(C244A)] 或缺失C末端72个氨基酸的rRgpB435。rRgpB具有催化活性，主要定位于两种背景菌株的外膜上。rRgpB(C244A) 无活性且附着于外膜，根据十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳结果，两种背景菌株均呈现典型的附着模式，但在YH522KAB中，前结构域未被去除。因此，在体内，RgpB的输出和膜附着独立于RgpA、RgpB或Kgp的蛋白水解活性。然而，对于涉及RgpB蛋白水解加工的成熟过程，需要RgpB、RgpA或Kgp的蛋白水解活性。C末端截短的rRgpB435未附着于外膜，在培养上清液和周质中以大部分无活性的、离散的71 kDa和48 kDa异构体形式存在。这些结果表明，C末端结构域对于外膜附着至关重要，可能参与输出及附着于细胞表面的协同过程。
