Katz Jannet, Yang Qiu-Bo, Zhang Ping, Potempa Jan, Travis James, Michalek Suzanne M, Balkovetz Daniel F
Department of Oral Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Infect Immun. 2002 May;70(5):2512-8. doi: 10.1128/IAI.70.5.2512-2518.2002.
Porphyromonas gingivalis has been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivalis can degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis (i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivalis cells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivalis cells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis.
牙龈卟啉单胞菌被认为是成人牙周炎的病原体。我们之前已经表明，牙龈卟啉单胞菌能够降解上皮细胞间连接复合物，因此表明该细菌可以通过细胞旁途径侵入下方的结缔组织。然而，这一过程中涉及的精确机制尚未阐明。本研究的目的是确定牙龈卟啉单胞菌的精氨酸和赖氨酸特异性牙龈蛋白酶（即分别为HRgpA、RgpB和Kgp）是否负责降解黏着连接中的细胞间黏附蛋白E-钙黏蛋白。此外，我们比较了完整牙龈蛋白酶HRgpA和Kgp与其单独催化结构域的降解能力。在这些研究中，将免疫沉淀的E-钙黏蛋白以及极化的Madin-Darby犬肾（MDCK）上皮细胞单层培养物与牙龈蛋白酶一起孵育，并通过蛋白质印迹分析评估E-钙黏蛋白的水解情况。牙龈卟啉单胞菌细胞与免疫沉淀的E-钙黏蛋白孵育会导致降解，而牙龈卟啉单胞菌细胞预先暴露于亮抑酶肽，尤其是乙酰-Leu-Val-Lys-醛（分别为精氨酸和赖氨酸特异性抑制剂）会降低这种活性。此外，用不同的牙龈蛋白酶孵育E-钙黏蛋白免疫沉淀物会导致该蛋白质有效且相似的水解。然而，当MDCK细胞单层暴露于牙龈蛋白酶时，Kgp在水解黏着连接中的E-钙黏蛋白分子方面最有效。在500 nM时，Kgp在降解E-钙黏蛋白方面比其催化结构域更有效，但在较低浓度（250 nM）时并非如此。这些结果表明，Kgp的血凝素结构域在降解中起作用，并且该活性存在一个临界阈值浓度。综上所述，这些结果提供了证据，表明牙龈蛋白酶，尤其是Kgp，参与上皮细胞黏着连接的降解，这可能在牙龈卟啉单胞菌侵入牙周结缔组织中起重要作用。
