Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system
- 作者：Yutaka Ishida ; JinPing Hu ; Eiko Sakai ; Tomoko Kadowaki ; Kenji Yamamoto ; Takayuki Tsukuba ; Yuzo Kato ; Koji Nakayama ; Kuniaki Okamoto
- 关键词：Arg-gingipain ; Lys-gingipain ; Periodontal diseases ; Porphyromonas gingivalis ; Recombinant expression
- 刊名：Archives of Oral Biology
- 出版年：2008
- 期刊代码：25_00039969
- 类别：med
- 出版时间：June 2008
- 卷：53
- 期：6
- 页码：538-544
- 文件大小：569 K
摘要
Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis.