Establishment of protein expression system in Porphyromonasi gingivalis

牙龈卟啉单胞菌蛋白表达系统的建立

• 批准号: 15591971
• 负责人: OKAMOTO Kuniaki
• 金额: $ 2.3万
• 依托单位: NAGASAKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591971/
• 关键词: Arg-gingipain; cysteine proteinase; Lys-gingipain; periodontitis disease; Porphysomonas gingivalis; transport; periodontal disease

Porphyromonas gingivalis(P.gingivalis) is a Gram-negative anaerobic bacterium and produces a number of proteinsaes. Among them, Arginine-specific cysteine proteinase(Arg-gingipain, Rgp) and lysine-specific cysteine proteinase(Lys-gingipain, Kgp) are implicated as major virulence factors in the development and progression of chronic periodontaitis. Rgp is encoded by two separate rgp genes (rgpA and rgpB), whereas Kgp is encoded by the single gene (kgp). The initial translation products of rgpA and kgp genes primarily consist of two functional parts : proteinase domain and adhesin domain. Furthermore, the C-terminal adhesin domains are derived into three or four subdomains (HGP44,HGP15,HGP17 and HGP27 for RgpA ; nHGP44,HGP15 and cHGP44 for Kgp). These C-terminal adhesin domains are highly homologous. In this study, we focused Kgp and established its expression system. Furthermore, we attempted to decide the active sites for Kgp. Although the active site of Rgp have been decided, that of Kgp was not clear due to have two cysteine residues in putative active site. Therefore, we introduced point mutation in the two putative active sites of Kgp. First, we constructed a plasmid that 248 cysteine (^<248>Cys) and 249 cysteine (^<249>Cys) were exchanged with alanine and introduced into Kgp-deficient strain KDP129. The mutation of both sites resulted in lack of emzymatic activity of Kgp, although the expression of Kgp was recognized by Western blot analysis. Next, we constructed a plasmid that point mutation plasmid against each active site and introduced into KDP129. These two mutants had the emzymatic activity of Kgp. These results suggested that ^<248>Cys and ^<249>Cys residues were important for the activity of Kgp.

牙龈卟啉单胞菌（Porphyromonas gingivalis，P.gingivalis）是一种革兰氏阴性厌氧菌，能产生多种蛋白酶。其中，精氨酸特异性半胱氨酸蛋白酶（Arg-gingipain，Rgp）和赖氨酸特异性半胱氨酸蛋白酶（Lys-gingipain，Kgp）被认为是慢性牙周炎发生发展的主要毒力因子。Rgp由两个独立的rgp基因（rgpA和rgpB）编码，而Kgp由单个基因（kgp）编码。rgpA和kgp基因的初始翻译产物主要由两个功能部分组成：蛋白酶结构域和粘附素结构域。此外，C-末端粘附素结构域衍生为三个或四个亚结构域（对于RgpA为HGP 44、HGP 15、HGP 17和HGP 27;对于Kgp为nHGP 44、HGP 15和cHGP 44）。这些C-末端粘附素结构域是高度同源的。本研究以Kgp为研究对象，建立了Kgp的表达系统。此外，我们试图确定Kgp的活性位点。虽然Rgp的活性位点已被确定，但Kgp的活性位点尚不清楚，因为在推测的活性位点上有两个半胱氨酸残基。因此，我们在Kgp的两个推定的活性位点引入点突变。首先，我们构建了248个半胱氨酸（^<248>Cys）和249个半胱氨酸（^<249>Cys）与丙氨酸交换的质粒，并将其导入Kgp缺陷型菌株KDP 129中。这两个位点的突变导致Kgp酶活性的缺乏，虽然Kgp的表达通过Western印迹分析被识别。接下来，我们构建了针对每个活性位点的点突变质粒，并将其导入KDP 129。这两个突变体均具有Kgp的酶学活性。这些结果表明，^<248>Cys和^<249>Cys残基对Kgp的活性是重要的。

项目成果

Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis

牙龈卟啉单胞菌中精氨酸和赖氨酸的作用

• 发表时间: 2004
• 期刊: Biol.Chem. 385
• 作者: Abe N.;et al.
• 通讯作者: et al.

Tsukuba T., et al.: "Association of cathepsin E deficiency with development of atopic dermatitis"J.Biochem.. 134. 893-902 (2003)

Tsukuba T.等：“组织蛋白酶E缺乏与特应性皮炎发生的关联”J.Biochem..134.893-902(2003)

The major structural components of two cell surface filaments of Porphyromonas gingivalis are matured through lipoprotein precursors

• DOI: 10.1111/j.1365-2958.2004.04105.x
• 发表时间: 2004-06-01
• 期刊: MOLECULAR MICROBIOLOGY
• 影响因子: 3.6
• 作者: Shoji, M;Naito, M;Nakayama, K
• 通讯作者: Nakayama, K

Shibata M., et al.: "Disruption of structural functional integrity of alpha 2-macroglobulin by cathepsin E"Eur.J.Biochem.. 270. 1189-1198 (2003)

Shibata M. 等人：“组织蛋白酶 E 破坏 α2-巨球蛋白的结构功能完整性”Eur.J.Biochem.. 270. 1189-1198 (2003)

Roles of Arg- and Lys-gingipains in Coaggregation of Porphyromonas gingivalis : Identification of Its Responsible Molecules in Translation Products tf rgpA, kgp and HagA Genes.

Arg-和Lys-gingipains在牙龈卟啉单胞菌共聚集中的作用：鉴定翻译产物tf rgpA、kgp和HagA基因中的责任分子。

• 发表时间: 2004
• 期刊: Biol.Chem. 385
• 作者: Abe N;Baba A;Takii R;Nakayama K;Kamaguchi A;Shibata Y;Abiko Y;Okamoto K;Kadowaki T;Yamamoto K.
• 通讯作者: Yamamoto K.