Studies on iron acquisition mechanism of periodontal pathogen

牙周病原菌获取铁机制的研究

• 批准号: 09671877
• 负责人: FUJIMURA Setsuo
• 金额: $ 0.32万
• 依托单位: Matsumoto Dental University, School of Dentistry
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671877/
• 关键词: p.gingivalis; periodontopathogen; iron acquisition; proteinases; プロテイナーゼ

Two arginine specific proteinases (RGP-A, RGP-B) and a lysine specific proteinase (KGP) were purified from the solubilized materials by a detergent of the envelope of Porphyromonas gingivalis ATCC 33277. The three enzymes were activated by reducing reagents. RGP-B was remarkably activated by glycyiglycine. Both RGP-A and RGP-B were inhibited by PCMB, NEM, EDTA, luepeptin, E64, and antipain. KGP was inhibited by PCMB, NEM, and TLCK.Moecular masses of RGP-A and RGP-B were 43 kDa and that of KGP was 48 kDa. Hydrolytic activities of RGP and KGP to the synthetic chromogenic substrates were limited to those containing arginine and lysine at the P-ipositions, respectively. Hemoglobin was almost completely digested when it was treated by RGP-B.Furthermore, it was found that a fraction of the hydrolytic products separated by Sepliadex G-100 gel filtration stimulated the growth ofresting cells of P.gingivlis derived by heme- depleted condition, indicating RGP-B plays a partial role to provide iron source from hemoglobin.P gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of N-terminal amino acid sequence indicated that 19 kDa protein was encoded by HGP15 of RGP.The HGP 15 domain protein was purified from an HGP15-overproducing E.coli and was found that to have the ability to bind hemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa hemoglobin binding protein. P.gingivalis wild-type strain showed pH-dependent hemoglobin adsorption, whereas its non-pigmented mutants that produced no HGPI5-related proteins showed deficiency in hemoglobin adsorption. These results indicate a close relationship among GPI5 production, adsorption, and heme accumulation of P gingivalis.

通过牙龈卟啉单胞菌 ATCC 33277 包膜的去污剂从溶解的材料中纯化出两种精氨酸特异性蛋白酶（RGP-A、RGP-B）和一种赖氨酸特异性蛋白酶（KGP）。这三种酶被还原剂激活。 RGP-B 被甘氨酰甘氨酸显着激活。 RGP-A 和 RGP-B 均被 PCMB、NEM、EDTA、luepeptin、E64 和抗痛剂抑制。 PCMB、NEM和TLCK均能抑制KGP。RGP-A和RGP-B的分子质量为43 kDa，KGP的分子质量为48 kDa。 RGP 和 KGP 对合成显色底物的水解活性分别仅限于 P 位含有精氨酸和赖氨酸的底物。 RGP-B处理后血红蛋白几乎被完全消化。此外，通过Sepliadex G-100凝胶过滤分离出的一部分水解产物还刺激了血红素耗尽条件下的P.gingivlis静息细胞的生长，表明RGP-B起到了部分作用，从血红蛋白中提供铁源。P.gingivalis细胞表达了显着的铁源。 在血琼脂平板上生长时为 19 kDa 蛋白质。 N端氨基酸序列分析表明，19 kDa的蛋白是由RGP的HGP15编码的。从过量产生HGP15的大肠杆菌中纯化出HGP 15结构域蛋白，并发现其具有以pH依赖性方式结合血红蛋白的能力。抗 HGP15 抗血清与 19 kDa 血红蛋白结合蛋白发生反应。牙龈卟啉单胞菌野生型菌株表现出pH依赖性血红蛋白吸附，而其不产生HGPI5相关蛋白的非色素突变体表现出血红蛋白吸附缺陷。这些结果表明牙龈卟啉单胞菌的 GPI5 产生、吸附和血红素积累之间存在密切关系。

项目成果

K.Nakayama, S.Fujimura: "Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the ....." Molecular Microbiology. 27/1. 51-61 (1998)

K.Nakayama、S.Fujimura：“血红蛋白受体蛋白是由半胱氨酸蛋白酶编码基因和......”在基因内编码的。”分子微生物学。

藤村節夫: "Comparative properties of envelope-asociated arginine・gingipains and lysine-gingipain of Porphyromonas gingivalis" FEMS(Microbiology Letters). 163・2. 173-179 (1998)

Setsuo Fujimura：“牙龈卟啉单胞菌的包膜相关精氨酸·牙龈蛋白酶和赖氨酸牙龈蛋白酶的比较特性”FEMS（微生物学快报）163·2（1998）。

藤村節夫: "Comparisons of the isozymes of arginine-specific proteinases in the culture supernatant of Porphyromonas gingivalis" 松本歯学. 24. 52-57 (1998)

Setsuo Fujimura：“牙龈卟啉单胞菌培养物上清液中精氨酸特异性蛋白酶同工酶的比较”Matsumoto Dentistry 24. 52-57 (1998)。

藤村節夫: "comparative properties of envelope-associated arginine-gingipains and lysine-gingipain of Porphyromonas gingivalis" FEMS(Microbiology Letters). 163. 173-179 (1998)

Setsuo Fujimura：“牙龈卟啉单胞菌的包膜相关精氨酸-牙龈蛋白酶和赖氨酸-牙龈蛋白酶的比较特性”FEMS（微生物学快报）163. 173-179（1998）。

中山浩次: "Haemogiolin receptor protein is in tragenically encoded by the cysteine proteinase encoding genes and the haemagglutinine‥‥" Molecular Microbiology. 27. 51-61 (1998)

Koji Nakayama：“血红蛋白受体蛋白是由半胱氨酸蛋白酶编码基因和血凝素以悲剧性方式编码的……”《分子微生物学》27. 51-61 (1998)。