Studies on iron acquisition mechanism of periodontal pathogen

用牙龈卟啉单胞菌（Porphyromonas gingivalis）ATCC 33277的外膜去污剂从溶解的材料中纯化了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。甘氨酰甘氨酸可显著激活RGP-B。RGP-A和RGP-B均被PCMB、NEM、EDTA、E64和镇痛药抑制。KGP可被PCMB、NEM和TLCK抑制，RGP-A和RGP-B的分子量为43 kDa，KGP的分子量为48 kDa。RGP和KGP对合成的显色底物的水解活性分别限于那些在P-1位含有精氨酸和赖氨酸的底物。用Sepliadex G-100凝胶过滤法分离的血红蛋白水解产物中有一小部分能刺激血红素缺乏的牙龈卟啉单胞菌静息细胞的生长，表明RGP-B在血红蛋白提供铁源方面起部分作用。N端氨基酸序列分析表明，RGP的HGP 15编码19 kDa的蛋白，从HGP 15的高产菌株中纯化得到HGP 15结构域蛋白，并发现其具有与血红蛋白结合的能力，并具有pH依赖性。抗HGP 15抗血清与19 kDa血红蛋白结合蛋白反应。牙龈卟啉单胞菌野生型菌株显示pH依赖性血红蛋白吸附，而其不产生HGPI 5相关蛋白的非色素突变体显示血红蛋白吸附不足。这些结果表明，GPI 5的生产，吸附和血红素积累的牙龈卟啉单胞菌之间的密切关系。

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.