Porphyromonas gingivalis の endopeptidase 遺伝子の同定および性状に関する研究

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Porphyromonas gingivalis is one of major etiologic agents of progressive periodontal disease and has several proteolytic enzymes implicated in invasion, tissue destruction and evasion of host antibacterial defenses. The aim in our laboratory is to fully clarify the role of enzymes in relation to pathogenesis of this bacterium. In the previous study, we determined the nucleotide sequence of clone pAL2 obtained from P. gingivalis 381 (Microbiology 141 : 2047-2052, 1995), which appeared to contain a DNA fragment encoding a proteolytic enzyme. An approximate 3.8kb DNA fragment (pAL2) was sequenced, the DNA sequence analysis revealed one complete ORF (pepO) and two incomplete ORFs in this fragment. ORF (pepO) encoded a protein (P. gingivalis PepO) of 690 amino acids with a calculated molecular weight of 78, 796. A comparison of the amino acid sequence of P. gingivalis PepO with other proteins in the SWISS-PROT database revealed a 31.7% identity with Endothelin-Converting Enzyme 1 (ECE 1), one of the NEP families. Polymerase chain reaction (PCR) was performed for amplification of pepO, and the PCR fragment was cloned into pET-3a in order to overexpress P. gingivalis PepO in E. coli BL21 (DE3). Furthemore, the overexpressed P. gingivalis PepO was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. The optimal pH of the purified enzyme was between 6.8 and 7.2, and the optimal temperature range was between 40℃ and 50℃. The activity was strongly inhibited by the NEP inhibitor phosphoramidon, but only slightly by the NEP inhibitor thiorphan, like ECE 1. The purified enzyme hydrolyzed metenkephalin, bradykinin and substance P, which were physiological peptides, like NEP from Lactococcus lactis. And the enzyme cleaved big endothelin (ET)-1 big ET-2 and big ET-3, so that generated ET1, ET2 and ET3. The ECE 1 is a key enzyme during processing for generating ET1 which acts as a mitogen as well as a vasoconstrictor for vascular smooth muscle cells. Therefore, these results suggest that P. gingivalis PepO might operate as a virulence factor of not only periodontitis but cardiovascular diseases.