| Co-Investigator(Kenkyū-buntansha) |
田中 隆治 サントリー株式会社, ヘルスケア事業部, 部長
AMANO Atsuo Osaka Univ.Graduate School of Dentistry, Professor, 大学院・歯学研究科, 教授 (50193024)
KAWABATA Shigetada Osaka Univ.Graduate School of Dentistry, Associate Professor, 大学院・歯学研究科, 助教授 (50273694)
TANAKA Ryuji Suntory Co., Director
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| Budget Amount *help |
¥13,100,000 (Direct Cost: ¥13,100,000)
Fiscal Year 2000: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 1999: ¥8,800,000 (Direct Cost: ¥8,800,000)
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| Research Abstract |
Fimbriae of Porphyromonas gingivalis are thought to play an important role in the colonization and invasion to periodontal tissues. In this study, we analyzed the interactions of P.gingivalis fimbriae with human hemoglobin, fibrinogen, salivary components (i.e., proline-rich protein [PRP], proline-rich glycoprotein [PRG] and statherin), and extracellular matrix proteins (laminin, elastin, fibronectin, type I collagen, thrombospondin and vitronectin) based on surface plasmon resonance (SPR) spectroscopy using a biomolecular interaction analyzing system (BIAcore). The BIAcore profiles demonstrated that fimbriac can specifically bind to all of the examined proteins with significant association constants [Ka]. Vitronectin showed the highest affinity to fimbriae [Ka=3.79×10^6(M^<-1>)]. A synthetic peptide which is a potent inhibitor to fimbrial bindings to salivary proteins was not significantly effective in the fimbrial interactions with all of the host proteins. These results suggest that
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the interactions between fimbriae and the ECM proteins occur with specific affinities which are not mediated by mechanisms identical to those of salivary proteins. Furthermore, we investigated the effects of fimbriae on the interactions between vitronectin and fibronectin and their receptors, αvβ3 and α5β1 integrins. αvβ3- or α5β1-overexpressing cell lines were established by transfection of human pcDNA3.1 vectors ligated with cDNAs encoding αvβ3 and α5β1 integrin subunits, into CHO cells. The number of P.gingivalis bound to CHOαvβ3 and CHOα5β1 were 2.5 and 1.6 times more than that to CHO cells, respectively. The overexpression of the integrins also promoted the binding of fimbriae to the cells, by 230% in CHOαvβ3 and by 140% in CHOα5β1. The molecular interactions between fibronectin/vitronectin and CHOα5β1/CHOαvβ3 were markedly inhibited by fimbriae, on BIACORE analysis. The effects of fimbriae on the ECM/ integrin-related cellular functions were further evaluated. CHOα5β1 and CHOαvβ3 were incubated in serum-depleted medium to reduce their attachments onto the polystyrene culture dishes. Subsequent addition of fibronectin or vitronectin (1μg/ml) markedly promoted the cellular attachments to the dish surfaces. The simultaneous addition of fimbriae clearly inhibited the cellular attachment in a dose dependent manner, and the highest dose of 30μg/ml of fimbriae achieved complete inhibition. Protamines (salmine prepared from sperm DNA of salmon, and clupeine from herring sperm) which are basic peptides rich in arginine were found to inhibit proteolytic activity of arginine-specific cysteine protease (RC-protease) from Porphyromonas gingivalis. Lineweaver-Burk plot analysis revealed that the protamines competitively inhibited the proteolytic activity with the cleavage of benzoyl-L-arginine p-nitroanilide, a synthetic substrate of RC-protease. Furthermore, the protamines were capable of binding strongly to P.gingivalis fimbriae, and inhibited the fimbrial interaction to immobilized fibronectin. These results clearly show that the protamines are a potent inhibitor for proteolytic and adhesive activities of P.gingivalis. Less
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