Preventive strategy of marginal periodontitis by antimicrobial and adhesion-inhibitory basic peptides and protamines.

牙龈卟啉单胞菌的菌毛被认为在牙周组织的定植和侵袭中起重要作用。本研究利用生物分子相互作用分析系统（BIAcore），基于表面等离子体共振（SPR）光谱分析了牙龈卟啉菌与人血红蛋白、纤维蛋白原、唾液成分（即富含脯氨酸的蛋白[PRP]、富含脯氨酸的糖蛋白[PRG]和statherin）以及细胞外基质蛋白（层粘连蛋白、弹性蛋白、纤维连接蛋白、I型胶原蛋白、凝血蛋白和玻璃体粘连蛋白）的相互作用。BIAcore图谱表明，纤维可以特异性结合所有具有显著结合常数的检测蛋白[Ka]。Vitronectin对菌毛的亲和力最高[Ka=3.79×10^6(M^<-1>)]。一种合成的肽是一种有效的抑制唾液腺蛋白与毛毡结合的抑制剂，但在毛毡与所有宿主蛋白的相互作用中却没有显著的效果。这些结果表明，更多的是菌毛与ECM蛋白之间的相互作用具有特异性亲和力，而不是由与唾液蛋白相同的机制介导的。此外，我们还研究了菌毛对玻璃体连接蛋白和纤维连接蛋白及其受体αvβ3和α5β1整合素相互作用的影响。将编码αvβ3和α5β1整合素亚基的cdna连接人pcDNA3.1载体转染CHO细胞，建立αvβ3或α5β1过表达细胞系。结合CHOαvβ3和CHOα5β1的牙龈假单胞菌数量分别是CHO细胞的2.5倍和1.6倍。整合素的过表达也促进了菌毛与细胞的结合，在CHOαvβ3和CHOα5β1中分别提高了230%和140%。BIACORE分析显示，纤维连接蛋白/玻璃体连接蛋白与CHOα5β1/CHOαvβ3的分子相互作用被菌毛明显抑制。进一步评估毛对ECM/整合素相关细胞功能的影响。CHOα5β1和CHOαvβ3在无血清培养基中孵育，以减少其在聚苯乙烯培养皿上的附着。随后加入纤维连接蛋白或玻璃体连接蛋白（1μg/ml）可显著促进细胞对培养皿表面的附着。同时添加菌毛明显抑制细胞附着，且呈剂量依赖性，最高剂量为30μg/ml的菌毛达到完全抑制。研究发现，富含精氨酸的碱性肽蛋白蛋白（从鲑鱼精子DNA中提取的盐胺和从鲱鱼精子中提取的克氏蛋白）可抑制牙龈卟啉单胞菌精氨酸特异性半胱氨酸蛋白酶（rc -蛋白酶）的蛋白水解活性。Lineweaver-Burk图分析显示，蛋白蛋白竞争性地抑制了rc蛋白酶合成底物苯甲酰- l-精氨酸对硝基苯胺的水解活性。此外，蛋白蛋白能够与牙龈假单胞菌的菌毛强结合，并抑制菌毛与固定纤维连接蛋白的相互作用。这些结果清楚地表明，蛋白蛋白是一种有效的抑制牙龈假单胞菌蛋白水解和粘附活性的抑制剂。少

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 … More 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