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

抗菌和粘附抑制碱性肽和鱼精蛋白预防边缘性牙周炎的策略。

• 批准号: 11557132
• 负责人: HAMADA Shigeyuki
• 金额: $ 8.38万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557132/
• 关键词: Porphyromonas gingivalis; fimbriae; marginal periodontitis; BIACORE; Binding; Integrin; Vitronectin; Protemines; BIAcore

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

牙龈卟啉单胞菌的菌毛被认为在牙龈卟啉单胞菌在牙周组织的定植和侵袭中起重要作用。在这项研究中，我们分析了牙龈卟啉单胞菌菌毛与人血红蛋白、纤维蛋白原、唾液组分（即，富含脯氨酸的蛋白质[PRP]、富含脯氨酸的糖蛋白[PRG]和唾液酸脱氢酶）和细胞外基质蛋白质（层粘连蛋白、弹性蛋白、纤连蛋白、I型胶原、血小板反应蛋白和玻连蛋白）。BIAcore图谱表明，菌毛可以特异性结合所有检测的蛋白质，具有显著的缔合常数[Ka]。玻连蛋白对菌毛的亲和力最高[Ka=3.79×10^6（M^<-1>）]。一种合成肽，这是一个有效的抑制剂，以菌毛结合唾液蛋白是不显着有效的菌毛与所有的宿主蛋白质的相互作用。这些结果表明 ...更多信息 菌毛和ECM蛋白之间的相互作用以特异性亲和力发生，所述亲和力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连蛋白和纤连蛋白及其受体αvβ3和α5β1整合素之间相互作用的影响。通过将编码αv β 3和α 5 β1整合素亚基的cDNA连接到人pcDNA3.1载体上，转染CHO细胞，建立α v β 3或α 5 β1过表达细胞系。与CHOαvβ3和CHOα5β1结合的牙龈卟啉单胞菌数量分别是与CHO细胞结合的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中为230%，在CHOα5β1中为140%。BIACORE分析表明，菌毛可显著抑制纤维连接蛋白/玻连蛋白和CHOα5β1/CHOαvβ3之间的分子相互作用。进一步评估了菌毛对ECM/整合素相关细胞功能的影响。将CHOα5β1和CHOαvβ3在去血清培养基中孵育，以减少其在聚苯乙烯培养皿上的附着。随后加入纤连蛋白或玻连蛋白（1μg/ml）显著促进细胞在培养皿表面的附着。同时加入菌毛可明显抑制细胞粘附，且呈剂量依赖性，最高剂量为30μg/ml时可完全抑制细胞粘附。发现鱼精蛋白（从鲑鱼的精子DNA制备的鲑鱼精蛋白和从鲱鱼精制备的鲱鱼精蛋白）是富含精氨酸的碱性肽，其抑制来自牙龈卟啉单胞菌的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作图分析表明，鱼精蛋白竞争性抑制RC蛋白酶的底物苯甲酰-L-精氨酸对硝基苯胺的水解活性。此外，鱼精蛋白能够与牙龈卟啉单胞菌菌毛强烈结合，并抑制菌毛与固定化纤连蛋白的相互作用。这些结果清楚地表明，鱼精蛋白是牙龈卟啉单胞菌的蛋白水解和粘附活性的有效抑制剂。少

项目成果

Nakagawa,I., et al.: "Distribution and molecular characterization of Porphyromonas gingivalis carrying a new type of fimA gene"Journal of Clinical Microbiology. 38. 1909-1914 (2000)

Nakakawa,I., et al.：“携带新型 fimA 基因的牙龈卟啉单胞菌的分布和分子特征”临床微生物学杂志。

Nakamura, T. et al.: "Specific interaction between Porphyromonas gingivalis fimbriae and human extracellular matric proteins"FEMS Microbiology Letters. 175・2. 267-272 (1999)

Nakamura，T.等人：“牙龈卟啉单胞菌菌毛和人类细胞外基质蛋白之间的特异性相互作用”FEMS微生物学快报175·2（1999）。

Amano, A. et al.: "Distribution of Porphyromonas gingivalis strains with fimA genetypes in periodontitis patients"Journal of Clinical Microbiology. 37・5. 1426-1430 (1999)

Amano, A. 等：“牙周炎患者中具有 fimA 基因型的牙龈卟啉单胞菌菌株的分布”临床微生物学杂志 37·5（1999）。

Amano,A., et al.: "Prevalence of specific genotypes of Porphyromonas gingivalis fimA and periodontal health status"Journal of Dental Research. 79. 1664-1668 (2000)

Amano,A., et al.：“牙龈卟啉单胞菌 fimA 特定基因型的患病率和牙周健康状况”牙科研究杂志。

Amano A, Nakamura T, Kimura S, Morisaki I, Nakagawa I, Kawabata S, Hamada S: "Molecular interactions of Porphyromonas gingivalis fimbriae with host proteins : kinetic analyses based on surface plasmon resonance."Infection and Immunity. 67(5). 2399-2405 (1

Amano A、Nakamura T、Kimura S、Morisaki I、Nakakawa I、Kawabata S、Hamada S：“牙龈卟啉单胞菌菌毛与宿主蛋白的分子相互作用：基于表面等离子体共振的动力学分析。”感染和免疫。