Kataoka K, Amano A, Kuboniwa M, Horie H, Nagata H, Shizukuishi S
Department of Preventive Dentistry, Osaka University Faculty of Dentistry, Suita, Japan.
Infect Immun. 1997 Aug;65(8):3159-64. doi: 10.1128/iai.65.8.3159-3164.1997.
Porphyromonas gingivalis fimbriae specifically bind salivary acidic proline-rich protein 1 (PRP1) through protein-protein interactions. The binding domains of fimbrillin (a subunit of fimbriae) for PRP1 were analyzed previously (A. Amano, A. Sharma, J.-Y. Lee, H. T. Sojar, P. A. Raj, and R. J. Genco, Infect. Immun. 64:1631-1637, 1996). In this study, we investigated the sites of binding of the PRP1 molecules to the fimbriae. PRP1 (amino acid residues 1 to 150) was proteolysed to three fragments (residues 1 to 74 [fragment 1-74], 75 to 129, and 130 to 150). 125I-labeled fimbriae clearly bound fragments 75-129 and 130-150, immobilized on a polyvinylidene difluoride membrane; both fragments also inhibited whole-cell binding to PRP1-coated hydroxyapatite (HAP) beads by 50 and 83%, respectively. However, the N-terminal fragment failed to show any effect. Analogous peptides corresponding to residues 75 to 89, 90 to 106, 107 to 120, 121 to 129, and 130 to 150 of PRP1 were synthesized. The fimbriae significantly bound peptide 130-150, immobilized on 96-well plates, and the peptide also inhibited binding of 125I-labeled fimbriae to PRP1-coated HAP beads by almost 100%. Peptides 75-89, 90-106, and 121-129, immobilized on plates, showed considerable ability to bind fimbriae. For further analysis of active sites in residues 130 to 150, synthetic peptides corresponding to residues 130 to 137, 138 to 145, and 146 to 150 were prepared. Peptide 138-145 (GRPQGPPQ) inhibited fimbrial binding to PRP1-coated HAP beads by 97%. This amino acid sequence was shared in the alignment of residues 75 to 89, 90 to 106, and 107 to 120. Six synthetic peptides were prepared by serial deletions of individual residues from the N and C termini of peptide GRPQGPPQ. Peptide PQGPPQ was as inhibitory as peptide GRPQGPPQ. Further deletions of the dipeptide Pro-Gln from the N and C termini of peptide PQGPPQ resulted in significant loss of the inhibitory effect. These results strongly suggest that PQGPPQ is the minimal active segment for binding to P. gingivalis fimbriae and that the moiety of the Pro-Gln dipeptide plays a critical role in expressing binding ability.
牙龈卟啉单胞菌菌毛通过蛋白质 - 蛋白质相互作用特异性结合唾液酸性富含脯氨酸蛋白1（PRP1）。先前已分析了菌毛蛋白（菌毛的一个亚基）与PRP1的结合结构域（A. 天野、A. 夏尔马、J.-Y. 李、H.T. 索贾尔、P.A. 拉杰和R.J. 根科，《感染与免疫》64:1631 - 1637，1996年）。在本研究中，我们调查了PRP1分子与菌毛的结合位点。PRP1（氨基酸残基1至150）被蛋白酶解为三个片段（残基1至74 [片段1 - 74]、75至129以及130至150）。固定在聚偏二氟乙烯膜上的125I标记菌毛能与片段75 - 129和130 - 150明显结合；这两个片段还分别使全细胞与PRP1包被的羟基磷灰石（HAP）珠的结合抑制了50%和83%。然而，N端片段未显示出任何作用。合成了与PRP1的残基75至89、90至106、107至120、121至129以及130至150对应的类似肽。固定在96孔板上的菌毛能与肽130 - 150显著结合，并且该肽还使125I标记菌毛与PRP1包被的HAP珠的结合抑制了近100%。固定在板上的肽75 - 89、90 - 106和121 - 129显示出相当强的结合菌毛的能力。为进一步分析残基130至150中的活性位点，制备了与残基130至137、138至145以及146至150对应的合成肽。肽138 - 145（GRPQGPPQ）使菌毛与PRP1包被的HAP珠的结合抑制了97%。该氨基酸序列在残基75至89、90至106以及107至120的比对中存在。通过从肽GRPQGPPQ的N端和C端逐个缺失残基制备了六个合成肽。肽PQGPPQ与肽GRPQGPPQ的抑制作用相同。从肽PQGPPQ的N端和C端进一步缺失二肽Pro - Gln导致抑制作用显著丧失。这些结果有力地表明PQGPPQ是与牙龈卟啉单胞菌菌毛结合的最小活性片段，并且Pro - Gln二肽部分在表达结合能力中起关键作用。
