Amano A, Shizukuishi S, Horie H, Kimura S, Morisaki I, Hamada S
Division of Special Care Dentistry, Osaka University Faculty of Dentistry, Suita, Japan.
Infect Immun. 1998 May;66(5):2072-7. doi: 10.1128/IAI.66.5.2072-2077.1998.
Porphyromonas gingivalis, a putative periodontopathogen, can bind to human saliva through its fimbriae. We previously found that salivary components from the submandibular and sublingual glands bind to P. gingivalis fimbriae and that acidic proline-rich protein (PRP) and statherin function as receptor molecules for fimbriae. In this study, we investigated the fimbria-binding components in parotid saliva. Fractionated human parotid saliva by gel-filtration chromatography was immobilized onto nitrocellulose membranes for the overlay assay following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The salivary components on the membrane were allowed to interact with fimbriae purified from P. gingivalis ATCC 33277, and the interacted fimbriae were probed with anti-fimbria antibodies. The fimbriae were shown to bind to two forms of proline-rich glycoproteins (PRGs) as well as to acidic PRPs and statherin. Moreover, fimbriae bound to several components of smaller molecular size which appeared to be acidic PRP variants and basic PRPs. Fimbriae bound strongly to the purified PRGs adsorbed onto hydroxyapatite (HAP) beads. In contrast, PRGs in solution failed to inhibit the fimbrial binding to the immobilized PRGs on the HAP beads. These findings suggest that the appearance of binding site(s) of PRGs can be ascribed to their conformational changes. We previously identified the distinct segments within PRP and statherin molecules that are involved in fimbrial binding. The peptides analogous to the binding regions of PRP and statherin (i.e., PRP-C and STN-C) markedly inhibit the binding of fimbriae to PRP and statherin immobilized on the HAP beads, respectively. The PRP-C significantly inhibited the binding of fimbriae to PRG-coated HAP beads as well as to PRP on HAP beads. The peptide did not affect the binding of fimbriae to statherin, whereas the STN-C showed no effect on the fimbrial binding to PRPs or PRGs. In the overlay assay, the PRP-C clearly diminished the interactions between the fimbriae and the various salivary components, including PRPs, the PRGs, and the components with smaller molecular sizes but not statherin. These results strongly suggest that fimbriae bind to salivary components (except statherin) via common peptide segments. It is also suggested that fimbriae bind to saliva through the two distinct binding domains of receptory salivary components: (i) PRGs and PRPs and (ii) statherin.
牙龈卟啉单胞菌是一种公认的牙周病原体，可通过其菌毛与人唾液结合。我们之前发现，颌下腺和舌下腺的唾液成分可与牙龈卟啉单胞菌菌毛结合，且富含脯氨酸的酸性蛋白（PRP）和富含组氨酸的糖蛋白（statherin）作为菌毛的受体分子发挥作用。在本研究中，我们调查了腮腺唾液中的菌毛结合成分。通过凝胶过滤色谱法分离的人腮腺唾液在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳后固定在硝酸纤维素膜上用于覆盖分析。使膜上的唾液成分与从牙龈卟啉单胞菌ATCC 33277纯化的菌毛相互作用，并用抗菌毛抗体检测相互作用的菌毛。结果显示，菌毛可与两种形式的富含脯氨酸的糖蛋白（PRG）以及酸性PRP和富含组氨酸的糖蛋白结合。此外，菌毛还与几种较小分子量的成分结合，这些成分似乎是酸性PRP变体和碱性PRP。菌毛与吸附在羟基磷灰石（HAP）珠上的纯化PRG强烈结合。相比之下，溶液中的PRG未能抑制菌毛与HAP珠上固定的PRG的结合。这些发现表明，PRG结合位点的出现可归因于其构象变化。我们之前确定了PRP和富含组氨酸的糖蛋白分子中参与菌毛结合的不同片段。与PRP和富含组氨酸的糖蛋白结合区域类似的肽（即PRP - C和STN - C）分别显著抑制菌毛与固定在HAP珠上的PRP和富含组氨酸的糖蛋白的结合。PRP - C显著抑制菌毛与PRG包被的HAP珠以及HAP珠上的PRP的结合。该肽不影响菌毛与富含组氨酸的糖蛋白的结合，而STN - C对菌毛与PRP或PRG的结合无影响。在覆盖分析中，PRP - C明显减少了菌毛与各种唾液成分之间的相互作用，包括PRP、PRG以及较小分子量的成分，但不包括富含组氨酸的糖蛋白。这些结果强烈表明，菌毛通过共同的肽段与唾液成分（富含组氨酸的糖蛋白除外）结合。还表明，菌毛通过受体唾液成分的两个不同结合域与唾液结合：（i）PRG和PRP以及（ii）富含组氨酸的糖蛋白。
