Amano A, Sharma A, Lee J Y, Sojar H T, Raj P A, Genco R J
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo 14214, USA.
Infect Immun. 1996 May;64(5):1631-7. doi: 10.1128/iai.64.5.1631-1637.1996.
Fimbriae (the oligomeric form of fimbrillin) are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. In the present study, we have identified the structural domains of P. gingivalis fimbrillin that mediate the binding to salivary proline-rich protein 1 (PRP1) and statherin. A series of synthetic fimbrillin peptides were used to localize the active fimbrillin domains involved in the binding to PRP1 and statherin. The binding of 125I-labeled 41-r-Fim (whole-length recombinant fimbrillin, amino acid [aa] residues 1 to 337) to PRP1-coated hydroxyapatite beads (HAP) was strongly inhibited by the fimbrillin C-terminal peptides corresponding to aa residues 266 to 286 and 318 to 337 (peptides 266-286, and 318-337, respectively), while the binding to statherin was inhibited by C-terminal peptides 266-286, 293-306 and 307-326. Peptide 126-146 also showed a weak inhibitory effect, about half that of other active peptides, on the binding to both PRP1 and statherin. P. gingivalis whole-cell binding to PRP1- or statherin-coated HAP was inhibited by more than 80% by the same active peptides. To confirm that the C-terminal portion of fimbrillin includes domains responsible for the binding, two C-terminally truncated variants of recombinant fimbrillin were generated and purified. These were designated 34.5-r-Fim, corresponding to aa residues 1 to 286, and 32-r-Fim, corresponding to aa residues 1 to 265. 125I-34.5-r-Fim revealed 35 and 34% loss of binding ability to PRP1 and statherin, respectively. 125I-32-r-Fim had significantly less binding ability to PRP1 and statherin than 125I-34.5-r-Fim, which was reduced 78 and 73%, respectively. Whole-cell binding to PRP1-, statherin-, or whole saliva-coated HAP was inhibited up to 100% by 41-r-Fim, while 32-r-Fim also showed considerable inhibition, possibly due to the region of aa 126 to 146. Collectively, these results suggest that there are separate and multiple binding sites for PRP1 and statherin in the P. gingivalis fimbrillin, and the combination of all of these binding sites may be indispensable in establishing stable bacterial adherence to saliva-coated surfaces in the oral cavity.
菌毛（菌毛蛋白的寡聚形式）被认为在牙龈卟啉单胞菌在口腔中的黏附和定植过程中起重要作用。在本研究中，我们确定了牙龈卟啉单胞菌菌毛蛋白中介导与富含脯氨酸的唾液蛋白1（PRP1）和statherin结合的结构域。使用一系列合成的菌毛蛋白肽来定位参与与PRP1和statherin结合的活性菌毛蛋白结构域。125I标记的41-r-Fim（全长重组菌毛蛋白，氨基酸[aa]残基1至337）与包被PRP1的羟基磷灰石珠（HAP）的结合受到对应于aa残基266至286和318至337的菌毛蛋白C末端肽（分别为肽266-286和318-337）的强烈抑制，而与statherin的结合则受到C末端肽266-286、293-306和307-326的抑制。肽126-146对与PRP1和statherin的结合也显示出较弱的抑制作用，约为其他活性肽的一半。牙龈卟啉单胞菌全细胞与包被PRP1或statherin的HAP的结合受到相同活性肽的抑制达80%以上。为了证实菌毛蛋白的C末端部分包含负责结合的结构域，生成并纯化了两种重组菌毛蛋白的C末端截短变体。这些被命名为34.5-r-Fim，对应于aa残基1至286，和32-r-Fim，对应于aa残基1至26
