Analysis of structure and function of saliva protein receptor domains for periodontopathogen

本研究的目的是确定唾液蛋白受体与牙龈卟啉单胞菌特异相互作用的氨基酸残基，并分析受体的功能。富含脯氨酸的蛋白(PRP)被蛋白降解后，用~&lt；125&gt；I标记的fimbrins和包被PRP的羟基磷灰石小球(HAP)，通过酶联免疫吸附试验和结合抑制实验，检测PRP片段与重组fimbrin的相互作用。与显示结合活性的片段相对应的类似肽被合成并用于确定结合结构域。表位定位实验表明，Pro-Gln-Gly-Pro-Pro-Gln是与菌毛结合的最小活性片段。合成的代表他瑟素类似物的多肽被用来定位他汀类药物的结合域。通过去掉C端和N端的单个氨基酸残基，合成了与苯布林具有结合活性的连续多肽。结合抑制实验表明，Leu-29-Tyr-30和Tyr-41-Thr-42-Phe-43是介导他汀类药物与牙龈假单胞菌结合的重要结合区。从腮腺唾液中提纯的富含脯氨酸的糖蛋白(PRG)也能与菌毛结合。与PRP结合区类似的多肽显著抑制菌毛与PRG包被的HAP的结合，而与他汀类结合区类似的多肽对菌毛与PRG的结合没有影响。这些结果表明，菌毛通过唾液成分的两个不同的结合域，即PRG/PRP和Statherin与唾液结合。

The objects of this study are to determine the amino acid residues of saliva protein receptors that interact specifically with Porphyromonas gingivalis, and to analyze the function of the receptors. After proline-rich protein (PRP) was proteolysed, the interaction of each PRP fragment with recombinant fimbrillin was examined by ELISA and binding inhibition experiment using ^<125>I-labeled fimbrillin and PRP-coated hydroxyapatite beads (HAP). Analogous peptides corresponding to the fragments which showed the binding activity were synthesized and used to determine the binding domain. Epitope mapping experiments showed that peptide Pro-Gln-Gly-Pro-Pro-Gln was minimal active segment for binding to P.gingivalis fimbriae. Synthetic peptides representing statherin analogs were used to localize the binding domains of statherin. Successive peptides were synthesized by deleting individual amino acid residues from the C and N termini of the peptide that showed the binding activity to fimbrillin. The binding inhibition experiments using the peptides indicated that Leu-29-Tyr-30 and Tyr-41-Thr-42-Phe-43 are important binding regions that mediate the binding of statherin to P.gingivalis. It was shown that fimbriae also bound to proline-rich glycoprotein (PRG) purified from parotid saliva. The peptide analogous to the binding region of PRP significantly inhibited the binding of fimbriae to PRG-coated HAP,while the peptide analogous to the binding region of statherin showed no effect on the fimbrial binding to PRG.The similar result is obtained by Overlay assay. These results suggest that fimbriae bind to saliva through the two distinct binding domains of receptory salivary components, PRG/PRP and statherin.