ACTIVE-SITES OF SALIVARY PROLINE-RICH PROTEIN FOR BINDING TO PORPHYROMONAS-GINGIVALIS FIMBRIAE

Porphyromonas gingivalis fimbriae specifically bind salivary acidic pr oline-rich protein 1 (PRP1) through protein-protein interactions, The binding domains of fimbrillin (a subunit of fimbriae) for PRP1 were an alysed previously (A. Amano, A. Sharma, J.-Y. Lee, H. T. Sojar, P. A. Raj, and R. J. Genco, Infect. Immun. 63:1631-1637, 1996), In this stud y, we investigated the sites of binding of the PRP1 molecules to the f imbriae, 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 ). I-125-labeled fimbriae clearly bound fragments 75-129 and 130-150, immobilized on a polyvinylidene difluoride membrane; both fragments al so inhibited whole-cell binding to PRP1-coated hydroxyapatite (HAP) be ads by 50 and 83%, respectively, However, the N-terminal fragment fail ed 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, immobi lized on 96-well plates, and the peptide also inhibited binding of I-1 25-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 res idues 130 to 150, synthetic peptides corresponding to residues 130 to 137, 138 to 145, and 146 to 150 were prepared. Peptide 138-145 (GRPQGP PQ) inhibited fimbrial binding to PRP1-coated HAP beads by 97%, This a mino acid sequence was shared in the alignment of residues 75 to 89, 9 0 to 106, and 107 to 120. Six synthetic peptides were prepared by seri al deletions of individual residues from the N and C termini of peptid e GRPQGPPQ, Peptide PQGPPQ was as inhibitory as peptide GRPQGPPQ, Furt her deletions of the dipeptide Pro-Gin from the N and C termini of pep tide PQGPPQ resulted in significant loss of the inhibitory effect, The se results strongly suggest that PQGPPQ is the minimal active segment for binding to P. gingivalis fimbriae and that the moiety of the Pro-G in dipeptide plays a critical role in expressing binding ability.