Identification of the binding domain of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase for Porphyromonas gingivalis major fimbriae.

Porphyromonas gingivalis forms communities with antecedent oral biofilm constituent streptococci. P. gingivalis major fimbriae bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) present on the streptococcal surface, and this interaction plays an important role in P. gingivalis colonization. This study identified the binding domain of Streptococcus oralis GAPDH for P. gingivalis fimbriae. S. oralis recombinant GAPDH (rGAPDH) was digested with lysyl endopeptidase. Cleaved fragments of rGAPDH were applied to a reverse-phase high-pressure liquid chromatograph equipped with a C18 column. Each peak was collected; the binding activity toward P. gingivalis recombinant fimbrillin (rFimA) was analyzed with a biomolecular interaction analysis system. The fragment displaying the strongest binding activity was further digested with various proteinases, after which the binding activity of each fragment was measured. The amino acid sequence of each fragment was determined by direct sequencing, mass spectrometric analysis, and amino acid analysis. Amino acid residues 166 to 183 of S. oralis GAPDH exhibited the strongest binding activity toward rFimA; confocal laser scanning microscopy revealed that the synthetic peptide corresponding to amino acid residues 166 to 183 of S. oralis GAPDH (pep166-183, DNFGVVEGLMTTIHAYTG) inhibits S. oralis-P. gingivalis biofilm formation in a dose-dependent manner. Moreover, pep166-183 inhibited interbacterial biofilm formation by several oral streptococci and P. gingivalis strains with different types of FimA. These results indicate that the binding domain of S. oralis GAPDH for P. gingivalis fimbriae exists within the region encompassing amino acid residues 166 to 183 of GAPDH and that pep166-183 may be a potent inhibitor of P. gingivalis colonization in the oral cavity.

The receptor-Galpha fusion protein as a tool for ligand screening: a model study using a nociceptin receptor-Galphai2 fusion protein.

As a model system to screen endogenous ligands for G(i)-coupled receptors, we have prepared and characterized a fusion protein of nociceptin receptor and alpha subunit of G(i2). We detected nociceptin binding to the fusion protein by measuring stimulation of [(35)S]GTPgammaS binding with an EC(50) of 2.0 nM and a gain of approximately five times. The stimulation by nociceptin of [(35)S]GTPgammaS binding to the fusion protein was clearly observed in the presence of an appropriate concentration of GDP, because the affinity for GDP was decreased in the presence of agonist. Full and partial agonists differed in their effects on apparent the affinity of the fusion protein for GDP: the IC(50) values for GDP to displace 100 pM [(35)S]GTPgammaS were estimated to be 2 micro M, 0.4 micro M, and 0.05 micro M in the presence of full agonist (nociceptin), partial agonist (F/G-NC), and antagonist (NBZH), respectively. We also detected the activity to stimulate [(35)S]GTPgammaS binding to the fusion protein in the brain extract derived from 2-3 g wet weight tissue without false-positive results. The active component was identified as endogenous nociceptin itself. These results indicate that the fusion protein of GPCR and Galpha(i) is useful for screening of endogenous ligands.

Determination of endogenous peptides in the porcine brain: possible construction of peptidome, a fact database for endogenous peptides.

Peptides play crucial roles in many physiological events. However, a database for endogenous peptides has not yet been developed, because the peptides are easily degraded by proteolytic enzymes during extraction and purification. In this study, we demonstrated that the data for endogenous peptides could be collected by minimizing the proteolytic degradation. We separated porcine brain peptides into 5250 fractions by 2-dimensional chromatography (first ion-exchange and second reversed-phase high-performance liquid chromatography), and 75 fractions of average peptide contents were analyzed in detail by mass spectrometers and a protein sequencer. Based on the analysis data obtained in this study, more than 10000 peptides were deduced to be detected, and more than 1000 peptides to be identified starting from 2 g of brain tissue. Thus, we deduce that it is possible to construct a database for endogenous peptides starting from a gram level of tissue by using 2-dimensional high-performance liquid chromatography coupled with a mass spectrometer.

Oligomerization process of the hemolytic lectin CEL-III purified from a sea cucumber, Cucumaria echinata.

CEL-III is a Ca(2+)-dependent lectin purified from a sea cucumber, Cucumaria echinata. This protein exhibits strong hemolytic activity as well as cytotoxicity toward some cultured cell lines. Hemolysis is caused by CEL-III oligomers formed in the cell membrane after binding to specific carbohydrate chains on the cell surface. We have found that the oligomerization of CEL-III is also induced by the binding of simple carbohydrates, such as lactose, in aqueous solution under high pH and high ionic strength conditions. From gel filtration analysis of the oligomerization of CEL-III, it was found that the formation of the CEL-III oligomer is effectively induced by the binding of lactose and lactulose, disaccharides containing a beta-galactoside structure. Electron micrographs of the resulting oligomers revealed them to exist as particles with a size of approximately 20-30 nm. The oligomerization process required more than 1 h, which is consistent with the increase in surface hydrophobicity as measured using a fluorescent probe, 8-anilinonaphthalene-1-sulfonate. However, a change in the far-UV CD spectra as well as small-angle X-ray scattering occurred within a few minutes, suggesting that a structural change in the protein takes place rapidly, but the following growth of the oligomer is a much slower process.