Hiromiki Kuwahara

Identification of the Binding Domain of Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase for Porphyromonas gingivalis Major Fimbriae

ABSTRACT 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.

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.

Small-angle X-ray scattering study on CEL-III, a hemolytic lectin from Holothuroidea Cucumaria echinata, and its oligomer induced by the binding of specific carbohydrate

Hemolytic lectin CEL‐III from a marine invertebrate Cucumaria echinata forms an oligomer upon binding of specific carbohydrate such as lactose at high pH values and in the presence of high concentrations of salt. In this study, using small‐angle X‐ray scattering, we characterized CEL‐III and its oligomer induced by the binding of lactose. The molecular mass of the oligomer was determined as 1019 kDa from its forward scattering value, compared with 47 490 Da for the monomer. This oligomer size is much larger than that estimated using SDS–polyacrylamide gel electrophoresis (SDS‐PAGE, 270 kDa). The monomer has a 24.6 Å radius of gyration and can be approximated by a rod which has a 20 Å radius and a height of 75 Å, while the oligomer has a 101.4Å radius of gyration. Together with the comparison of the radii of gyration and the forward scattering of the cross‐section of the monomer and oligomer, it is suggested that in aqueous solution the oligomer comprises three or four molecules of a smaller unit which was observed by SDS‐PAGE (270 kDa), held by a relatively weak interaction. The scattering profile also suggests that the oligomer has a hole in its central axis which might be associated with the formation of ion‐permeable pores in the erythrocyte membrane by CEL‐III during the hemolytic process.