title: Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide.
abstract: Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.
authors: Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Sakamoto 1-7-1, Nagasaki 852-8588, Japan.
authors: Eiko
authors: E
authors: Sakai
authors: Mariko
authors: M
authors: Naito
authors: Keiko
authors: K
authors: Sato
authors: Hitoshi
authors: H
authors: Hotokezaka
authors: Tomoko
authors: T
authors: Kadowaki
authors: Arihide
authors: A
authors: Kamaguchi
authors: Kenji
authors: K
authors: Yamamoto
authors: Kuniaki
authors: K
authors: Okamoto
authors: Koji
authors: K
authors: Nakayama
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000187:drug effects; D000080867:Gingipain Cysteine Endopeptidases; D006021:Glycophorins / Q000378:metabolism; D006384:Hemagglutination / Q000187:drug effects; D006386:Hemagglutination Tests; D006388:Hemagglutinins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D020349:Surface Plasmon Resonance
chemical_list: D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006021:Glycophorins; D006388:Hemagglutinins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases