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

Eiko Sakai, Mariko Naito, Keiko Sato, Hitoshi Hotokezaka, Tomoko Kadowaki, Arihide Kamaguchi, Kenji Yamamoto, Kuniaki Okamoto, Koji Nakayama

Research output: Contribution to journalArticle

22 Citations (Scopus)

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 HGP44720-1081, a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44720-1138, did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44720-1138 but not in HGP44 720-1081, could bind HGP44720-1081 in a dose-dependent manner and effectively inhibited HGP44720-1081-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 HGP44720-1081-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44720-1081-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44720-1081 could bind to asialoglycophorin with a dissociation constant of 3.0 × 10-7 M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.

Original languageEnglish
Pages (from-to)3977-3986
Number of pages10
JournalJournal of Bacteriology
Volume189
Issue number11
DOIs
Publication statusPublished - Jun 2007

Fingerprint

Porphyromonas gingivalis
Hemagglutination
Hemagglutinins
Glycophorin
Erythrocytes
Peptides
Genes
Erythrocyte Membrane
Proteins
Gram-Negative Anaerobic Bacteria
Chronic Periodontitis
Surface Plasmon Resonance
Heme
Amino Acid Sequence
Peptide Hydrolases
Porphyromonas gingivalis argingipain
Bacteria
Membranes
Growth

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

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. / Sakai, Eiko; Naito, Mariko; Sato, Keiko; Hotokezaka, Hitoshi; Kadowaki, Tomoko; Kamaguchi, Arihide; Yamamoto, Kenji; Okamoto, Kuniaki; Nakayama, Koji.

In: Journal of Bacteriology, Vol. 189, No. 11, 06.2007, p. 3977-3986.

Research output: Contribution to journalArticle

Sakai, Eiko ; Naito, Mariko ; Sato, Keiko ; Hotokezaka, Hitoshi ; Kadowaki, Tomoko ; Kamaguchi, Arihide ; Yamamoto, Kenji ; Okamoto, Kuniaki ; Nakayama, Koji. / 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. In: Journal of Bacteriology. 2007 ; Vol. 189, No. 11. pp. 3977-3986.
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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 HGP44720-1081, a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44720-1138, did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44720-1138 but not in HGP44 720-1081, could bind HGP44720-1081 in a dose-dependent manner and effectively inhibited HGP44720-1081-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 HGP44720-1081-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44720-1081-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44720-1081 could bind to asialoglycophorin with a dissociation constant of 3.0 × 10-7 M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.",
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AU - Naito, Mariko

AU - Sato, Keiko

AU - Hotokezaka, Hitoshi

AU - Kadowaki, Tomoko

AU - Kamaguchi, Arihide

AU - Yamamoto, Kenji

AU - Okamoto, Kuniaki

AU - Nakayama, Koji

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AB - 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 HGP44720-1081, a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44720-1138, did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44720-1138 but not in HGP44 720-1081, could bind HGP44720-1081 in a dose-dependent manner and effectively inhibited HGP44720-1081-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 HGP44720-1081-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44720-1081-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44720-1081 could bind to asialoglycophorin with a dissociation constant of 3.0 × 10-7 M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.

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