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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

Division of Oral Pathopharmacology, Department of Developmental and Reconstructive Medicine,¹ Division of Microbiology and Oral Infection, Department of Molecular Microbiology and Immunology,² Division of Orthodontics and Dentofacial Orthopedics, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan,³ Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan,⁴ Department of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, Sapporo, Japan⁵

Received 2 November 2006/ Accepted 13 March 2007

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.

Published ahead of print on 23 March 2007.