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 inter-domain regional peptide

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

^* To whom correspondence should be addressed. Email: knak{at}nagasaki-u.ac.jp.

	Abstract

Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, has the ability to agglutinate human erythrocytes. In general, hemagglutination can be considered as the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of the 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 1083-1102, 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 inter-domain regional amino acid sequence may function as an intra-molecular suppressor of hemagglutinating activity. Analyses with 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 exogenously added, inhibited HGP44_720-1081-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and decrease in HGP44_720-1081-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44_720-1081 could bind to asialoglycophorin at a dissociation constant of 3.0 x 10^-7 M. These results indicate that the target molecule of HGP44 on the erythrocyte membrane appears to be glycophorin A.