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

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 × 10 −7 M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.