BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 249, 38–43 (1998) ARTICLE NO. RC988958 Hemoglobin-Binding Protein Purified from Porphyromonas gingivalis Is Identical to Lysine-Specific Cysteine Proteinase (Lys-Gingipain) Masae Kuboniwa,* Atsuo Amano,† ,1 and Satoshi Shizukuishi* *Department of Preventive Dentistry and †Division of Special Care Dentistry, Osaka University Faculty of Dentistry, Suita, Osaka, Japan Received June 8, 1998 developed specific iron acquisition systems to counter- The functional protein that binds to human hemo- act nutritional deprivation by controlling iron avail- globin (hemoglobin-binding protein; HBP) was puri- ability in the host. Many aerobic and facultative anaer- fied from Porphyromonas gingivalis cells. The analyses obic bacteria produce siderophores, low-molecular- of the amino-terminal sequence and amino acid com- mass iron chelators, that remove iron complexed to position revealed that HBP is identical to lysine-spe- host iron-carrying proteins and deliver it to bacterial cific cysteine proteinase (51 kDa Lys-gingipain; KGP) cells. Some bacterial genera that are adapted to hu- of P. gingivalis 381. It is a novel finding that KGP has mans can use heme, hemoglobin, transferrin, lactofer- binding affinity to hemoglobin. The binding activity of rin, and hemopexin iron directly without the intercon- HBP was enhanced by acidic or anaerobic conditions. nection of siderophores (2, 3, 4). Arg-gingipain, a member of the gingipain family, of P. Porphyromonas gingivalis is a gram-negative black- gingivalis exhibited no ability to bind to hemoglobin. pigmented anaerobe considered to be a putative peri- The recombinant protein of KGP (r-KGP) generated in odontopathogen (5). The availability of iron in gingival Escherichia coli showed both hemoglobin-binding and proteolytic activities. The treatment of r-KGP by pro- crevicular fluid would be crucial for the growth and tein disulfide isomerase effectively enhanced binding virulence of this organism, which produces no sidero- to hemoglobin, whereas the proteinase activity was de- phores (6). P. gingivalis can utilize hemin as an iron creased. The treated r-KGP significantly inhibited the source and also seems to store hemin on its cell surface, binding of hemoglobin to the whole cell extracts in a which causes the black pigmentation of its colonies (7, dose-dependent manner. These results suggest that 8). Hemin has been shown to regulate many major bac- the hemoglobin binding of P. gingivalis is mediated by terial components (6) including a 26 kDa outer mem- KGP through active domain(s) distinct from that for brane protein suspected to bind and transport hemin proteinase activity.  1998 Academic Press into the cell (9, 10, 11, 12). P. gingivalis can utilize other iron sources such as lactoferrin, transferrin and hemoglobin, which are known constituents of crevicu- lar fluid and probably support the growth of several In a host, iron is extracellularly bound to transferrin periodontopathogens in vivo (15, 16). The iron uptake and lactoferrin, and the majority of iron is intracellu- system from hemoglobin has been shown to be distinct larly contained within ferritin, hemosiderin and he- from that of hemin acquisition (17). Whole cells, outer min-containing compounds such as hemoglobin and membrane components and the lipopolysaccharide of myoglobin. Free iron is kept at an extremely low level, P. gingivalis have been reported to bind hemoglobin far below that needed for bacterial growth, resulting and hemin (12, 18, 19). It was recently reported that in the limitation of bacterial infection (1). Since iron is the 19 kDa protein isolated from the P. gingivalis enve- an essential nutrient for most bacteria, pathogens have lope expressed binding ability to human hemoglobin (20). The gene corresponding to the 19 kDa protein (HGP15) has been reported (21). However, it is still 1 Address correspondence to Atsuo Amano, D.D.S., Ph.D., Division unclear what components are involved in the iron up- of Special Care Dentistry, Osaka University Faculty of Dentistry, take system from hemoglobin. 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. Fax: /81-6-879- 2284. E-mail: amanoa@dent.osaka-u.ac.jp. In this study, we isolated and purified a novel hemo- 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 38