Porphyrin-mediated binding to hemoglobin by the HA2 domain of cysteine proteinases (gingipains) and hemagglutinins from the periodontal pathogen Porphyromonas gingivalis

Abstract

Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogen Porphyromonas gingivalis. We therefore examined the potential role of the dominant P. gingivalis cysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd = 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki = 2.5 microM). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd = 2.1 nM) was also inhibited by protoporphyrin IX (Ki = 10 microM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released by P. gingivalis cells in a batch culture, in parallel with proteinase activity. Cysteine proteinases from P. gingivalis appear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition in P. gingivalis may allow precise targeting of this critical metabolic aspect for periodontal disease prevention.

FIG. 1

Domain structure and homologies between the gingipains RGP-1 and KGP. CAT represents the putative catalytic domain. Shaded areas represent regions of >98% amino acid identity between the two gingipains. Each fraction represents the degree of identity for each RGP-1 domain. Approx. kDa, approximate molecular mass in kilodaltons.

FIG. 2

Hemoglobin binding by rHA2, RGP-1, and KGP. (a) Microtiter wells were coated with hemoglobin and then incubated with threefold dilutions of purified rHA2 at 2.5 μg/ml (⧫), RGP-1 at 5 μg/ml (○), or KGP at 5 μg/ml (▴). Association of rHA2 with hemoglobin was measured with MAb 5A1, followed by substrate development after binding of a secondary anti-mouse AP-conjugated antibody. (b) Hemoglobin binding by native, but not denatured, gingipains. Wells were coated with hemoglobin and then incubated overnight with dilutions of either RGP-1 (●), KGP (▴), RGP-1 denatured by boiling (○), or KGP denatured by boiling (▵). For this experiment, native or denatured gingipains that bound to hemoglobin were recognized by MAb IIB2, which specifically detects both native and denatured gingipains. Primary antibody IIB2 was followed by substrate development after binding of a secondary anti-mouse AP-conjugated antibody. These data are representative of three separate experiments. Abs, absorbance.

FIG. 3

Binding of the HA2 domain to the heme moiety. (a) Binding of rHA2 to dilutions of hemin (⧫), hemoglobin (Hgb) (○), or hemoglobin degraded by proteinase K (▴). Microtiter wells were coated with dilutions of samples, and then overnight binding of rHA2 to coated wells was detected with MAb 5A1, followed by substrate development after binding of a secondary anti-mouse AP-conjugated antibody. The absence of contaminating protein within 90 μg of the hemin preparation and the absence of nondegraded subunits of hemoglobin remaining after proteinase K treatment were verified by SDS-PAGE (data not shown). (b) Binding of rHA2 to hemin. Microtiter wells were coated with hemin, and overnight binding of rHA2 dilutions was detected with MAb 5A1 as described above. These data are representative of two separate experiments. Abs, absorbance.

FIG. 4

Inhibition of hemin or hemoglobin binding. Microtiter wells were coated overnight with hemin (a) or hemoglobin (b). rHA2 in E. coli lysate (100-fold dilution) (×), RGP-1 at 65 ng/ml (●) or KGP at 65 ng/ml (▴) was preincubated with dilutions of 300 μM protoporphyrin IX for 1 h and then transferred to the ligand-coated plates for overnight incubation. Binding of rHA2 or the gingipains to coated wells was detected with MAb 5A1 or IIB2, respectively, followed by substrate development after binding of a secondary anti-mouse AP-conjugated antibody. These data are representative of two separate experiments. The absence of contaminating protein in a 90-μg protoporphyrin IX preparation was verified by SDS-PAGE and Coomassie dye binding (data not shown). ABS, absorbance.

FIG. 5

Directed porphyrin binding by rHA2. Microtiter wells were coated with 100 mM ethylene diamine (pH 4.7) and then incubated with hemin, protoporphyrin IX, or hematoporphyrin at 90 μg/ml overnight in 50% dimethyl formamide in the presence (+) or absence (−) of 10 mM carbodiimide. Wells were washed four times with water, and then the amount of porphyrin bound to the wells was determined by measuring absorbance (Abs) at 414 nm (striped bars). Wells were blocked with PBS/Tween and then incubated with rHA2 at 125 ng/ml overnight. Binding of rHA2 to coated wells was detected with MAb 5A1, followed by substrate development after binding of a secondary anti-mouse AP-conjugated antibody (solid bars). Error bars represent standard deviations of absorbance measurements. Diagrams of the chemical structures of hemin, protoporphyrin IX, and hematoporphyrin are presented adjacent to the corresponding data.

FIG. 6

Measurement of high-affinity binding of MAb 5A1 with rHA2, denatured but not native gingipains, and gingipains from the culture supernatant. (a) RGP-1 (●), KGP (▴), or rHA2 in crude E. coli lysate (■) was used to coat microtiter wells and incubated with serial dilutions of MAb 5A1. (b) Dilutions of RGP-1 (○), KGP (▵), or heat-denatured RGP-1 (●) or KGP (▴) were used to coat microtiter wells with threefold dilutions from 10 μg/ml and then incubated with MAb 5A1. (c) Purified rHA2 (■) or purified high-molecular-weight aggregates of gingipain domains isolated from culture supernatant (●) were used to coat microtiter wells and incubated with serial dilutions of MAb 5A1. These data are representative of three separate experiments. Abs, absorbance.

FIG. 7

Immunoreactivity of synthetic peptides with MAb 5A1. ELISA demonstrating selective immunoreactivity of MAb 5A1 with peptide 1. Peptide 1 (■) or 2 (▵) was used to coat microtiter plates at a concentration of 5 μg/ml, incubated overnight, and then incubated with dilutions of MAb 5A1. These data are representative of two separate experiments. Abs, absorbance.

FIG. 8

Expression of HA2-related immunoreactive hemoglobin-binding protein from P. gingivalis. Aliquots of P. gingivalis culture medium were removed daily during a period of 8 days (d), immediately separated into a cell pellet and culture supernatant, and then frozen until use. The OD₆₆₀ and purity of the culture were measured daily. The cell pellets were dispersed evenly into 1 ml of PBS/N₃. (a and b) Arg- and Lys-specific proteinase activities, respectively, of the cell-free culture supernatant (□) and cellular fraction (▵) were measured as described in Materials and Methods. Measurements of the cellular fractions were normalized to the culture densities (OD₆₆₀) recorded daily. (c) The HA2 domain (1/243 dilution; □) and the HA2 domain associated with hemoglobin binding (1/81 dilution; ■) in culture supernatants were measured by ELISA and a ligand-binding assay, respectively, as described in Materials and Methods. In P. gingivalis whole-cell fractions, the HA2 domain (1/243 dilution; ▵) and the HA2 domain associated with hemoglobin binding (1/9 dilution; ▴) were measured by ELISA and a ligand-binding assay, respectively, as described in Materials and Methods. Measurements of the cell-associated fractions were normalized to the culture densities (OD₆₆₀) recorded daily. The corresponding background immunoreactivity with murine anti-human CD-19 immunoglobulin G was subtracted from each measurement. These data are representative of two separate experiments in which the patterns of expression were similar. Abs, absorbance.