The phosphocholine-binding pocket on C-reactive protein is necessary for initial protection of mice against pneumococcal infection

Abstract

Human C-reactive protein (CRP) protects mice from lethal Streptococcus pneumoniae infection when injected into mice within the range of 6 h before to 2 h after the administration of pneumococci. Because CRP binds to phosphocholine-containing substances and subsequently activates the complement system, it has been proposed that the antipneumococcal function of CRP requires the binding of CRP to phosphocholine moieties present in pneumococcal cell wall C-polysaccharide. To test this proposal experimentally, in this study, we utilized a new CRP mutant incapable of binding to phosphocholine. Based on the structure of CRP-phosphocholine complexes, which showed that Phe(66), Thr(76), and Glu(81) formed the phosphocholine-binding pocket, we constructed a CRP mutant F66A/T76Y/E81A in which the pocket was blocked by substituting Tyr for Thr(76). When compared with wild-type CRP, mutant CRP bound more avidly to phosphoethanolamine and could be purified by affinity chromatography using phosphoethanolamine-conjugated Sepharose. Mutant CRP did not bind to phosphocholine, C-polysaccharide, or pneumococci. Mutant CRP was free in the mouse serum, and its rate of clearance in vivo was not faster than that of wild-type CRP. When either 25 μg or 150 μg of CRP was administered into mice, unlike wild-type CRP, mutant CRP did not protect mice from lethal pneumococcal infection. Mice injected with mutant CRP had higher mortality rates than mice that received wild-type CRP. Decreased survival was due to the increased bacteremia in mice treated with mutant CRP. We conclude that the phosphocholine-binding pocket on CRP is necessary for CRP-mediated initial protection of mice against lethal pneumococcal infection.

FIGURE 1.

One subunit of CRP. A, structure of one of the five subunits of WT CRP bound to PCh (Protein Data Bank (PDB) ID 1B09) is shown. The side chains of Phe⁶⁶, Thr⁷⁶ and Glu⁸¹ involved in the formation of the PCh-binding pocket are highlighted. Calcium ions (Ca) are shown as cyan balls. B, molecular modeling of F66A/T76Y/E81A mutant CRP. The PDB coordinates of mutant CRP were generated from the PDB file 1B09 using SYBYL (Tripos, Inc.). The side chains of Phe⁶⁶, Thr⁷⁶ and Glu⁸¹ are substituted with Ala, Tyr, and Ala, respectively. One of the five subunits is shown. The figures were rendered using PyMOL (66).

FIGURE 2.

Overall pentameric structure of CRP. A, elution profiles of WT and mutant CRP from the Superose 12 gel filtration chromatography column are shown. WT CRP (1.0 mg) in TBS containing 2 mm CaCl₂ was applied to the equilibrated column and eluted with the same buffer. Mutant CRP (0.95 mg) in TBS containing 5 mm EDTA (see “Experimental Procedures”) was applied to the equilibrated column and eluted with the same buffer. Sixty fractions (0.25 ml) were collected, and protein was measured (A₂₈₀) to determine the elution volume of CRP from the column. A representative of three experiments is shown. B, denaturing SDS-PAGE (4–20% gel) of CRP (5 μg). A representative gel, stained with Coomassie Brilliant Blue, is shown. C, microtiter wells were coated with anti-CRP mAb HD2.4. The unreacted sites in the wells were blocked with gelatin. Purified CRP diluted in TBS-Ca was then added to the wells. Bound CRP was detected by using rabbit polyclonal anti-CRP antibody and HRP-conjugated donkey anti-rabbit IgG. Color was developed, and the absorbance was read at 405 nm. A representative of three experiments is shown.

FIGURE 3.

PCh-binding site of CRP. A and B, microtiter wells were coated with PCh-BSA (A) and PnC (B). The unreacted sites in the wells were blocked with gelatin. Purified CRP diluted in TBS-Ca was then added to the wells. Bound CRP was detected by using anti-CRP mAb HD2.4 and HRP-conjugated goat anti-mouse IgG. Color was developed, and the absorbance was read at 405 nm. A representative of four experiments is shown. Similar results were obtained when polyclonal anti-CRP antibody was used to detect ligand-bound CRP (data not shown). C, microtiter wells were coated with anti-CRP mAb EA4.1. The unreacted sites in the wells were blocked with gelatin. Purified CRP diluted in TBS-Ca was then added to the wells. Bound CRP was detected by using rabbit polyclonal anti-CRP antibody and HRP-conjugated donkey anti-rabbit IgG. Color was developed, and the absorbance was read at 405 nm. A representative of three experiments is shown.

FIGURE 4.

Binding of CRP to pneumococci. A, microtiter wells were coated with pneumococci. The unreacted sites in the wells were blocked with gelatin. Purified CRP diluted in TBS-Ca was then added to the wells. Bound CRP was detected by using anti-CRP mAb HD2.4 and HRP-conjugated goat anti-mouse IgG. Color was developed, and the absorbance was read at 405 nm. A representative of four experiments is shown. Similar results were obtained when polyclonal anti-CRP antibody was used to detect ligand-bound CRP (data not shown). B, a representative denaturing SDS-PAGE gel (4–20%) stained with Coomassie Brilliant Blue is shown. Lane 1, purified WT CRP (5 μg); lane 2, purified mutant CRP (5 μg); lane 3, pneumococci alone; lane 4, pneumococci after mixing with WT CRP; lane 5, pneumococci after mixing with mutant CRP.

FIGURE 5.

Binding of CRP to PEt. Microtiter wells were coated with avidin in TBS. The unreacted sites in the wells were blocked with gelatin. Biotinylated PEt diluted in TBS was then added to the wells. After washing the wells with TBS, purified CRP diluted in TBS-Ca was added to the wells. Bound CRP was detected by using anti-CRP mAb HD2.4 and HRP-conjugated goat anti-mouse IgG. Color was developed, and the absorbance was read at 405 nm. A representative of four experiments is shown. Similar results were obtained when polyclonal anti-CRP antibody was used to detect PEt-bound CRP (data not shown).

FIGURE 6.

Survival curves of mice infected with S. pneumoniae with or without 25 and 150 μg of either WT or mutant CRP. CRP was injected first; bacteria (5 × 10⁷ cfu) were injected 30 min later. Deaths were recorded three times a day for 7 days. The data are combined from two separate experiments with six mice in each group in both experiments. The p values for the difference in the survival curves between groups A and B, A and C, B and C, A and D, A and E, and D and E are <0.005, 0.79, <0.005, <0.005, 0.31, and <0.005, respectively.

FIGURE 7.

Bacteremia in mice treated with or without 25 and 150 μg of either WT or mutant CRP. Blood samples were collected from each surviving mouse shown in Fig. 6 for the first 5 days after infection. Bacteremia was determined by plating. Each dot represents one mouse. The horizontal line in each group of mice represents the median value of bacteremia in that group. A bacteremia value of >10⁸ indicates a dead mouse. The p values for the difference between groups A and B, A and C, B and C, A and D, A and E, and D and E, on day 1, are 0.011, 0.74, 0.025, <0.005, 0.09, and 0.17, respectively. The p values for the difference between groups A and B, A and C, B and C, A and D, A and E, and D and E, on day 2, are <0.005, 0.72, <0.005, <0.005, 0.39, and 0.02, respectively.

FIGURE 8.

Clearance of CRP from mouse circulation. Mice were injected with 100 μg of CRP in TBS containing 2 mm CaCl₂. Blood was collected at various time points, sera were separated, and the concentration of CRP was measured.

FIGURE 9.

Repurification of mutant CRP from purified mutant CRP-spiked mouse serum. A representative denaturing SDS-PAGE gel (4–20%) stained with Coomassie Brilliant Blue is shown. Lane 1, molecular mass markers; lane 2, purified mutant CRP (5 μg); lane 3, EDTA eluate (25 μl, A₂₈₀ 1.13) from the PEt affinity chromatography column through which mouse serum containing mutant CRP was passed in the presence of CaCl₂; lane 4, EDTA eluate (25 μl, A₂₈₀ 0.29) from the PEt affinity chromatography column through which mouse serum alone was passed in the presence of CaCl₂.