C-Reactive Protein (CRP) and Leptin Receptor in Obesity: Binding of Monomeric CRP to Leptin Receptor

Abstract

While leptin deficiency or dysfunction leads to morbid obesity, obese subjects are characterized paradoxically by hyperleptinemia indicating lack of response to leptin. C-reactive protein (CRP) has been suggested to be a key plasma protein that could bind to leptin. To examine whether CRP interferes with leptin action, mediated through its cell surface receptor, docking studies of CRP with the extracellular domain of the leptin receptor were done employing bioinformatics tools. Monomeric CRP docked with better Z-rank score and more non-bond interactions than pentameric CRP at the CRH2-FNIII domain proximal to the cell membrane, distinct from the leptin-docking site. Interaction of CRP with leptin receptor was validated by solid phase binding assay and co-immunoprecipitation of CRP and soluble leptin receptor (sOb R) from human plasma. Analysis of the serum levels of leptin, CRP, and sOb R by ELISA showed that CRP levels were significantly elevated (p < 0.0001) in non-morbid obese subjects (n = 42) compared to lean subjects (n = 32) and correlated positively with body mass index (BMI) (r = 0.74, p < 0.0001) and leptin (r = 0.8, p < 0.0001); levels of sOb R were significantly low in obese subjects (p < 0.001) and showed a negative correlation with BMI (r = -0.26, p < 0.05) and leptin (r = -0.23, p < 0.05) indicating a minimal role for sOb R in sequestering leptin.

Keywords: leptin; leptin receptor; monomeric C-reactive protein; obesity; protein–protein docking.

Figure 1

Docking of C-reactive protein (CRP) to leptin receptor. Protein–protein docking was done by employing Z Dock Protocol in Discovery Studio as described in the text. The image of the pose with the best Z-rank score in the largest cluster is shown in (A) leptin receptor, PDB ID 3V6O, (CRH2 domain), (deep blue) docked (blind) with CRP monomer, PDB Id 1GNH, (single chain) (green) and (B) enlarged image of docked amino acid skeleton.

Figure 2

Docking of leptin to leptin receptor. Protein–protein docking was done between leptin and leptin receptor (CRH2, PDB ID 3V6O) as described in legends to Figure 1. The image of the pose with the best Z-rank score in the largest cluster is shown in (A) leptin, PDBID-1AX8, (pink) docked (blind) with leptin receptor, PDB ID 3V6O, (CRH2 domain) (deep blue) and (B) enlarged image of docked amino acid skeleton.

Figure 3

Docking of C-reactive protein (CRP) to modeled leptin receptor. Protein–protein docking was done between modeled leptin receptor (CRH2–FNIII) and CRP (monomer) (PDB Id 1GNH) as described in Figure 1. The image of the pose with the best Z-rank score in the largest cluster is shown in (A) leptin receptor modeled [CRH2 (deep blue) –Fn III domains (light blue)] docked with CRP monomer (green) and (B) enlarged image of docked amino acid skeleton.

Figure 4

Docking of C-reactive protein (CRP) to leptin–leptin receptor complex. Leptin (PDB Id 1AX8) –leptin receptor (CRH2–FNIII) complex was prepared, the complex was energy minimized, prepared for further docking as described in Section “Materials and Methods” and docked with CRP (PDB Id 1GNH). Protein–protein docking was done as described in Figure 1. The image of the pose with the best Z-rank score in the largest cluster is shown. (A) Leptin (pink) was docked (site specific) to modeled leptin receptor [CRH2 (deep blue)–Fn III (light blue)], CRP monomer (green). (B) Enlarged image of docked amino acid skeleton.

Figure 5

Solid phase binding assay of leptin receptor to C-reactive protein (CRP). (A) Concentration dependence: different concentrations of monomeric CRP (mCRP) from serum (s-mCRP), recombinant CRP (R-mCRP), native serum CRP (n-CRP), and heat treated (h-mCRP) were coated on to 96-well micro titer plate incubated with leptin receptor (100 ng of Ob R) and the amount of Ob R bound was quantitated by ELISA using anti-Ob R antibody and HRP-conjugated secondary antibody as described in text. Values given are the average of 3–6 experiments done in duplicate ± SD. (B) Effect of different substances: mCRP (0.6 µg/ml) was coated on to 96-well micro titer plate and binding of leptin receptor in the presence of different cations (1 µM each of MnCl₂, MgCl₂, and CaCl₂), or different amino acids (1 µM each of His, Tyr, Asp, and Glu) or in buffer pH 5.0 was assayed. Binding of leptin receptor to wells coated with BSA (100 µg/ml), lysozyme (100 µg/ml) native CRP (nCRP), or heat-treated CRP (0.6 µg/ml) was also analyzed. Results are expressed as percent of untreated mCRP control. Each value represents mean ± SD of three experiments.

Figure 6

Co-precipitation of soluble leptin receptor (sOb R) and C-reactive protein (CRP) from serum. (A) Co-precipitation of CRP with sOb R: sOb R was immunoprecipitated from fresh human serum and the amount of CRP present in the immunoprecipitate was assayed by ELISA. Total CRP in serum was also assayed. Scatter plot represents results of multiple experiments in replicates. (mean ± SD of total CRP was 32.18 ± 1.24 and that of co-precipitated CRP was 1.74 ± 0.17).(B) Co-precipitation of soluble leptin receptor with CRP: CRP was immunoprecipitated from freshly isolated human serum and the amount of soluble leptin receptor present in the immunoprecipitate was determined by ELISA. Total soluble leptin receptor in serum was also determined. [mean ± SD of total soluble leptin receptor (sOb R) was 59.6 ± 5.6 and that of co-precipitated sOb R was 0.67 ± 0.14]. (C) Co-precipitation of CRP from serum spiked with different concentrations of soluble leptin receptor. To 50 µl of freshly isolated serum, different concentrations (50, 100 ng) of leptin receptor were added, incubated for 30 min, immunoprecipitated leptin receptor, and estimated the amount of CRP co-precipitated by ELISA as above. Scatter plot represents results of multiple assays and shows an increase in co-precipitated CRP with increase in concentrations of added leptin receptor. (D) Immunoblot analysis of co-precipitated CRP. sOb R was immunoprecipitated from human serum, subjected to 7.5% SDS-PAGE followed by Western blotting, probed with anti-CRP, and located by ECL as described in the text. Lane 1: sOb R co-precipitate. Lane 2: immune-precipitated CRP from sOb R co-precipitate. Lane 3: immunoprecipitated CRP from whole serum. M: molecular weight markers.

Figure 7

Correlation of leptin, C-reactive protein (CRP), and soluble leptin receptor (sOb R) with body mass index (BMI) analyses of correlation of BMI with serum levels of leptin, CRP, and sOb R as well as that between leptin and CRP was done using Spearman coefficient of correlation. A value of p < 0.05 was considered significant. Dark circles represent individual values. (A) BMI and leptin (n = 78, r = 0.79), (B) BMI and CRP (n = 78, r = 0.74), (C) leptin and CRP (n = 77, r = 0.73), and (D) BMI and sOb R (n = 79, r = −0.26).

Figure 8

Co-precipitation of leptin receptor and C-reactive protein (CRP) from serum of obese and lean subjects. (A) Soluble leptin receptor (sOb R) was immunoprecipitated from the serum of lean (n = 6) (body mass index, BMI <23) and obese (n = 5) (BMI >25) male subjects and CRP estimated by ELISA as shown in Figure 3. (B) CRP was immunoprecipitated from the serum of lean and obese male subjects and estimated sOb R by ELISA. Values given are the mean ± SD. p > 0.05 not significant.