Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein

Abstract

There are many controversies surrounding the biological activities of native C-reactive protein (nCRP) and its various modified forms such as monomerized and biotinylated CRP (mCRP and bCRP). No simple methods have been described to distinguish among these forms. By adapting established electrophoresis methods, we have developed a useful quality control method with which we have investigated the structural and functional characteristics of these forms of CRP. Under all electrophoresis conditions, biotinylation altered the electrophoretic mobility of CRP. nCRP was sensitive to sodium dodecyl sulphate (SDS)-induced monomerization, and only mCRP was susceptible to digestion by trypsin or neutrophil-derived serine proteases. bCRP and mCRP but not nCRP bound to cells, suggesting that chemical modification by biotin and denaturation had altered the structural integrity of CRP. Neither nCRP nor mCRP had the ability to induce secretion of chemokines, nor did they increase intracellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

Figure 1

Analysis of native, biotinylated and urea/ethylenediaminetetraacetic acid (EDTA)-chelated C-reactive protein (CRP) under different electrophoretic conditions. Native CRP (N), biotinylated CRP (B) and monomerized CRP (M), mixed with sample buffer, and with or without heating or reduction, as indicated, were subjected to polyacrylamide electrophoresis (PAGE) [with or without sodium dodecyl sulphate (SDS) (standard SDS PAGE conditions) or with 1/20th SDS (1/20 of standard SDS) as indicated]. Gels were stained with Coomassie brilliant blue. (a) Not heated, + SDS; (b) heated, + SDS; (c) not heated, 1/20th SDS; (d) not heated, no SDS; (e) heated, reduced, + SDS. Results are representative of at least three experiments.

Figure 3

Susceptibility of C-reactive protein (CRP) forms to enzymatic digestion. Native CRP (a) and monomerized CRP (b; generated by heat treatment) at 50 µg/ml were incubated for 1 hr at 37° with different trypsin:CRP molar ratios (as indicated) in the presence of 2 mm CaCl₂. (c) Native CRP (N), biotinylated CRP (B) and monomerized CRP (M; generated by heat treatment) were incubated with freeze/thaw (F) or phorbol myristic acid (PMA) (P)-activated neutrophil supernatants, or buffer (C) for 18 hr at 37°. (d) Monomerized CRP, generated by heat treatment, was incubated with the supernatant of PMA-stimulated neutrophils in the presence or absence of various inhibitors for 18 hr at 37°. Lane 1, monomerized CRP control incubated in the absence of enzymes. Other lanes, neutrophil supernatants plus: (lane 2) buffer control; (lane 3) 10 mm ethylenediaminetetraacetic acid (EDTA); (lane 4) 10 µm 1,10-phenanthroline; (lane 5) 10 µm phenylmethylsulphonyl fluoride (PMSF); (lane 6) 1·2 mm 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride (AEBSF); (lane 7) 10 µm E64; (lane 8) 50 µg/ml elastase inhibitor II. Proteins were analysed on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) (without heating) followed by western blot analysis using a monoclonal (a, b) or a polyclonal (c, d) anti-CRP antibody. Results are representative of at least three experiments.

Figure 2

Heat stability of C-reactive protein (CRP). Native CRP (100 µg/ml) was incubated at different temperatures in a polymerase chain reaction (PCR) machine with a peltier heating block for 1 hr (a) or at 70° for various periods of time (b), mixed with sample buffer and, without heating, run on polyacrylamide electrophoresis (PAGE) gels containing 1/20th sodium dodecyl sulphate (SDS). Gels were stained with Coomassie brilliant blue. Results are representative of at least three experiments.

Figure 4

Comparison of native, biotinylated and monomeric C-reactive protein (CRP) binding to human umbilical cord endothelial cells (HUVEC). HUVEC were incubated with different forms of CRP (100 µg/ml): (a) native CRP; (b) monomeric CRP [generated by urea/ethylenediaminetetraacetic acid (EDTA) chelation]; (c) and (d) biotinylated CRP. Cells were washed and analysed by flow cytometry for the binding of CRP using a monoclonal anti-CRP antibody and goat anti-mouse immunoglobulin fluorescein isothiocyanate (GAM-FITC) (a–c) or Strep-Al488 (d). Results are expressed as fluorescence histograms, with thin, medium and thick lines representing background fluorescence, buffer control and cells incubated with CRP, respectively. Results are representative of at least three experiments carried out in triplicate.

Figure 5

Comparison of the effects of C-reactive protein (CRP) from different sources on human umbilical cord endothelial cell (HUVEC) activation. HUVEC were incubated with CRP buffer control, 10 ng/ml tumour necrosis factor (TNF)-α, or 100 µg/ml of native CRP (nCRP), monomerized CRP (mCRP) [urea/ethylenediaminetetraacetic acid (EDTA)-chelated], Calbiochem recombinant CRP (Cal) or dialysed Calbiochem CRP (DCal). After 24 hr, cell culture media were analysed for interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 content by enzyme-linked immunosorbent assay (ELISA) and HUVEC were analysed for intracellular adhesion molecule (ICAM)-1 expression by flow cytometry. Results are expressed as mean IL-8 or MCP-1 concentrations or median fluorescence intensities (MFI) ± standard error of the mean for experiments carried out in triplicate. Results are representative of at least three experiments. Data points marked with asterisks were significantly different from the control treatment (P < 0·001).