How C-Reactive Protein Structural Isoforms With Distinctive Bioactivities Affect Disease Progression

Abstract

C-reactive protein (CRP) is a widely known, hepatically synthesized protein whose blood levels change rapidly and pronouncedly in response to any tissue damaging event associated with an inflammatory response. The synthesis and secretion of CRP is stimulated by interleukin-6, an early pleiotropic cytokine released by macrophages, endothelial, and other cells that are activated when localized normal tissue structures are compromised by trauma or disease. Serum CRP levels can change rapidly and robustly from 10-100-fold within 6-72 h of any tissue damaging event. Elevated blood levels correlate with the onset and extent of both activated inflammation and the acute phase biochemical response to the tissue insult. Because its functional bioactivity as the prototypic acute phase reactant has eluded clear definition for decades, diagnosticians of various conditions and diseases use CRP blood levels as a simple index for ongoing inflammation. In many pathologies, which involves many different tissues, stages of disease, treatments, and responses to treatments, its interpretive diagnostic value requires a deeper understanding of the localized tissue processes and events that contribute signals which regulate protective or pathological host defense bioactivities. This report presents concepts that describe how local tissue activation events can lead to a non-proteolytic, conformational rearrangement of CRP into a unique isoform with distinctive solubility, antigenicity, binding reactivities and bioactivities from that protein widely known and measured in serum. By describing factors that control the expression, tissue localization, half-life and pro-inflammatory amplification activity of this CRP isoform, a unifying explanation for the diagnostic significance of CRP measurement in disease is advanced.

Keywords: CRP - C-reactive protein; MCRP; blood kinetics; conformational isoforms; inflammation; modified/monomeric (mCRP).

Figure 1

Key structural features of serum-soluble pentameric CRP. (A) illustrates the location and orientation of Phosphocholine (PC) binding sites (one per subunit, PC groups shown in gray and involving residues L64, F66, and T76 of each subunit (there are 206 amino acids in each subunit), calcium ions (two per subunit shown in orange, juxtaposed to each PC binding sites and involving residue E147), inter-subunit contact residues (shown in yellow and involving 40YTE42, 115PRVRKSLKK123 AND 197EVFTKP202 and which non-covalently contribute to pCRP pentameric quaternary structure), the one per subunit intrachain disulfide bond (shown in red and covalently linking C36–C97 in each subunit) and the location and orientation of the cholesterol binding residues (shown in blue and involving 35VCLHFYTELSSTR47) in relation to the PC-binding sites and the inter-subunit contact residues (PDB code: 1B09) [residues identified in Shrive et al. (1)]. (B) illustrates the orientation of these same residues when the discoid protein is laid flat (i.e., side view). This view illustrates that all PC binding sites are on one face of the flattened discoid structure and shows how a single helical secondary structure (involving residues 168PDEINTIYL176) is oriented on the top of the opposite face of the discoid protein.

Figure 2

Conversion of pCRP to mCRP induces inflammatory signaling. Monoacyl phosphatidylcholine (aka Lyso-PC or LPC) generated by phospholipase A₂ (PLA₂) in the lipid bilayer, or by oxidation of lipid acyl chains by reactive oxygen species, promotes the binding and dissociation of pentameric CRP (pCRP) to monomeric CRP (mCRP). Formation of mCRP exposes a cholesterol binding sequence such that mCRP enters cholesterol-rich lipid rafts, activating intracellular signaling pathways involving NF-κβ-regulated translation of proteins involved in pro-inflammatory responses.

Figure 3

Temporal depiction of the hypothesized conversion of pCRP into mCRP and the appearance of pCRP in blood after activation or an acute phase response. Schematic representation for the relative amounts of pCRP released from hepatocytes in response to cytokine signals (e.g., IL-6) and its appearance in the blood. (A) Depicts that while pCRP is hepatically secreted within minutes of acute phase response signaling (dotted red line) measured plasma levels of CRP display a lag (i.e., 6–12 h) (blue solid line). Hepatically released CRP can be from both pre-synthesized CRP stored in vesicles, or from de novo synthesis. In the first 12 h. of an inciting stimulus, the pCRP that is released from hepatocytes, but which is not quantified in blood is converted at the site of tissue damage into mCRP (gray shaded area). (B) Depicts a generalized time course for the hepatic secretion of pCRP (red dotted line), the relative amount of pCRP measured in blood (solid blue line as may occur during acute inflammation associated with major tissue damage; dotted blue line as may occur with low grade, chronic inflammation with lesser tissue damage). Plasma concentration of pCRP in healthy individuals (i.e., baseline CRP levels of < 10 μg/ml) is shown as a solid orange line. The shaded yellow area depicts a time period in which pCRP is secreted but rapidly consumed so that it is not quantified in plasma. The consumption of pCRP into mCRP would involve membrane binding to activated cells (e.g., endothelial cells, platelets, leukocytes) and entry into lipid rafts where mCRP activates signaling pathways to stimulate immediate, amplified pro-inflammatory responses to the threat.

Figure 4

Effect of calcium on the apparent molecular weight of pentameric CRP. Sephadex 200 dextran gel filtration chromatography resin was equilibrated in 25 mM Tris-HCl, 0.15 M NaCl buffer (pH 7.4) (TBS) containing 2, 5, or 10 mM Calcium chloride (TBS-Ca), 10 mM EDTA (TBS-EDTA), or 10 mM Citrate (TBS-citrate). Purified pCRP (1 mg) was pre-equilibrated in each column buffer and was chromatographed. Control proteins including IgG (Mw 160,000), Transferrin (Mw 79,500), BSA (Mw 66,500), Chymotrypsin (Mw 24,000) and Lysozyme (Mw 14, 300) were similarly pre-equilibrated and chromatographed. The elution profile for CRP was also assessed using 5 mM CaCl₂ containing 1 mM Phosphocholine hapten (PC) as a specific ligand known to bind each subunit of pCRP as a function of calcium. All standard proteins chromatographed with the same elution volume in each of the buffers studied. The correlation coefficient (R²) of the Mw Vs elution volume standard curve was 0.9766. The apparent Mw of pCRP in 2 mM CaCl₂ was 115,093 (2 mM CaCl2 is a physiological concentration of calcium; this Mw agrees closely with the calculated Mw of pCRP based on its amino acid sequence). The apparent Mw of pCRP reduced to 96,828 in 5 mM CaCl₂ and 86,160 in 10 mM CaCl₂. When either calcium chelator was used, the apparent Mw of pCRP increased to 133, 122. When PC hapten was bound to pCRP in 5 mM CaCl₂ (5 PC sites/pCRP), the pCRP did not appear to contract but assumed a Stoke's radius similar to that observed in when calcium was chelated. These data indicate pCRP will compact or expand from its calculated Mw (115,235) (https://www.ncbi.nlm.nih.gov/protein/NP_001315986.1?report=fasta&from=19, accessed on March 30^th, 2020) as a function of calcium and/or or chelation, and as a function of ligand binding.