Structure and Function

There is increasing evidence that C-reactive protein (CRP), an inflammatory protein, may play a role in the inflammation and wound healing processes. During the cutaneous wound healing process a number of cytokines and inflammatory mediators are secreted by inflammatory cells, including TNFα, IL-6 and migration inhibitory factor (MIF), alongside an increased expression of C-reactive protein (Black et al, 2004).

CRP is a homopentameric acute-phase inflammatory protein that exhibits elevated expression during inflammatory conditions such as rheumatoid arthritis, some cardiovascular diseases and infection (Du Clos & Mold, 2004). As an acute phase protein this means that the plasma concentration of the protein increases or decreases by at least 25% during inflammatory disorders (Gabay & Kushner, 1999). The highest concentrations of CRP are found in the serum and it has been shown that during some infections levels can increase up to 1000-fold (Thompson et al, 1999). However when the stimuli ends, values decrease exponentially over 18-20 hours, close to the half-life of CRP (Ridker, 2003). CRP plasma levels have been reported to increase from around 1µg/ml to over 500µg/ml within 24-72 hours of severe tissue damaging effects such as trauma and progressive cancer (Ciubotaru et

al, 2005). IL-6 is considered to be the main inducer of the CRP gene, with IL-1

enhancing the effect (Szalai et al, 1998). However even though IL-6 is necessary for the induction of the CRP gene, it cannot achieve this alone (Weinhold et al, 1997).

CRP is a phylogenetically/highly conserved plasma protein that was initially discovered in 1930 by Tillet and Francis while investigating the sera of patients suffering the acute stage of Pneumococcus infection (Tillet & Francis, 1930). CRP was named for its specificity of calcium binding for phosphocholine (PCh), which is also a part of the activation of the complement pathway of innate immunity (Volankis, 2001). CRP has many homologs in vertebrates and some invertebrates (Black et al, 2006) and is a member of the pentraxin family which also includes

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other structurally related molecules such as serum amyloid A (Gewurz et al, 1982). Transcriptional induction of the CRP gene mainly occurs in the hepatocytes in the liver in response to increased levels of inflammatory cytokines, especially IL-6 (Boras et al, 2014). The role of CRP is that it can activate the classical complement pathway through the recognition of phosphorycholine and activating C1q in the pathway (Schwalbe et al, 1992).

There are many factors that can alter baseline CRP levels including age, gender, smoking status, weight, lipid levels and blood pressure (Hage & Szalai, 2007). The average levels of CRP in the serum in a healthy Caucasian is around 0.8mg/L but this baseline can vary greatly in individuals due to other factors, including polymorphisms in the CRP gene (Devaraj et al, 2006). The human CRP gene can be found at 1q23.2 on the long arm of chromosome 1 and to date there have been no allelic variations or genetic deficiencies discovered for this gene, though some polymorphisms have been identified (Hage & Szalai, 2007). The baseline levels of CRP are regulated by genetic variations in the number of

dinucleotide repeats found in the intronic region of the gene, with the suggestion that up to 50% of baseline variance is due to these variations (Eisenhardt et al, 2009).

There is no significant seasonal variation in base-line CRP concentration; however, twin studies show a significant heritable component in base-line CRP values that is independent of age and body mass index (BMI) (Pepys & Hirschfield, 2003). Pankow et al (2001) found evidence that inter-individual variation in blood CRP levels is 35-40% heritable. Raised CRP levels are typically associated with disease but liver failure is one condition observed to impair CRP production. Very few drugs reduce elevated CRP levels unless they treat the underlying pathology that is causing the acute-phase stimulus (Pepys & Hirschfield, 2003).

10 1.2.2. Isoforms of C-reactive Protein

The pentameric protein, termed native CRP (nCRP) is characterised by a discoid configuration of five identical non-covalently bound subunits, each 206 amino acids long with a molecular mass of about 23kDa (Figure 1.1). These five subunits lie in the same orientation around a central pore and arranged in a characteristic ‘lectin fold’ with a two-layered beta sheet (Eisenhardt et al, 2009). Each subunit lies with the PCh binding site facing the ‘recognition’ face of the nCRP molecule (Boncler & Watala, 2009). The molecule has a ligand binding face which has a characteristic feature of having two calcium ions per protomer. These ions are greatly important for the stability and binding of ligands. The ‘opposite’ face

interacts with the C1q aspect of the complement pathway as well as interacting with Fc receptors (Du Clos & Mold, 2004).

The pentameric protein is synthesised primarily in liver hepatocytes but has also been reported to be synthesised in other cell types such as smooth muscle cells (Calabró et al, 2003), macrophages (Devaraj et al, 2009), endothelial cells (Pasceri

et al, 2000), lymphocytes (Kuta & Baum, 1986) and adipocytes (Calabró et al,

2005). CRP is first synthesised as monomers and then assembled into the pentamer Figure 1.1 – Structure of human pentameric C-reactive protein as viewed down the 5-fold symmetry axis. The effector face is on top with the opposite face where calcium and PCh binding sites underneath (Taken from Shrive et al, 1996)

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in the endoplasmic reticulum of the cell. In hepatocytes, the pentameric protein is retained in the endoplasmic reticulum by binding to two carboxylesterases, gp60a and gp50b (Macintyre et al, 1994). While in a resting state, the CRP is released slowly however in response to an increase in cytokine levels, the binding to these carboxylesterases decreases and secretion time is reduced, thus allowing an influx of CRP to be seen in response to an inflammatory stimulus such as bacterial

infection or injury (Du Clos & Mold, 2004). The stimulation of CRP synthesis mainly occurs in response to pro-inflammatory cytokines, most notably IL-6 and to a lesser degree IL-1 and TNF-α (Zhang et al, 1996).

Pentameric CRP can be irreversibly dissociated, with the resultant free subunits (Figure 1.2) termed monomeric (or modified) CRP (mCRP). The dissociation of nCRP into free subunits has been observed at either high concentrations of urea (Potempa et al, 1987) or high temperatures in the absence of calcium (Potempa et

al, 1983), although the precise process is not fully documented. Taylor and Van den

Berg (2006) found that by increasing the temperature of the nCRP to 90°C allowed for total dissociation of the protein, but near 100% monomerisation can also be achieved at 70°C, with the process starting after 10 minutes and taking nearly an hour to complete. This process was sped up to 5 minutes at 63°C when there was an absence of calcium. Dissociation has also occurred under exposure of nCRP to lysophosphatidylcholine (LPC), a lipid messenger exposed on cell membranes, which is generally found in apoptotic cells or activated platelets (Kim et al., 2002).

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The mCRP molecules are distinguished from nCRP by their different

antigenic, biological and electrophoretic activities (Kresl et al, 1998) and by the fact they express different neoepitopes (Khreiss et al, 2004). The two isoforms of CRP have been shown to have distinct biological functions in the inflammatory process. For example, Khreiss et al (2004) provided evidence that nCRP suppresses

adherence of platelets to neutrophils, whereas mCRP enhances these adhesive interactions. This difference in function can be explained by that the two isoforms having different types of Fcγ-receptor involved in the signalling process; mCRP utilises the low-affinity immune complex binding immunoglobulin G (IgG) FcγRIIIb (CD16b) on neutrophils, FcγRIIIa (CD16a) on monocytes while nCRP binds to the low-affinity IgG receptor FcγRIIa (CD32) (Bharadvaj et al, 1999; Stein et al, 2000; Tebo & Mortensen, 1990).

Evidence shows that nCRP tends to exhibit a more anti-inflammatory function relative to the mCRP isoform. It is suggested that this occurs by the nCRP binding at sites of tissue injury to limit the generation of the membrane attack complex (MAC) and C5a, thus inhibiting complement activation (Thiele et al, 2014). On the other hand, mCRP had marked pro-inflammatory properties in vitro and in

Figure 1.2- Structure of human monomeric C-reactive protein (mCRP). The β- strands are labelled A-N with the positions of key amino acid residues indicated. Also two ligated calcium ions are shown as spheres. (Taken from Shrive et al, 1996)

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vivo by promoting monocyte chemotaxis as well as the recruitment of circulating

leukocytes to areas of inflammation via Fcy-RI and Fcy-RIIa signalling (Thiele et al, 2014).