C-reactive Protein

Jennifer Kao and Melissa Martin, '07

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I. Introduction

C-reactive protein (CRP) is a highly conserved plasma protein that participates in the systemic response to inflammation (Pepys 1981). It acts as a pattern recognition molecule that can bind to specific molecular configurations typically exposed during cell death or found on the surfaces of pathogens (Marnell et al. 2005). Thus, CRP contributes to host defense and plays a crucial role in the first line of innate host defense.

Synthesis of CRP rapidly and dramtically increases within hours after tissue injury or infection, making it useful in determining disease progress or the effectiveness of treatment (Pepys 1981). If the patient recovered, the substance is again undetectable. It has also been used to gauge the inflammatory response in chronic diseases, such as vasculitis and rheumatoid arthritis, and found in the blood of patients with febrile diseases (Marnell et al. 2005).

An association between minor CRP elevation and future major cardiovascular events has been recognized, leading to a recent recommendation by the Centers for Disease Control and the American Heart Association that patients at risk for heart disease might benefit from measurement of CRP (Volanakis 2001). Heart Risk The risk factors for cardiovascular disease such as age, obesity, smoking, and diet are all factors that would increase CRP levels in the body.

This tutorial will largely focus on the structure of CRP, its ligands, the effector molecules with which it interacts, and its apparent functions.

II. General Structure

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CRP is part of the pentraxin family of proteins having five identical, non-covalently associated subunits < > that form a symmetrical ring. The outside diameter of the pentamer is 102 Å, the diameter of the inner core is 30 Å, and the diameter of the protomer is 36 Å (Volanakis 2001). The bulk of each 206 amino acid subunit consists of two anti-parallel Beta Sheets with an alpha helix on the effector face of the protein< >(Black et al. 2005). Amino acids 175-185 form part of the edge of a deep cleft in the protein around which C1q and FcgR receptor binding occurs on the effector face < > (Volanakis 2001). The recognition face contains the phosphocholine binding site which consists of two coordinated calcium ions next to a hydrophobic pocket in which the phosphocholine rests < >.

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III. Stabilization of Protomers

There are five identical non-covalently bound subunits which make up the CRP molecule. Interprotomer interations include three salt bridges and involve the 115-123 loop of one protomer < > and the 40-42 and 197-202 regions of adjaent protomers < >. The subunits are not all on the same plane, but rotated by 15-20° around an axis parallel to the central alpha-helix < >. This rotation allows the alpha-helices to lie closer together to the pentameric 5-fold axis, and brings the bound Ca2+ further away from it. PC is bound in a shallow surface pocket on each subunit, interacting with the two protein-bound calcium ions by the phosphate group and with Glu81 < > via the choline moiety (Volanakis 2001).

IV. Phosphocholine Binding Site

The ligand, phosphocholine, does not normally appear on the surface of cells, but is exposed by damage to the cell caused by phospholipases (Marnell et al. 2005). Activation
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Phosphocholine binds to C-Reactive Protein in a calcium dependent manner < > which then begins the classical complement pathway. Two binding sites of equal affinity to calcium exist on the recognition face consisting of residues Asp60, Asn61, Glu138, Asp140, and the main chain carbonyl of Gln139 for the first calcium ion and residues Glu138, Asp140, Gln150, and Glu147 for the second calcium ion < >. The two calcium ions interact with the oxygens of the phosphate group and the choline group which rests in a hydrophobic pocket formed by residues Phe66, Leu64, Thr76, and Glu81 < >. The face of Phe66 is exposed < >, allowing it to have hydrophobic interactions with the methyl groups of the choline. Glu81 interacts with the positively charged nitrogen on choline < > (Volanakis 2001).

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V. Clq Activation

The binding site for Clq is found of the effector phase, or opposite side the phosphocholine binding site. After CRP has been complexed with a ligand, Clq is able to bind and activate the complement activation pathway (Volanakis 2001). Activation of this pathway is very important since it plays a role in the killing of microorganisms and protects CRP from pathogenic bacteria such as S. pneumoniae and H. influenzae (Marnell et al., 2005).

The binding-site for Clq is located at the open shallow end of a cleft, where a depression is formed. The boundaries of the pocket are formed by the loops 86-92 and 112-114 on the protomer's C-terminus, and Tyr175 on the other < >. Residues Asp112 and Tyr75 are the contact residues for the Clq < >. Data showed that substitution of these residues with Ala results in significantly reduced affinity for CRP. Glu88 causes a conformational changed in CRP which is needed before complementation activation can occur, whereas Asn158and His38 are needed for the proper geometry at the binding site < >. Substitution of Ala for Lys114 resulted in more Clq-binding and increased complement activation < > (Volanakis 2001).

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VI. Receptor Binding

CRP binds to receptors such as IgG, FcgRI and FcgRII on the surface of phagocytic cells. The exact location of the binding sites of these molecules is not yet known other than that it binds on the effector face < > in the cleft of the molecule. However, the biology is currently being studied. FcgR receptors contain immonoreceptor tyrosine-based activation motifs (ITAM), which are activated by clustering on the cell surface (Marnell et al 2005). Crosslinking of FcgRI and FcgRIIa stimulates phagocytosis, where crosslinking of FcgRIIb inhibits phagocytosis and blocks activating signals. To stimulate phagocytosis, CRP bound on the recognition face to phosphocholine exposed on a damaged cell then binds to FcgRI and FcgRIIa on the effector face(Marnell 2005).

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VII. Structure of CRP is different in diseased patients

CRPs were purified from patients with six different pathological diseases. Small, but substantial, changed was seen in the shape of the C-reactive Protein. Human CRP is glycosylated in some pathological conditions (Dat et al. 2003).

Analysis of the structure showed the systematic absence of two peptide fragments, one at the N-terminus (loop 1-6) in all patients, the other near the C-terminus (loop 189-191) in patients with osteogenic sarcoma and Cushing's syndrome < > . In an undiseased individual, glycosylation sites are inacessible due to the presence of the N-terminal . The loss of these two fragments < > exposed two potential glycosylation sites on a cleft door (Dat et al. 2003). The functional areas of the pentraxin structure remains the same since the Ca2+ and phosophocholine sites are on the opposite site of the pentraxin molecule. < >

VIII. In Vivo

In mice CRP has been shown to protect against bacterial infection, have an anti-inflammatory effect, and clear cellular debris due to its ability to bind to damaged cell membranes and nuclear material (Black 2004).

CRP protected mice against bacterial infection by strains of bacteria with phosphocholine-rich surfaces such as S. pneumoniae and H. aemophilus influenza. It was also shown to protect mice against bacteria that has no surface phosphocholine, but does have phosphoethanolamine, another CRP ligand, on its surface (Marnell et al. 2005). The binding of CRP to these ligands is presumed to then activate the classical complement pathway. This interaction did not require FcyRs in mice infected with S. pneumoniae (Volanakis 2001).

CRP also plays a role in anti-inflammatory processes, which are shown to require FcgR receptors. Experiments have shown that CRP is protective in inflammatory conditions in mice. People with low levels of CRP are more likely to develop Systemic lupus erythematosus (SLE), or Lupus. When mice with a model form of SLE were injected with CRP they had delayed or reversed development of the disease (Marnell et al. 2005). CRP in treating diseases Also, the protein delays the onset of an animal model of multiple sclerosis. The use of C-Reactive protein to treat these diseases in humans is now being researched and offers hope to many afflicted with these diseases.

IX. References

Black, Steven, Kushner, Irving, and David Samols. 2004. C-reactive Protein: A Mini-Review. The Journal of Biological Chemistry 279:48487-48490.

Dat, T., Sen, A.K., Kempf, T., Pramanik, S.R. and C. Mandal. 2003. Induction of glycosylation in human C-reactive protein under different pathological conditions Biochemical J 373:345-55.

Marnell, Lorraine, Mold, Carolyn, and Terry W. Du Clos. 2005. C-reactive protein: Ligands, receptors and role in inflammationr. Clinical Imuunology. 117: 104-111. Volanakis, E., John. 2001. Human C-reactive Protein: expression, structure, and function.. The Journal of Biomedical Sciences. 38:189-197.
V. Pepys, M. B. (1981) C-reactive protein fifty years on. Lancet i, 653–657

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