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C-Reactive Proteins

C-Reactive Proteins. Amy Alread December 07, 2004. What are C-reactive proteins ?. C-reactive protein (CRP) is an acute phase reactant made in the liver. Preserved through evolution from Limulus polyhemus to man.

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C-Reactive Proteins

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  1. C-Reactive Proteins Amy Alread December 07, 2004

  2. What are C-reactive proteins ? • C-reactive protein (CRP) is an acute phase reactant made in the liver. • Preserved through evolution from Limulus polyhemus to man. • Named for its “capacity to precipitate the somatic C-polysaccharide of Streptococcus pneumoniae”

  3. CRP is released into the serum as a result of inflammation or tissue damage caused by infection, trauma, surgery, or other inflammatory disorders. • The concentration level of CRP in the serum could be useful in detecting infection or disease and indicating the effectiveness of treatments of diseases. • Concentration levels in the serum could be a good indicator of heart disease, stroke or other diseases involving inflammation. • Some high sensitivity assays have been developed to detect small quantities of CRP in the blood.

  4. Synthesis • Synthesized primarily in hepatocytes. • The CRP gene is located on the short arm of chromosome 1. • The gene contains one intron. • Interleukin-6 and other cytokines regulate CRP at the transcriptional level.

  5. CRP Structure • Member of pentraxins • Five noncovalently linked protomers in a symmetrical cyclic configuration. • Each protomer is a polypeptide chain consisting of 206 amino acid residues and a molecular weight of approximately 21500.

  6. The secondary structure consists of about 50% β-sheet, 12% α-helix, 24% β-turn and 14% random coil. • The lectin fold looks like a flattened jellyroll composed of double-layered β-sheet. This is the attachment site of the phosphocholine or other ligand. • The binding site, located on the concave face, is composed of two calcium molecules held about 4Å apart. They interact with amino acid side chains to keep them in place.

  7. The ligand-binding site grabs the phosphocholine or other ligands with the Phe66 and the Glu88 to stabilize it. The Phe66 interacts hydrophobically with the methyl groups on the phospocholine and the Glu88 interacts with the positively charged nitrogen on the choline.

  8. The other face of the pentamer contains the binding site for C1q which activates complement. The head of the C1q molecule fits nicely in the pore of the pentamer and two of the five protomers interact with it to activate the cascade.

  9. What does CRP bind to? • CRP has the highest affinity for phophocholine on bacteria or as a mixture of sphingomyelin and phosphatidylcholine in eukaryotic membranes. • CRP can also recognize self ligands such as plasma lipoproteins, damaged cell membranes, several phospholipids, small nuclear ribonucleoprotein components, and apoptotic cells. • Some extrinsic ligands that CRP bind are glygan, phospholipids, capsular or cell body components of bacteria, fungi, and parasites, as well as plant particles.

  10. The large amount of self ligands CRP can bind presents some overlapping of ligands that do not need to be destroyed. • This may be one of the causes of cardiovascular and other diseases.

  11. Activation • Once CRP has bound to a ligand, C1q attaches and activates the complement cascade. • CRP is also able to bind FcγRI and FcγRII. • These two immunoglobulin receptors create responses from phagocytic cells.

  12. CRP and Cardiovascular Disease • CRP is known to bind to oxidized LDL. • When CRP binds to LDL in plaques it creates an inflammatory response bringing in complement proteins and other inflammatory reactants. • Inflammation in the plaque could cause blockage leading to a heart attack or stroke.

  13. Other Diseases CRP might have correlation to • Strokes • Periodontal Disease • rheumatic fever • rheumatoid arthritis • Cancer • diabetes • Alzheimer’s disease • Systemic lupus erythematosus (SLE)

  14. Circulating CRP concentration levels • High circulating CRP levels put an apparently healthy individual at an increased risk of congestive heart failure. • Risk • Low <1.0 mg/L • Average 1.0-3.0 mg/L • High >3.0 mg.L • When an individual’s concentration is 5.0 mg/l or above, an almost 3-fold increased risk of heart failure is present.

  15. Factors influencing Normal CRP levels • Gender • Body Mass Index • Ethnicity • Exercise • Alcohol Consumption

  16. Radial Immunodiffusion Assay • Used for the quantitative determination of human CRP in serum. • Human CRP is added to agar well plates of decreasing amounts of human anti-CRP antibodies. • Sera diffuses through the agar and forms a precipitation ring with the antibodies. • The diameter of the ring is compared with a standard to determine the amount of CRP in the blood • Disadvantage: Low sensitivity

  17. High Sensitivity CRP Enzyme Immunoassay Test (hs-CRP ELISA) • Able to detect small quantities of CRP in the serum unlike the RID assay. • Solid phase enzyme-linked immunosorbent assay, sandwich method • Mouse antibodies against human CRP are attached to the wall of the well plate. • Human CRP and goat antibody against human CRP are added to the well plate. • A sandwich structure is formed with the two antibodies and the human CRP. • All unattached antibodies are washed away. • Tetramethylbenzidine is added to the plate resulting in a blue color. • HCL is then added to change the blue color to yellow. This is the stopping agent. • The amount of CRP is determined by the intensity of the color through spectrophotometric devices at 450nm.

  18. Future of CRP • It has been know for a long time that CRP is a marker of inflammation. • The correlation between heart disease and CRP suggests that CRP might be pathogenic. • It is unknown whether CRP is pathogenic or just an indicator of inflammation. • The best way to test the role CRP in pathogenesis would be a drug that suppresses CRP binding and inflammatory response. • This would help indicate where CRP is just a marker or and actual cause of disease. • Since we know information about the 3-D structure of CRP we are in a good position to create this drug.

  19. Conclusion • CRP is an acute phase reactant in response to inflammation. • It is suspected to play a role in diseases such as cardiovascular disease. • The levels of CRP in the blood help indicate one’s risk for disease. The higher the level, the higher the risk for disease. • The levels are measured by high-sensitivity tests such as the ELISA. • Researchers are working to find out more about CRP’s pathogenesis.

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