Pentraxins. SAP and C reactive protein(CRP) are closely related plasma proteins that comprise the highly conserved pentraxin family of homopentameric molecules, belonging to the ‘lectin fold’ superfamily1,2, with specific calcium dependent ligand binding properties. Human CRP is the classical non-specific acute phase protein while human SAP is a stable constitutive plasma protein which does not increase in concentration during the early acute phase response but may rise modestly during chronic inflammation3. Despite much information about their properties and behaviour in humans and other species, neither the normal physiological functions nor the actual roles of either of these proteins in pathophysiology of disease are known for certain. No deficiency of either protein in humans, or even any structural polymorphism, has yet been reported, and even the glycan moiety of human SAP is remarkably invariant4. No mouse CRP knockout has been reported but the mouse SAP knockout is fertile, healthy and lives a normal span, although it has altered innate immunity and impaired capacity to develop amyloidosis5,6.
Human CRP is a rapidly responsive non-specific acute phase protein with a fast plasma half life of 19 h and a dynamic range between less than 0.05 mg/l and more than 500 mg/l. Despite stable phylogenetic conservation of protein sequence, there are marked differences between CRPs in different species in normal concentration, behaviour as acute phase proteins, fine ligand binding specificity, and secondary effects of ligand binding including precipitation, agglutination and complement activation7,8. In order to be plausible, any in vivo role proposed for CRP, whether specifically human or phylogenetically conserved, should be consistent with these facts.
Pentraxin binding to agarose. The characteristic functional property of all pentraxins is their calcium dependent binding to specific anion containing ligands. Human CRP binds with greatest affinity to phosphocholine9 while human SAP binds to phosphoethanolamine10 and to the cyclic pyruvate acetal of galactose, a trace constituent of agarose, a galactan polysaccharide derived from seaweed11,12. However, as has been known for decades, human CRP also binds weakly to agarose13, whereas rat CRP binds more avidly14. CRP binding may reflect recognition of trace sulphate as well as pyruvate constituents of agarose, the abundance of both of which varies significantly between different batches of this biological product, and thus between different lots from the same manufacturer as well as between different manufacturers.
Beaded agarose is widely used as a solid phase matrix for coupling of ligands and for affinity chromatography. When binding by pentraxins to ligands immobilised on agarose is observed, it is absolutely essential to demonstrate that the interaction is with the ligand and not the matrix. All such studies must therefore include the identical activated agarose beads, either coupled with a genuinely non reactive ligand, or simply blocked with the same blocking compound, most commonly Tris or ethanolamine, as used for the ligand coupled beads. Specific binding to the coupled ligand is then indicated by a significant difference between binding under identical circumstances to the test and control beads. Since we first reported the binding of pentraxins to agarose 30 years ago11,13, omission of this obvious and essential control procedure has led to several false conclusions about binding reactivity of different pentraxins, the most egregious being the claim that SAP was a fourth subcomponent, C1t, of the C1 component of the complement system15,16. The claim that CRP binds to leptin is likely to reflect this same systematic error, since binding between these two proteins could not be reproduced, regardless of which one was immobilised and which one was in the fluid phase. Human CRP also did not have any effect on the suppression of appetite and loss of weight induced by human leptin in mice.