The phosphocholine and the polycation-binding sites on rabbit C-reactive protein are structurally and functionally distinct.
C-reactive protein (CRP) is an acute phase protein in humans and rabbits that has the ability to bind a number of biologically important ligands including phosphocholine (PCh), histones, and polycations. In addition to this recognition function, ligand-complexed or aggregated CRP is capable of activating the classical complement pathway. We have generated two strains of transgenic mice in order to study CRP-binding to PCh and consequent complement activation. Based on crystallographic and mutagenesis studies in human CRP (huCRP), we mutated Phe66 and Glu81 in the rabbit CRP (rbCRP) gene and generated a strain of transgenic mice (F66Y/E81K), which expressed this variant form of rbCRP. We also mutated Tyr175 in rbCRP to generate transgenic mice which expressed a variant form of rbCRP (Y175A). In vitro, F66Y/E81K rbCRP purified from serum had dramatically reduced binding to PCh. Additionally F66Y/E81K rbCRP not only maintained its ability to bind polycations and histones, but also bound more avidly to specific histones and lysine polymers than wild type (wt) rbCRP. Y175A rbCRP was not able to activate complement when bound to pneumococcal C-polysaccharide (PnC), but was, along with F66Y/E81K and wild type rbCRP, able to activate complement when bound to a small lysine polymer or when directly adsorbed to a solid phase. This complement activation presumably occurs through the classical complement pathway as the three rbCRPs, adsorbed to a solid phase, bound C1q. Taken together, our results demonstrate that the PCh-binding and the polycation-binding sites on rbCRP are distinct but possibly overlapping. The conformational changes in the C1q-binding site of CRP to activate complement depend on the nature of the ligand and on the location of the ligand-binding site.
Black
Steven
S
Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
Agrawal
Alok
A
Samols
David
D
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002097:C-Reactive Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D015922:Complement C1q / Q000378:metabolism; D003171:Complement Pathway, Classical; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000032:analysis; D051379:Mice; D008822:Mice, Transgenic; D010767:Phosphorylcholine / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism; D000071228:Polyelectrolytes; D011107:Polylysine / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D011817:Rabbits; D012710:Serum Albumin, Bovine
D002097:C-Reactive Protein; D002412:Cations; D015922:Complement C1q; D006657:Histones; D008239:Lysine; D010767:Phosphorylcholine; D011073:Polyamines; D000071228:Polyelectrolytes; D011107:Polylysine; D011135:Polysaccharides, Bacterial; D012710:Serum Albumin, Bovine; C027158:polysaccharide C-substance (Streptococcus)