Carbohydrate-binding properties of human neo-CRP and its relationship to phosphorylcholine-binding site.
Binding characteristics of two types of ligands for human neo-C-reactive protein (neo-CRP), which is a conformationally altered but physiologically relevant form of CRP, were studied fluorometrically by probing CRP immobilized on a polystyrene surface with europium-labeled ligands. Two Eu-ligands used were bovine serum albumin derivatives that contain on average 40 residues of ligand structures, one derivative containing phosphorylcholine (PC) and the other lactosyl residues. The PC-containing ligands required the presence of calcium for binding, whereas galactose-containing derivatives bound in the absence of calcium. The optimal pH for the PC-dependent binding was broad (pH 6-8), whereas the best binding pH for the galactose-dependent binding was around 6. The carbohydrate-mediated binding is rather nonspecific: the binding site prefers galactose configuration, but other hexoses can be accommodated. The two best monosaccharide inhibitors at this site were galactose-6-phosphate and galacturonic acid, suggesting the importance of having a negatively charged group at C-6 position of galactose. In fact, the phosphate-binding site is common to both PC and sugar phosphates, and the choline- and the sugar-binding sites are probably located on either side of the phosphate-binding site. Binding characteristics of Eu-labeled PC-BSA to neo-CRP are quite similar to that found for native CRP in solution phase [Lee et al. (2002) J. Biol. Chem., 277, 225-232], whereas binding of sugar phosphates by neo-CRP shows considerably less stringent requirements compared to native CRP. For instance, galactose-alpha1-phosphate was not inhibitory at all in the native CRP binding assay, whereas it was a good inhibitor in the neo-CRP assay.
Lee
Reiko T
RT
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA. reiko.lee@jhu.edu
Lee
Yuan C
YC
D001665:Binding Sites; D002097:C-Reactive Protein / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D000939:Epitopes / Q000032:analysis; D005690:Galactose / Q000378:metabolism*; D005695:Galactosephosphates / Q000494:pharmacology; D006603:Hexuronic Acids / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism*; D008024:Ligands; D004369:Pentetic Acid / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism*; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000378:metabolism
D000939:Epitopes; D005695:Galactosephosphates; D006603:Hexuronic Acids; D008024:Ligands; D010710:Phosphates; C076110:phosphorylcholine-bovine serum albumin; D010767:Phosphorylcholine; D012710:Serum Albumin, Bovine; C007819:galacturonic acid; C038269:galactose-6-phosphate; D004369:Pentetic Acid; D002097:C-Reactive Protein; D007785:Lactose; D002118:Calcium; D005690:Galactose