A C-reactive protein mutant that does not bind to phosphocholine and pneumococcal C-polysaccharide.
C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe(66) and Glu(81) contribute to the formation of the PCh binding site of CRP. We used site-directed mutagenesis to analyze the contribution of Phe(66) and Glu(81) to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe(66) is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation.
Agrawal
Alok
A
Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA. axa144@po.cwru.edu
Simpson
Melanie J
MJ
Black
Steven
S
Carey
Marianne P
MP
Samols
David
D
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001666:Binding Sites, Antibody / Q000235:genetics; D002097:C-Reactive Protein / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D016466:CHO Cells; D019556:COS Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004983:Ethanolamines / Q000737:chemistry; D005353:Fibronectins / Q000737:chemistry; D005822:Genetic Vectors / Q000138:chemical synthesis; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D010767:Phosphorylcholine / Q000737:chemistry*; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013296:Streptococcus pneumoniae / Q000737:chemistry; D014162:Transfection
D000911:Antibodies, Monoclonal; D004983:Ethanolamines; D005353:Fibronectins; D011135:Polysaccharides, Bacterial; C027158:polysaccharide C-substance (Streptococcus); D010767:Phosphorylcholine; D012710:Serum Albumin, Bovine; D018698:Glutamic Acid; D010649:Phenylalanine; C005448:phosphorylethanolamine; D002097:C-Reactive Protein