title: The phosphocholine-binding pocket on C-reactive protein is necessary for initial protection of mice against pneumococcal infection.
abstract: Human C-reactive protein (CRP) protects mice from lethal Streptococcus pneumoniae infection when injected into mice within the range of 6 h before to 2 h after the administration of pneumococci. Because CRP binds to phosphocholine-containing substances and subsequently activates the complement system, it has been proposed that the antipneumococcal function of CRP requires the binding of CRP to phosphocholine moieties present in pneumococcal cell wall C-polysaccharide. To test this proposal experimentally, in this study, we utilized a new CRP mutant incapable of binding to phosphocholine. Based on the structure of CRP-phosphocholine complexes, which showed that Phe(66), Thr(76), and Glu(81) formed the phosphocholine-binding pocket, we constructed a CRP mutant F66A/T76Y/E81A in which the pocket was blocked by substituting Tyr for Thr(76). When compared with wild-type CRP, mutant CRP bound more avidly to phosphoethanolamine and could be purified by affinity chromatography using phosphoethanolamine-conjugated Sepharose. Mutant CRP did not bind to phosphocholine, C-polysaccharide, or pneumococci. Mutant CRP was free in the mouse serum, and its rate of clearance in vivo was not faster than that of wild-type CRP. When either 25 μg or 150 μg of CRP was administered into mice, unlike wild-type CRP, mutant CRP did not protect mice from lethal pneumococcal infection. Mice injected with mutant CRP had higher mortality rates than mice that received wild-type CRP. Decreased survival was due to the increased bacteremia in mice treated with mutant CRP. We conclude that the phosphocholine-binding pocket on CRP is necessary for CRP-mediated initial protection of mice against lethal pneumococcal infection.
authors.affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
authors.forename: Toh B
authors.initials: TB
authors.lastname: Gang
authors.forename: David J
authors.initials: DJ
authors.lastname: Hammond
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Singh
authors.forename: Donald A
authors.initials: DA
authors.lastname: Ferguson
authors.forename: Vinod K
authors.initials: VK
authors.lastname: Mishra
authors.forename: Alok
authors.initials: A
authors.lastname: Agrawal
mesh_terms: D000818:Animals; D016470:Bacteremia / Q000097:blood / Q000382:microbiology / Q000473:pathology; D001665:Binding Sites; D002097:C-Reactive Protein / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D010714:Phosphatidylethanolamines; D010767:Phosphorylcholine / Q000378:metabolism*; D011008:Pneumococcal Infections / Q000097:blood / Q000382:microbiology* / Q000517:prevention & control*; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000502:physiology*; D016019:Survival Analysis
chemical_list: D050505:Mutant Proteins; D010714:Phosphatidylethanolamines; D021122:Protein Subunits; D010767:Phosphorylcholine; C483858:phosphatidylethanolamine; D002097:C-Reactive Protein