Mapping of the complement C9 binding domain in paramyosin of the blood fluke Schistosoma mansoni.
Schistosomes are believed to evade complement-mediated damage by expression of complement inhibitory proteins. Our previous results [Deng, J., Gold, D., LoVerde, P.T., Fishelson, Z., 2003. Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin. Infect. Immun. 71, 6402-6410.] have demonstrated that paramyosin (Pmy) of the blood fluke S. mansoni binds to the human complement proteins C8 and C9, inhibits complement activation at the terminal stage and protects the parasite from complement-mediated damage. In order to locate the Pmy binding site to C8 and C9, various fragments of Pmy cDNA were PCR-cloned into a pET28a bacterial expression vector. Recombinant His-tagged Pmy fragments were expressed in BL21 Escherichia coli and purified over a nickel-nitrilotriacetic acid column. Binding assays by Western blotting with monoclonal anti-His antibody demonstrated that PmyCC (Pmy amino acids (744)Asp-(866)Met) was the only Pmy fragment that bound to human C8 and C9. Functional analyses demonstrated that PmyCC inhibited hemolysis of rabbit erythrocytes and of antibody-sensitized sheep erythrocytes by human complement. Importantly, PmyCC inhibited in vitro killing of trypsin-sensitized schistosomula of S. mansoni by human complement. In the presence of PmyCC, Zn(2+)-induced C9 polymerization was inhibited. Most of the immunodominant B-cell antigenic epitopes of Pmy are present in the PmyCC region, as antibodies collected from mice immunized with recombinant Pmy bound primarily to PmyCC. Taken together, this study has mapped the complement regulatory domain in Pmy, capable of binding to C8 and C9 and preventing polyC9 formation, to its C-terminal region.
Deng
Jiusheng
J
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Gold
Daniel
D
LoVerde
Philip T
PT
Fishelson
Zvi
Z
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D000947:Antigens, Helminth / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D003167:Complement Activation / Q000276:immunology; D003185:Complement C8 / Q000276:immunology / Q000378:metabolism; D003186:Complement C9 / Q000276:immunology / Q000378:metabolism*; D003165:Complement System Proteins / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D015801:Helminth Proteins / Q000276:immunology / Q000378:metabolism; D006461:Hemolysis / Q000276:immunology; D006801:Humans; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012550:Schistosoma mansoni / Q000276:immunology / Q000378:metabolism*; D012756:Sheep; D014335:Tropomyosin / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000947:Antigens, Helminth; D003185:Complement C8; D003186:Complement C9; D000939:Epitopes; D015801:Helminth Proteins; D011994:Recombinant Proteins; D014335:Tropomyosin; D003165:Complement System Proteins