Novel purification scheme and functions for a C3-binding protein from Streptococcus pneumoniae

To isolate microbial proteins capable of binding the third component of com plement (C3), we coupled the free sulfhydryl group of methylamine-inactivat ed C3 to a thiolSepharose matrix. This simple technique facilitated the pur ification of the first C3-binding protein isolated from a bacterium (Strept ococcus pneumoniae). Both metastable (native) and thioester-disrupted C3 we re recognized by this protein; binding of C3 was noncovalent, independent o f thioester conformation, and preferential for the C3 cc-chain. Sequencing of amino-terminal and internal peptides from the C3-binding protein disclos ed a proline-rich region spanning approximately 20 amino acids and a signal peptide that had not been previously reported. The gene was isolated from a library of genomic DNA from laboratory strain CP1200 by screening with a 1200 bp PCR product amplified from degenerate oligonucleotides encoding the amino terminal sequence and the internal proline-rich sequence. The open r eading frame spanned 1692 bp; all peptide sequences were identified in the translated gene product, which also contained at least three choline-bindin g repeats at the carboxy-terminus. The gene was conserved and the translate d protein was functionally active in pneumococcal clinical isolates of sero types 1, 3, 4, 14, and 19F. Serum from a patient recovering from acute pneu mococcal infection contained IgG antibodies specific for this protein by im munoblot. Wide conservation among clinical isolates, saturable binding of C 3, and the ability to stimulate the human immune response have not previous ly been reported for this choline-binding protein. A similar biochemical ap proach should enable the identification of other C3-binding proteins in mic roorganisms able to elude complement-mediated host defense.