Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8 gamma is not essential.

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the cytolytic membrane attack complex (MAC) on bacterial cell membranes. It is an oligomeric protein composed of a disulfide-linked C8 alpha-gamma heterodimer and a non-covalently associated C8 beta chain. Previous studies revealed that C8 alpha and C8 beta have distinct roles in the formation of the MAC on simple cells such as erythrocytes and that both subunits are essential for cell lysis. These studies also determined that C8 gamma is not required for expression of MAC hemolytic activity. To determine if these conclusions are applicable to more biologically relevant systems, the C8 subunits were examined for their ability to support complement-mediated killing of Gram-negative bacteria. Results indicate: (1) C8 alpha-gamma, C8 alpha, C8 beta and C8 gamma have no independent bactericidal activity; (2) bacterial killing requires C8 beta and either C8 alpha-gamma or C8 alpha; (3) C8 alpha is an effective substitute for C8 alpha-gamma in bacterial killing; and (4) C8 gamma enhances, but is not required for C8 bactericidal activity. Together, these data suggest that C8 alpha and C8 beta have correspondingly similar roles in MAC-mediated lysis of erythrocytes and bacterial killing. Furthermore, they provide the first direct evidence that C8 gamma is not required for complement-mediated killing of Gram-negative bacteria.

Crystal structure of human complement protein C8gamma at 1.2 A resolution reveals a lipocalin fold and a distinct ligand binding site.

C8gamma is a 22-kDa subunit of human C8, which is one of five components of the cytolytic membrane attack complex of complement (MAC). C8gamma is disulfide-linked to a C8alpha subunit that is noncovalently associated with a C8beta chain. In the present study, the three-dimensional structure of recombinant C8gamma was determined by X-ray diffraction to 1.2 A resolution. The structure displays a typical lipocalin fold forming a calyx with a distinct binding pocket that is indicative of a ligand-binding function for C8gamma. When compared to other lipocalins, the overall structure is most similar to neutrophil gelatinase associated lipocalin (NGAL), a protein released from granules of activated neutrophils. Notable differences include a much deeper binding pocket in C8gamma as well as variation in the identity and position of residues lining the pocket. In C8gamma, these residues allow ligand access to a large hydrophobic cavity at the base of the calyx, whereas corresponding residues in NGAL restrict access. This suggests the natural ligands for C8gamma and NGAL are significantly different in size. Cys40 in C8gamma, which forms the disulfide bond to C8alpha, is located in a partially disordered loop (loop 1, residues 38-52) near the opening of the calyx. Access to the calyx may be regulated by movement of this loop in response to conformational changes in C8alpha during MAC formation.