Topology of the membrane-bound form of complement protein C9 probed by glycosylation mapping, anti-peptide antibody binding, and disulfide modification.

The two N-linked oligosaccharides in native human C9 were deleted by site-specific mutagenesis. This aglycosyl-C9 did not differ from its native form in hemolytic and bactericidal activity. A new N-glycosylation site (K311N/E313T) was introduced into the turn of a helix-turn-helix [HTH] fold that had been postulated to form a transmembrane hairpin in membrane-bound C9. This glycosylated form of human C9 was as active as the native protein suggesting that the glycan chain remains on the external side of the membrane and that translocation of this hairpin is not required for membrane anchoring. Furthermore, flow cytometry provided evidence for the recognition of membrane-bound C9 on complement-lysed ghosts by an antibody specific for the HTH fold. A new N-glycosylation site (P26N) was also introduced close to the N-terminus of C9 to test whether this region was involved in C9 polymerization, which is thought to be required for cytolytic activity of C9. Again, this glycosylated C9 was as active as native C9 and could be induced to polymerize by heating or incubation with metal ions. The two C-terminal cystines within the MACPF domain could be eliminated partially or completely without affecting the hemolytic activity. Free sulfhydryl groups of unpaired cysteines in such C9 mutants are blocked since they could not be modified with SH-specific reagents. These results are discussed with respect to a recently proposed model that, on the basis of the MACPF structure in C8alpha, envisions membrane insertion of C9 to resemble the mechanism by which cholesterol-dependent cytolysins enter a membrane.