Mutation of recombinant complement component C9 reveals the significance of the N-terminal region for polymerization.
Complement component C9 binds to C5b-8 sites on target cells and polymerizes to form the membrane attack complex (MAC). The aim of the work reported here was to discover which region within C9 was responsible for protecting the globular protein against self-polymerization. Computer prediction modelling highlighted the domain at the N-terminus of C9, which was then investigated by site-directed mutagenesis. The mutated proteins were expressed using insect cells infected with baculovirus. Removal of 16, 20 or 23 amino acids at the N-terminus of C9 resulted in inactivation due to self-polymerization. In contrast, removal of 4, 8 or 12 amino acids resulted in a C9 that did not polymerize spontaneously, had two to threefold enhanced lytic activity on erythrocytes, and had increased binding to C5b-8 sites on rat neutrophils. These results suggest that the domain within the first 16 amino acids at the N-terminus of C9 is crucial in preventing the self-polymerization of the globular protein. We have also found that C9 contains a motif (27WSEWS31) common to a family of cytokine receptors that is similar to a tryptophan-rich motif (WEWWR) of the membrane pore formers, thiol-activated cytolysins. Mutation of this motif in C9 resulted in polymerized protein, consistent with this site keeping the N-terminus in a protected conformation and preventing premature self-polymerization.
Taylor
K M
KM
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK.
Trimby
A R
AR
Campbell
A K
AK
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D010856:Columbidae / Q000276:immunology; D003186:Complement C9 / Q000737:chemistry* / Q000276:immunology; D003198:Computer Simulation; D004912:Erythrocytes / Q000276:immunology; D006461:Hemolysis; D006801:Humans; D046911:Macromolecular Substances; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000276:immunology; D011108:Polymers; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship
D003186:Complement C9; D046911:Macromolecular Substances; D011108:Polymers; D011994:Recombinant Proteins