Molecular determinants of oligomer formation and complement fixation in mannose-binding proteins.

Rat serum mannose-binding protein (MBP-A) functions as part of the innate immune system by targetting complement toward potentially pathogenic microorganisms. In order to examine the molecular basis for complement activation, rat MBP-A has been overproduced in Chinese hamster ovary cells. Recombinant protein is post-translationally modified in the same way as the native lectin. Hydrodynamic studies indicate that MBP-A consists predominantly of covalent oligomers containing one to four copies of a subunit that comprises a trimer of polypeptides. These oligomers are non-interconverting and do not assemble into higher order structures at concentrations in excess of those normally found in serum. Disulfide bonds formed between cysteine residues at the N-terminal end of the collagen-like domain link polypeptides to form covalent oligomers. Analysis of wild-type MBP-A and MBP-A containing the substitution Cys6 --> Ser suggests that polypeptides within each trimeric structural unit are mostly linked by disulfide bonds between cysteine residues at positions 13 and 18 arranged in an asymmetrical configuration. Disulfide bonds involving Cys6 connect polypeptides within separate trimers. Analysis of chimeras between MBP-A and rat liver MBP (MBP-C) indicates that residues within the N-terminal region of the collagenous domain and the cysteine-rich domain of MBP-A enable assembly of trimers into higher order oligomers. The activity of MBP-A in a hemolytic complement fixation assay using mannan-coated sheep erythrocytes was approximately 20-fold greater than the activity of MBP-C. Analysis of the MBP chimeras and isolated oligomers of MBP-A reveals that the larger oligomers are more efficient at complement activation. These data indicate that the overall complement fixing activity of MBP-A is a function of the individual molecular activities of oligomers and their relative abundance within the serum.