Mutations in the putative lipid-interaction domain of complement C9 result in defective secretion of the functional protein.

ABSTRACT

Complement protein C9 assembles with C5, C6, C7, C8 on the surface of target cells to form the lytic membrane attack complex (MAC). During MAC assembly and insertion into the target membrane, the hydrophilic, globular C9 partially unfolds to expose a hydrophobic lipid interaction domain. Several copies of amphiphilic C9 subsequently polymerize to form the characteristic ring-like MAC. Using a combined photoaffinity label and computer modeling approach, two amphipathic helices in a segment encompassing the amino acids 293-334 have been predicted to interact with membrane lipids. To elucidate the mechanism of C9 lipid binding and insertion, site-directed mutagenesis was used to change the amphipathic character of the helices. While some conservative amino acid replacements such as Thr307 by a Leu were tolerated and yielded fully active C9 when expressed in COS cells, successive changes of Leu305 into Val, Ala, and Glu on the hydrophobic site of the first helix gave rise to only partly or not secreted C9. All non-conservative amino acid replacements introduced on either side of the helices resulted in non-secreted C9 that was subsequently degraded intracellularly, indicating the importance of the correct folding of the presumptive transmembrane domain during biosynthesis. A natural secretion-incompetent mutant was found in which Val293, located in the proposed lipid-binding region, was lacking. Taken together, these findings suggest that the high incidence of homozygous C9 deficiencies may be due to a blockage in intracellular transport and secretion due to point mutations in this 'hot spot' region of the molecule.