Structure/function studies of human decay-accelerating factor

The decay-accelerating factor (DAF) contains four complement control protei n repeats (CCPs) with a single N-linked glycan positioned between CCPs 1 an d 2. In previous studies we found that the classical pathway regulatory act ivity of DAF resides in CCPs 2 and 3 while its alternative pathway regulato ry activity resides in CCPs 2, 3 and 4. Molecular modelling of the protein predicted that a positively charged surface area on CCPs 2 and 3 (including KKK125-127) and nearby exposed hydrophobic residues ((LF148)-F-147) on CCP 3 may function as ligand-binding sites. To assess the roles of the N-linked glycan and the above two sets of amino acids in the function of DAF, we mu tated N-61 to Q, KKK125-127 to TTT and (LF148)-F-147 to SS. Following expre ssion of the mutated cDNAs in Chinese hamster ovary cells, the glycosylphos phatidylinositol (GPI)-anchored mutant proteins were affinity purified and their functions were assessed. In initial assays, the proteins were incorpo rated into sheep and rabbit erythrocytes and the effects of the mutations o n regulation of classical and alternative C3 convertase activity were quant ified by measuring C3b deposition. Since DAF also functions on C5 convertas es, comparative haemolytic assays of cells bearing each mutant protein were performed. Finally, to establish if spatial orientation between DAF and th e convertases on the cell surface played any role in the observed effects, fluid-phase C3a generation assays were performed. All three assays gave equ ivalent results and showed that the N-linked glycan of DAF is not involved in its regulatory function; that (LF148)-F-147 in a hydrophobic area of CCP 3 is essential in both classical and alternative pathway C3 convertase regu lation; and that KKK125-127 in the positively charged pocket between CCPs 2 and 3 is necessary for the regulatory activity of DAF on the alternative p athway C3 convertase but plays a lesser role in its activity on the classic al pathway enzyme.