Characterization of the active sites in decay-accelerating factor

Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activi ty resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positive ly charged surface area on CCP2 extending into CCP3, and hydrophobic moieti es between CCPs 2 and 3 as being primary convertase-interactive sites. To m ap the residues providing for the activity of DAF, we analyzed the function s of 31 primarily alanine substitution mutants based in part on this model. Replacing R-69, R-96, R-100, and K-127 in the positively charged CCP2-3 gr oove or hydrophobic F-148 and L-171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K-126 and F -169 and of R-206 and R-212 in downstream CCP4 selectively reduced alternat ive pathway activity without affecting classical pathway activity. Rhesus m acaque DAF has all the above human critical residues except for F-161, whic h is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assa ys. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF f unctions similarly on the two enzymes. When CCP2-3 of DAF were oriented acc ording to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatia l relationship.