A DETAILED ANALYSIS OF THE C5A ANAPHYLATOXIN EFFECTOR DOMAIN - SELECTION OF C5A PHAGE LIBRARIES ON DIFFERENTIATED U937 CELLS

We have used a phage-display-based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two differ ent libraries were constructed. In library I, known binding positions 67, 68, 72 and 74 of human complement C5a (hC5a) and in library II, po sitions 69-73 of hC5a without C-terminal Arg74 (des-Arg74-C5a) were ra ndomly mutated. In more than 80% (position 72) or 90% (positions 68 an d 74) of all cases, the original residues of hC5a were selected from l ibrary I, demonstrating that the phage system can be used to define bi nding points within the C5a molecule. Surprisingly, in more than 90% o f all clones, a Phe residue was enriched at position 67 instead of the original His residue which, however, did not affect the binding affin ity or the signalling activity. In library II, Leu was preferentially selected at positions 70-72 and Tyr at position 73, while no enrichmen t of an individual amino acid was observed at position 69. Mutants wit h (a) Leu in positions 71 and 72 (b) Ser or Leu in position 70 and (c) Arg or Tyr in position 73, showed a 4-10-fold higher binding affinity as compared to des-Arg74-[Ala27, Phe67]C5a. The binding affinity was indistinguishable from that of hC5a. In consequence, not only position 72 but also positions 70, 71 and 73 are able to interact with the C5a receptor, whereas position 69 is not. Intriguingly one mutant with a high binding affinity but without signalling activity was selected. Th us, random mutagenesis of phage-displayed C5a was proven to be a power ful strategy to define receptor-binding points and to select C5aR anta gonists based on the structure of the natural ligand.