PMID: 9523709Apr 2, 1998Paper

A detailed analysis of the C5a anaphylatoxin effector domain: selection of C5a phage libraries on differentiated U937 cells

European Journal of Biochemistry
M HenneckeJ Köhl

Abstract

We have used a phage-display-based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two different libraries were constructed. In library I, known binding positions 67, 68, 72 and 74 of human complement C5a (hC5a) and in library II, positions 69-73 of hC5a without C-terminal Arg74 (des-Arg74-C5a) were randomly mutated. In more than 80% (position 72) or 90% (positions 68 and 74) of all cases, the original residues of hC5a were selected from library I, demonstrating that the phage system can be used to define binding points within the C5a molecule. Surprisingly, in more than 90% of all clones, a Phe residue was enriched at position 67 instead of the original His residue which, however, did not affect the binding affinity or the signalling activity. In library II, Leu was preferentially selected at positions 70-72 and Tyr at position 73, while no enrichment of an individual amino acid was observed at position 69. Mutants with (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 ...Continue Reading

Citations

Oct 18, 2003·Biochemical Pharmacology·Stuart A CainPeter N Monk
Oct 24, 2000·Journal of Immunological Methods·S A CainP N Monk
Oct 30, 2008·Clinica Chimica Acta; International Journal of Clinical Chemistry·Kathleen DeiterenAnne Marie Lambeir
Dec 31, 2002·Journal of Receptor and Signal Transduction Research·Oliver Hartley

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