Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor

Peter BUBECK, Joachim GRÖTZINGER, Monica WINKLER, Jörg KÖHL, Axel WOLLMER, Andreas KLOS, Wilfried BAUTSCH*

*Korrespondierende/r Autor/-in für diese Arbeit
    37 Zitate (Scopus)


    To check and clarify existing data on receptor‐interacting residues in the human C5a anaphylatoxin, we tested mutant C5a proteins obtained by site‐directed mutagenesis of a recombinant human C5a (rhC5a) cDNA clone for structural and functional integrity. Amino acid positions in three different regions of the molecule were investigated: Arg74 at the C‐terminus, Arg40 and Pro45 located in the core region, and Lys14 and Lys19, Lys20 in the N‐terminus. Des‐Arg74‐rhC5a displayed only a residual 3–4% functional activity in the myeloperoxidase‐release assay from human granulocytes while retaining the three‐dimensional solution structure of wild‐type (wt)–rhC5a as shown by circular dichroism (CD) spectroscopy. Des‐Arg74‐rhC5a was able to activate the human C5a receptor transiently expressed in Xenopus oocytes, but was inactive in the heterologous guinea pig (gp) ileum‐contraction assay. These results reveal profound differences between the guinea pig and human C5a‐receptor ligand‐binding characteristics. Exchange of the core residue Arg40 by a glycine did not significantly affect functional C5a activity, in contrast to a previous observation [Mollison, K. W., Mandecki, W., Zuiderweg, E. P., Fayer, L., Fey, T. A., Krause, R. A., Conway, R. G., Miller, L., Edalji, R. P., Shallcross, M. A., Lane, B., Fox, J. L., Greer, J. & Carter, G. W. (1989) Identification of receptor‐interacting residues in the inflammatory complement protein C5a by site‐directed mutagenesis, Proc. Natl Acad. Sci. USA 86, 292–296], nor did exchange of the conserved Pro45 residue by the C3a analogue glutamic acid, a mutation expected to alter the whole geometry of the loop connecting helix III – helix IV (including Arg40) of the C5a molecule. Thus, participation of this loop in receptor interaction appears unlikely. While exchange of the N‐terminal Lys14 residue by alanine did not significantly affect functional activity, a double replacement of Lys19 and Lys20 by alanine residues reduced activity more than 30‐fold. These results confirm Lys19 and/or Lys20 as a putative receptor‐interacting site, although we could not obtain a CD spectrum of this important mutant due to poor expression.

    ZeitschriftEuropean Journal of Biochemistry
    Seiten (von - bis)897-904
    PublikationsstatusVeröffentlicht - 01.02.1994


    Untersuchen Sie die Forschungsthemen von „Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor“. Zusammen bilden sie einen einzigartigen Fingerprint.