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

Eur J Biochem. 1994 Feb 1;219(3):897-904. doi: 10.1111/j.1432-1033.1994.tb18571.x.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Circular Dichroism
  • Cloning, Molecular
  • Complement C5a / chemistry
  • Complement C5a / genetics*
  • Complement C5a / metabolism
  • DNA, Complementary / chemistry
  • Female
  • Granulocytes / enzymology
  • Guinea Pigs
  • Humans
  • Male
  • Molecular Sequence Data
  • Muscle, Smooth / physiology
  • Mutagenesis, Site-Directed*
  • Oligonucleotides / chemistry
  • Oocytes / physiology
  • Peroxidase / metabolism
  • Receptor, Anaphylatoxin C5a
  • Receptors, Complement / chemistry
  • Receptors, Complement / genetics
  • Receptors, Complement / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Xenopus laevis


  • DNA, Complementary
  • Oligonucleotides
  • Receptor, Anaphylatoxin C5a
  • Receptors, Complement
  • Recombinant Proteins
  • Complement C5a
  • Peroxidase