Site-specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor.
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.
Bubeck
P
P
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Grötzinger
J
J
Winkler
M
M
Köhl
J
J
Wollmer
A
A
Klos
A
A
Bautsch
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D015936:Complement C5a / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D005260:Female; D006098:Granulocytes / Q000201:enzymology; D006168:Guinea Pigs; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry; D009865:Oocytes / Q000502:physiology; D009195:Peroxidase / Q000378:metabolism; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D018076:DNA, Complementary; D009841:Oligonucleotides; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011994:Recombinant Proteins; D015936:Complement C5a; D009195:Peroxidase