A recombinant hybrid anaphylatoxin with dual C3a/C5a activity.
By site-directed mutagenesis of a human complement factor C5a cDNA clone, we have designed a hybrid anaphylatoxin in which three amino acid residues in the C-terminal sequence of human C5a were exchanged to create the native C-terminal human C3a (hC3a) sequence Leu-Gly-Leu-Ala-Arg. This hybrid anaphylatoxin rC5a-(1-69)-LGLAR exhibited true C3a and C5a activity when tested in the guinea pig ileum contraction assay. Quantitative measurements of ATP release from guinea pig platelets revealed about 1% intrinsic C3a activity for this hybrid, while the C5a activity was essentially unchanged. Competitive binding assays confirmed that the rC5a-(1-69)-LGLAR mutant was able to displace radioiodinated rhC5a with a KI of approx. 40 nM and hC3a with a KI of approx. 3.7 microM from guinea pig platelets. Since the C-termini of both human C3a and C5a anaphylatoxins are known to interact with their respective receptors, we conclude that the same peptidic sequence, LGLAR, is able to bind to and activate two different receptors, the C3a receptor as well as the C5a receptor. This clone provides a novel tool for the identification of further receptor-binding residues in both anaphylatoxins, since any mutants may be tested for altered C3a and C5a activity simultaneously.
Bautsch
W
W
Institute of Medical Microbiology, Hanover Medical School, Germany.
Kretzschmar
T
T
Stühmer
T
T
Kola
A
A
Emde
M
M
Köhl
J
J
Klos
A
A
Bitter-Suermann
D
D
D000255:Adenosine Triphosphate / Q000097:blood; D000595:Amino Acid Sequence; D000706:Anaphylatoxins / Q000235:genetics / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism; D015926:Complement C3a / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D015936:Complement C5a / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D006168:Guinea Pigs; D007082:Ileum / Q000502:physiology; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D016297:Mutagenesis, Site-Directed; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology
D000706:Anaphylatoxins; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011994:Recombinant Proteins; D015926:Complement C3a; D015936:Complement C5a; D000255:Adenosine Triphosphate