Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells.
The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35-40 was randomly mutated and the resulting C5a[35-40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A35E36R37A38S39R40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q35E36R37I38L39N40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.
Kola
A
A
Institute of Medical Microbiology, Medical School Hannover, Germany.
Baensch
M
M
Bautsch
W
W
Klos
A
A
Köhl
J
J
D015703:Antigens, CD / Q000378:metabolism*; D001435:Bacteriophages; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D015937:Complement C5a, des-Arginine / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism*; D020298:U937 Cells
D015703:Antigens, CD; D015937:Complement C5a, des-Arginine; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement