Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells

The human anaphylatoxin Cia is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies sugge st that several residues within the core and the C-terminus mediate the int eraction with the C5a receptor. However, the contribution of particular cor e residues to receptor binding remained to be clarified. By means of the ph age display technique, the loop between positions 35-40 was randomly mutate d and the resulting C5a[35-40] fusion phage library affinity selected on C5 a 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 mutan t C5a[A(35)E(36)R(37)A(37)S(39)R(40)], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor b inding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q(35)E(36)R(37)I(38)L(39)N(40)] 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, Arg 37 as well as Arg40 appear to be anchor residues in binding to the C5a rece ptor. (C) 1999 Elsevier Science Ltd. All rights reserved.