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

• Published in: Molecular immunology Vol. 36; no. 2; pp. 145 - 152
• Main Authors: Kola, A, Baensch, M, Bautsch, W, Klos, A, Köhl, J
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.02.1999
• Subjects: Affinity enrichment; Antigens, CD - metabolism; Bacteriophages; Binding Sites - genetics; Binding, Competitive; Complement C5a receptor; Complement C5a, des-Arginine - genetics; Complement C5a, des-Arginine - metabolism; Complement factor C5a; Gene Library; Humans; Phage display; Receptor, Anaphylatoxin C5a; Receptors, Complement - metabolism; U937 Cells; BSA, bovine serum albumin; Complement C5a receptor; C5a, the complement component C5a; [Ala27]C5a, human complement C5a with a Cys27Ala replacement; ELISA, enzyme linked immunosorbent assay; C5, the complement component C5; mAb, monoclonal antibody; Complement factor C5a; cfu, colony forming units; Bt 2cAMP, N 62′- O-dibutyryladenosine 3′,5′-cyclic monophosphate; [Ala27]C5a[35–40], the human complement C5a core library randomly mutated between amino acid residues 35–40; Phage display; IMAC, immobilized metal affinity chromatography; Affinity enrichment; bp, base pair; PCR, polymerase chain reaction; 125I-rhC5a, 125I-labeled recombinant human C5a; IPTG, isopropyl- β- d-thiogalactoside
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/S0161-5890(99)00019-X

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[A 35E 36R 37A 38S 39R 40], 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[Q 35E 36R 37I 38L 39N 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, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.