A detailed analysis of the C5a anaphylatoxin effector domain : selection of C5a phage libraries on differentiated U937 cells

Abstract

We have used a phage-display-based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two different libraries were constructed. In library I, known binding positions 67, 68, 72 and 74 of human complement C5a (hC5a) and in library II, positions 69Ϫ73 of hC5a without C-terminal Arg74 (des-Arg74-C5a) were randomly mutated. In more than 80% (position 72) or 90% (positions 68 and 74) of all cases, the original residues of hC5a were selected from library I, demonstrating that the phage system can be used to define binding points within the C5a molecule. Surprisingly, in more than 90% of all clones, a Phe residue was enriched at position 67 instead of the original His residue which, however, did not affect the binding affinity or the signalling activity. In library II, Leu was preferentially selected at positions 70Ϫ72 and Tyr at position 73, while no enrichment of an individual amino acid was observed at position 69. Mutants with (a) Leu in positions 71 and 72 (b) Ser or Leu in position 70 and (c) Arg or Tyr in position 73, showed a 4Ϫ10-fold higher binding affinity as compared to des-Arg74-[Ala27, Phe67]C5a. The binding affinity was indistinguishable from that of hC5a. In consequence, not only position 72 but also positions 70, 71 and 73 are able to interact with the C5a receptor, whereas position 69 is not. Intriguingly, one mutant with a high binding affinity but without signalling activity was selected. Thus, random mutagenesis of phage-displayed C5a was proven to be a powerful strategy to define receptor-binding points and to select C5aR antagonists based on the structure of the natural ligand.Keywords : complement 5a; complement 5a receptor; filamentous phage ; phage display; cell panning.During complement activation, a panel of proinflammatory fide bonds. The flexible C-terminus points away from the core into solution. mediators is generated, one of which is the C5a anaphylatoxin. It exerts a variety of biologic activities (for review, seeThe functional activities of C5a are mediated by a specific receptor (C5aR), which belongs to the family of seven-transwhich explain its causal involvement in both acute and chronic inflammatory diseases, such as sepsis or rheumatoid arthritis (for membrane domain GTP-binding-protein (G-protein)-coupled receptors [1]. The involvement of both, the C-terminus and Nreview, see [2]). To understand the functional properties of this terminus of the C5a molecule in receptor binding and activation molecule in more detail, a series of structural studies has been has been demonstrated using C-terminal and N-terminal deletion performed. NMR spectroscopy of bovine [3] and human C5a [4] mutants [6, 7]. Singular and multiple mutations at the C-termiand fluorescence spectroscopy of human C5a (hC5a) mutants nus, the disulfide-linked core and the N-terminus revealed 3Ϫ4 Leu72 and Arg74. The importance of Arg40, however, has been E-mail: koehl.joerg@mh-hannover.de questioned in a recent study [11]. Abbreviations. ABICAP, antibody immuno column for ana...

New developments in C5a receptor signaling

“…Deletion mutation analysis suggests that both the N- and C-terminal residues are involved in receptor binding and activation. 48 Our studies have suggested that there are several discontinuous regions of C5a that interact with C5aR. 10 Further, studies have shown that C-terminal synthetic peptides as short as eight aa are able to interact and induce C5aR-mediated function, albeit at much higher concentrations of the peptide than the intact C5a molecule.…”

“…10 Further, studies have shown that C-terminal synthetic peptides as short as eight aa are able to interact and induce C5aR-mediated function, albeit at much higher concentrations of the peptide than the intact C5a molecule. 48 …”

C5a Mutants Are Potent Antagonists of the C5a Receptor (CD88) and of C5L2

“…Two portions of 2.5 l each were electroporated into Escherichia coli TG1 cells (Stratagene) using a Bio-Rad pulser set at 25 microfarads, 2.5 kV, and 200 ⍀. Immediately after the pulse, 1 ml of freshly prepared SOC medium (0.5% yeast extract, 2% Tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 20 mM MgSO 4 , 20 mM glucose) was added, and the cells were grown for 1 h at 37°C and plated on TYE plates (15 g/liter Bacto agar, 10 g/liter Tryptone, 10 g/liter yeast extract, 5 g/liter NaCl) supplemented with 100 g/ml ampicillin and 1% glucose (TYEϩampϩgluc) as described (26). To score the total number of independent transformants, 100 l of appropriate dilutions were plated onto TYEϩampϩgluc plates.…”

“…Bacteria were grown overnight with shaking at room temperature. The next day, the periplasmic fraction was prepared by freezing and thawing as described (26). CNBr-activated Sepharose (Amersham Biosciences) was coated with C5a-specific mAb 561 (36) according to the manufacturer's instructions.…”

“…Cellular Activation and Inhibition Assays-The release of N-acetyl-␤-D-glucosaminidase from CD88-transfected RBL cells was determined as described (26). The ED 50 of rhC5a (Sigma-Aldrich) and C5a mutants was determined as the concentration leading to half-maximal enzyme release.…”