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

Abstract

The anaphylatoxin C5a exerts a plethora of biologic activities critical in the pathogenesis of systemic inflammatory diseases. Recently, we reported on a C5a mutant, jun/fos-A8, as a potent antagonist for the human and mouse C5a receptor (CD88). Addressing the molecular mechanism accounting for CD88 receptor antagonism by site-directed mutagenesis, we found that a positively charged amino acid at position 69 is crucial. Replacements by either hydrophobic or negatively charged amino acids switched the CD88 antagonist jun/fos-A8 to a CD88 agonist. In addition to CD88, the seven-transmembrane receptor C5L2 has recently been found to provide high affinity binding sites for C5a and its desarginated form, C5adesArg 74 . A jun/fos-A8 mutant in which the jun/ fos moieties and amino acids at positions 71-73 were deleted, A8 ⌬71-73 , blocked C5a and C5adesArg 74 binding to CD88 and C5L2. In contrast, the cyclic C5a C-terminal analog peptide AcF-[OP-D-ChaWR] inhibited binding of the two anaphylatoxins to CD88 but not to C5L2, suggesting that the C5a core segment is important for high affinity binding to C5L2. Both receptors are coexpressed on human monocytes and the human mast cell line HMC-1; however, C5L2 expression on monocytes is weaker as compared with HMC-1 cells and highly variable. In contrast, no C5L2 expression was found on human neutrophils. A8 ⌬71-73 is the first antagonist that blocks C5a and C5adesArg 74 binding to both C5a receptors, CD88 and C5L2, making it a valuable tool for studying C5L2 functions and for blocking the biological activities of C5a and C5adesArg 74 in mice and humans.

C5L2 Is a Functional Receptor for Acylation-stimulating Protein

“…The maximal effect of ASP is obtained at concentrations about 20ϫ the K d (1). This is similar to C5a with the C5a receptor, where the K d is about 2 nM, and maximal stimulation is obtained at about 40 nM (35).…”

T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4 + T cells

“…To determine whether autocrine engagement of the C5a receptors on T cells regulates T H 1 induction, we activated human CD4 + T cells with immobilized antibodies to CD3, CD3 and CD28, or CD3 and CD46 in the presence or absence of (i) a specific antagonist to C5aR1 [PMX53 (17)]; (ii) the C5aR1/C5aR2 receptor double antagonist A8 Δ71-73 [dRA (18)], targeting only C5aR2 (as the C5aR1 is expressed intracellularly); or (iii) a specific C5aR2 agonist (19). All reagents were cell-impermeable.…”

Ligand Specificity of the Anaphylatoxin C5L2 Receptor and Its Regulation on Myeloid and Epithelial Cell Lines

“…Northern blot data suggest that C5L2 is coexpressed with C5aR on various cells and tissues, such as neutrophils/macrophages, mast cells, immature dendritic cells, as well as in the brain, lung, heart, kidney, liver, ovary, or testis (8 -12). However, other data concerning C5L2 expression on neutrophils are conflicting (10,11). C5L2 binds C5a with nearly the same affinity as the C5aR and C5a-desArg 74 with a 20-fold higher affinity compared with C5aR (13).…”