Reevaluation of the C3a active site using short synthetic C3a analogues

In 1980 the C‐terminal pentapeptide LGLAR (C3a 73–77) was described (Caporale, L. H. et al. J. Biol. Chem. 1980. 255: 10758) as the minimal sequence inducing a C3a‐specific activity. We have synthesized C3a‐analogue peptides connected to non‐peptidic acyl residues known to potentiate biological activity. Starting from the acylated hexapeptide fluorenylmethoxycarbonyl(Fmoc)‐aminohexanoyl(Ahx)‐ALGLAR, a related series of shorter peptides was synthesized. C3a‐specific activity was measured as ATP release from guinea pig platelets. Even the tripeptide LAR, acylated with Fmoc‐Ahx, exhibited C3a‐specific activity. With 0.34% C3a activity, it was even more potent than the native LGLAR sequence which has 0.01% activity. N‐terminal extension of the acylated tripeptide LAR by adding one to three alanines increased activity tenfold up to 3.26% (Fmoc‐Ahx‐AAALAR), while N‐terminal addition of three glycine residues (Fmoc‐Ahx‐GGGLAR) only increased activity to 0.83% of native C3a. Furthermore, a stimulus‐specific desensitization could be observed. Fmoc‐Ahx‐R and Fmoc‐Ahx‐AR exhibited neither activity nor desensitizing capacity, but the addition of four alanines to the dipeptide AR led to a sequence (Fmoc‐Ahx‐AAAAAR) with a C3a‐specific activity of 0.14%. Even arginine prolonged N‐terminally with five glycines (Fmoc‐Ahx‐GGGGGR) exhibited some C3a‐specific activity so that for biological activity only the appropriate presentation of arginine seems to be essential.