Inhibitory effect of peptides derived from the N-terminus of lipocortin 1 on arachidonic acid release and proliferation in the A549 cell line: identification of E-Q-E-Y-V as a crucial component

1 The ability of the glucocorticoid-induced protein lipocortin 1 (LC1) to inhibit arachidonic acid release and cell proliferation in A549 cells may be mimicked by a sequence taken from the N-terminal, LCl13-25 (FIENEEQEYVQTV). We have now synthesized and tested for biological activity a library of 25 smaller peptides derived from this sequence. 2 Peptides were tested in two assays: A549 cells were prelabelled with tritiated arachidonic acid and thapsigargin (50 nM) and EGF (10 nM) used to stimulate the release of this fatty acid. Cell proliferation was determined by counting cell numbers following 3 day incubation with these peptides, or controls. 3 Many of the peptides were highly insoluble but could be more readily dissolved in aqueous solution in the presence of commercial liposomes or phosphatidyl serine (5 mu M). Since neither of these agents alone had any effect on arachidonic acid release or cell proliferation, all peptides were tested in the presence of 5 mu M phosphatidyl serine. Under these conditions LCl13-15 was active in both assay systems with an IC40 of 40.7 and 57.0 mu M respectively. 4 Deletion of amino acids from the C-terminus of the peptide progressively diminished (2-3 fold) the molar potency of LCl13-25 in both assays: after the removal of Val(22) biological activity was virtually undetectable or very weak (<30% of LCl13-25). 5 Removal of amino acids from the N-terminus also lead to a progressive reduction (3-5 fold) in the molar potency of the peptides and biological activity became undetectable, or very weak, after the removal of Glu(18). 6 All active peptides contained the core sequence EQEYV(Glu-Gln-Glu-Tyr-Val) which seems to represent a crucial component of the pharmacophore, although this sequence on its own was inactive and the shortest peptide with significant activity was LCl18-25(EQEYVQTV). 7 Methoxylation of Tyr(21) abolished the ability of LCl18-25 to inhibit cell proliferation and arachidonic acid release. A cyclized version of LCl18-25 was also tested and found to be inactive. 8 LCl18-25 (178 mu M) inhibits cPLA(2) activation in A549 cells as judged by a band-shift assay, whereas equimolar concentrations of an inactive peptide LCl19-25 were without effect in this assay system. 9 Several possible mechanisms whereby these peptides act are discussed in the light of LCl biology and of the effect of glucocorticoids on cell function.