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, LC1(13-25) (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 microM). 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 microM phosphatidyl serine. Under these conditions LC1(13-25) was active in both assay systems with an IC40 of 40.7 and 57.0 microM respectively. 4. Deletion of amino acids from the C-terminus of the peptide progressively diminished (2-3 fold) the molar potency of LC1(13-25) in both assays: after the removal of Val22 biological activity was virtually undetectable or very weak (< 30% of LC1[13-25]). 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 Glu18. 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 LC1(18-25) (EQEYVQTV). 7. Methoxylation of Tyr21 abolished the ability of LC1(18-25) to inhibit cell proliferation and arachidonic acid release. A cyclized version of LC1(18-25) was also tested and found to be inactive. 8. LC1(18-25) (178 microM) inhibits cPLA2 activation in A549 cells as judged by a band-shift assay, whereas equimolar concentrations of an inactive peptide LC1(19-25) were without effect in this assay system. 9. Several possible mechanisms whereby these peptides act are discussed in the light of LC1 biology and of the effect of glucocorticoids on cell function.