Stabilization of Leukotriene A4 by Epithelial Fatty Acid-binding Protein in the Rat Basophilic Leukemia Cell

Abstract

Leukotriene A 4 (LTA 4 ) is a chemically unstable triene epoxide product of 5-lipoxygenase metabolism of arachidonic acid. Despite this chemical reactivity and its synthesis at the perinuclear membrane, LTA 4 is enzymatically converted into the cysteinyl leukotrienes and leukotriene B 4 . Furthermore, LTA 4 participates in transcellular biosynthesis and is thus transferred between cells as an intact molecule. A cytosolic fatty acid-binding protein present in the rat basophilic leukemia cells was identified using mass spectrometry. This protein was determined to be the stabilizing factor present in the cell cytosol responsible for increasing the effective chemical half-life of LTA 4 . Rat epithelial fatty acid-binding protein (E-FABP) was isolated using partial protein purification and immunoprecipitation. In-gel digestion with trypsin followed by peptide fingerprint analysis using matrix-assisted laser desorption ionization mass spectrometry and sequencing the major tryptic peptide obtained from liquid chromatography/mass spectrometry/mass spectrometry analysis identified E-FABP in the active fraction. Semi-quantitative Western blot analysis indicated that E-FABP in the cytosolic fraction of RBL-1 cells was present at ϳ1-3 pmol/10 6 cells. E-FABP (9 M) was tested for its ability to stabilize LTA 4 , and at 37°C E-FABP was able to increase the half-life of LTA 4 from the previously reported half-life less than 3 s to a halflife of ϳ7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA 4 to be available for further enzymatic processing in various cellular regions.

Determination of leukotriene A4 stabilization by S100A8/A9 proteins using mass spectrometry

“…However, a significant increase in the ability of the S100A8/A9 protein to stabilize LTA 4 was seen with the addition of an excess of Ca 2+ to the reaction mixtures. In a concentration-dependent manner, a S100A8/A9 mixture at a concentration of 2.6 M and 1.7 M increased the half-life of LTA 4 (5 M) to 37 min and 22 min, respectively, showing the dependency of the binding protein concentration on LTA 4 stabilization as observed previously for fatty acid binding proteins ( 19 ). Even the isolated 40 g/ml cytosolic protein mixture increased the LTA 4 halflife to 13 min ( Fig.…”

“…Albumin was the fi rst protein identifi ed that was capable of increasing the half-life of LTA 4 to the order of minutes ( 12 ). Subsequent work found various fatty acid binding proteins that were also capable of stabilizing LTA 4 ( 19,20 ). molecular weight of 10.8 kDa and the S100A9 monomer a mass of 13.2 kDa ( 26 ).…”

“…However, even under these conditions, hydrolysis of LTA 4 could still occur. The studies of albumin and fatty acid binding proteins relied on unique HPLC retention time and UV spectrophotometry to monitor LTA 4 utilizing the distinct chromophore at 280 nm from the epoxide triene moiety ( 12,19,20 ). In the study with fatty acid binding proteins, LC/MS/MS analysis was also used to both specifi cally monitor LTA 4 and to increase the sensitivity of the assay in relation to the UV analysis ( 20 ).…”

“…Serum albumin increases the half-life of LTA 4 to more than 20 min at 25°C at protein concentrations found in plasma ( 12 ). Fatty acid binding proteins were later studied and found to also increase LTA 4 half-life ( 19,20 ). However, neither of these proteins is found in large concentrations within neutrophils.…”

Fatty acid binding proteins stabilize leukotriene A4

“…This protein was identified as the epithelial fatty acid binding protein (E-FABP) (13). There is increasing evidence that the FABP family may be involved in the intracellular trafficking of eicosanoid lipid mediators.…”