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

Leukotriene A4 (LTA4) 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, LTA4 is enzymatically converted into the cysteinyl leukotrienes and leukotriene B4. Furthermore, LTA4 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 LTA4. 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 ∼1–3 pmol/106 cells. E-FABP (9 μm) was tested for its ability to stabilize LTA4, and at 37 °C E-FABP was able to increase the half-life of LTA4 from the previously reported half-life less than 3 s to a half-life of ∼7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA4 to be available for further enzymatic processing in various cellular regions. Leukotriene A4 (LTA4) 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, LTA4 is enzymatically converted into the cysteinyl leukotrienes and leukotriene B4. Furthermore, LTA4 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 LTA4. 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 ∼1–3 pmol/106 cells. E-FABP (9 μm) was tested for its ability to stabilize LTA4, and at 37 °C E-FABP was able to increase the half-life of LTA4 from the previously reported half-life less than 3 s to a half-life of ∼7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA4 to be available for further enzymatic processing in various cellular regions. Leukotrienes are a family of biologically active metabolites of arachidonic acid known to play important roles in multiple physiological and pathophysiological processes by acting as lipid mediators through specific G protein-coupled receptors (1Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (853) Google Scholar, 2Evans J.F. Prostaglandins Other Lipid Mediat. 2002; 68–69: 587-597Crossref PubMed Scopus (62) Google Scholar). The biosynthesis of these 20 carbon fatty acids is regulated within cells that express 5-lipoxygenase, the first committed enzyme of the leukotriene cascade. The 5-lipoxygenase catalyzes two separate reactions; the first is abstraction of a hydrogen atom from carbon 7 of arachidonic acid and the insertion of molecular oxygen at carbon 5 to generate 5-(S)-hydroperoxyeicosatetraenoic acid (HpETE). 1The abbreviations used are: HpETE, 5-(S)-hydroperoxyeicosatetraenoic acid; LTA4, leukotriene A4; LTB4, leukotriene B4; LTC4, leukotriene C4; RBL, rat basophilic leukemia; FABP, fatty acid-binding protein; E-FABP, epithelial FABP; A-FABP, adipocyte FABP; HPLC, high pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; TOF, time-of-flight; LC, liquid chromatography. The second reaction involves abstraction of a hydrogen atom from carbon 10 of HpETE followed by internal rearrangement of double bonds, loss of the hydroxyl group, and formation of the chemically reactive conjugated triene epoxide, leukotriene A4 (LTA4) (3Shimizu T. Radmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 689-693Crossref PubMed Scopus (263) Google Scholar, 4Ford-Hutchinson A.W. Gresser M. Young R.N. Annu. Rev. Biochem. 1994; 63: 383-417Crossref PubMed Scopus (417) Google Scholar). LTA4 is a substrate for two enzymes that form the biologically active leukotrienes. LTA4 hydrolase converts LTA4 into leukotriene B4 (LTB4), a chemotactic factor for human neutrophils (5Haeggstrom J.Z. Am. J. Respir. Crit. Care Med. 2000; 161: S25-S31Crossref PubMed Scopus (67) Google Scholar). Leukotriene C4 synthase converts LTA4 into the glutathione adduct leukotriene C4 (LTC4), which is a myotropic agent (6Penrose J.F. Spector J. Baldasaro M. Xu K. Boyce J. Arm J.P. Austen K.F. Lam B.K. J. Biol. Chem. 1996; 271: 11356-11361Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Once LTA4 is formed and released from the active site of 5-lipoxygenase, a competitive nonenzymatic reaction with water can also lead to the hydrolysis of the epoxide in a reaction in buffer measured to have a half-life of less than 30 s at 37 °C (7Fitzpatrick F.A. Morton D.R. Wynalda M.A. J. Biol. Chem. 1982; 257: 4680-4683Abstract Full Text PDF PubMed Google Scholar) through formation of a carbocation intermediate (8Borgeat P. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3213-3217Crossref PubMed Scopus (394) Google Scholar). The products of this nonenzymatic hydrolysis reaction include biologically inactive but chemical stable isomers of LTB4 including the Δ6-trans-5,12-dihydroxyeicosatetraenoic acid and 5,6-dihydroxyeicosatetraenoic acid (8Borgeat P. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3213-3217Crossref PubMed Scopus (394) Google Scholar). In neutrophils, 5-lipoxygenase is found in the cytosol (9Rouzer C. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Abstract Full Text PDF PubMed Google Scholar). However, in other cell types, including the rat basophilic leukemia (RBL) cell, 5-lipoxygenase is found in the nucleus (10Brock T.G. Paine III, R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar). Upon stimulation of RBL cells, 5-lipoxygenase translocates to the nuclear envelope, where together with 5-lipoxygenase-activating protein (11Dixon R.A.F. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (651) Google Scholar) and arachidonic acid, which is released by cytosolic phospholipase A2 (12Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar), the biosynthesis of LTA4 takes place. Studies of the fate of LTA4 produced within the human neutrophil revealed that greater than 50% of this lipid generated after cell activation is released to participate in the process of transcellular metabolism (13Sala A. Bolla M. Zarini S. Muller-Peddinghaus R. Folco G. J. Biol. Chem. 1996; 271: 17944-17948Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Although many of the details of this process are unclear, it is now established that LTA4 produced in the neutrophil can appear within other cell types that express synthetic enzymes for the biologically active leukotrienes. For example, erythrocytes, which express LTA4 hydrolase but do not express 5-lipoxygenase, have been shown to convert LTA4 into LTB4 (14McGee J. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1349-1353Crossref PubMed Scopus (133) Google Scholar). Endothelial cells and platelets, which express LTC4 synthase but have no 5-lipoxygenase, have been shown to convert LTA4 derived from the neutrophil into LTC4 (15Feinmark S.J. Cannon P.J. J. Biol. Chem. 1986; 264: 16466-16472Abstract Full Text PDF Google Scholar, 16Maclouf J.A. Murphy R.C. J. Biol. Chem. 1988; 263: 174-181Abstract Full Text PDF PubMed Google Scholar). Because of the chemical instability of LTA4, it is clear that some mechanism must protect LTA4 from exposure to water, preventing the nonenzymatic hydrolysis during transit between cells. Both the chemical instability and the extent of transcellular biosynthesis taking place in multi-cellular compartments suggest that LTA4 is stabilized by binding to an intracellular protein that protects LTA4 from water. Previous work has shown that serum albumin from various species can increase the half-life of LTA4 to more than 20 min at concentrations that are found in plasma (7Fitzpatrick F.A. Morton D.R. Wynalda M.A. J. Biol. Chem. 1982; 257: 4680-4683Abstract Full Text PDF PubMed Google Scholar). However, intracellular proteins that function to protect LTA4 from hydrolysis have not been identified (17Peters Jr., P. All About Albumin. Academic Press, New York1996: 76-132Google Scholar). The purpose of our study was to critically test for the presence of LTA4 stabilizing proteins within RBL cells and identify the stabilizing proteins. RBL-1 cells were chosen for these studies because this cell line expresses 5-lipoxygenase and therefore is likely to have a protein that functions in lipid stabilization (18Goetze A.M. Fayer L. Bouska L. Bornemeier D. Carter G.W. Prostaglandins. 1985; 29: 689-701Crossref PubMed Scopus (42) Google Scholar). In addition, leukotriene biosynthesis has been extensively studied and characterized in these cells (19Jakschik B.A. Kuo C.G. Prostaglandins. 1983; 25: 767-782Crossref PubMed Scopus (44) Google Scholar). Our findings now identify epithelial fatty acid-binding protein as an important molecule for stabilizing LTA4 in these cells. Materials—LTA4 ethyl ester was a generous gift from Dr. Joseph Mancini at Merck-Frosst Canada (Pointe-Claire, Canada). All other eicosanoids were obtained from the Cayman Chemical Company (Ann Arbor, MI). LTA4-free acid was prepared as previously described (20Carrier D.J. Bogri T. Cosentino G.P. Guse I. Rakhit S. Singh K. Prostaglandins Leukot. Essent. Fatty Acids. 1988; 34: 27-30Abstract Full Text PDF PubMed Scopus (25) Google Scholar). Anti-epithelial fatty acid-binding protein (anti-E-FABP) and anti-adipocyte fatty acid-binding protein (anti-A-FABP) antibodies were prepared as previously described (21Hertzel A.V. Bennars-Eiden A. Bernlohr D.A. J. Lipid Res. 2002; 43: 2105-2111Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Nonspecific rabbit IgG and triethylamine were purchased from Aldrich. All other solvents and reagents were HPLC grade and were purchased from Fisher. Cytosol Preparation—Rat basophilic leukemia cells (RBL-1) were cultured at the National Cell Culture Center (Minneapolis, MN). The cells were grown in suspension to a density between 0.85 × 106 and 1.5 × 106 cells/ml in Joklik's medium supplemented with 10% fetal bovine serum. The cells were harvested by centrifugation at 2500 × g. The pellet was resuspended and washed twice with phosphate-buffered saline. The final cell pellet was snap frozen and stored at –70 °C until use. Cytosol preparation was performed as previously described (22Zarini S. Murphy R.C. J. Biol. Chem. 2003; 278: 11190-11196Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Protein Purification—Ammonium sulfate precipitation was performed as previously described (23England S. Seifter S. Deutscher M.P. Guide to Protein Purification. Academic Press, San Diego, CA1990: 285-297Google Scholar) to remove any proteins insoluble at 30% saturation (w/v) or soluble above 75% saturation (w/v). The pellet from this centrifugation was resuspended in a total volume of 2 ml of 50 mm NaOAc, pH 5.0, and injected onto a Superose 12 prep grade column (16 × 30 mm) (Amersham Biosciences) at a flow rate of 1 ml/min. A mixture of known proteins was chromatographed under the same conditions to create a molecular mass calibration curve. The active fractions from the size exclusion chromatography were pooled and exchanged into 25 mm Tris, pH 7.2, using a desalting column (Econo-Pac® 10DG disposable desalting column; Bio-Rad). These fractions were injected onto an anion exchange column (Econo-Pac® High Q, 5-ml bed with a flow rate of 2 and a from to 1 in 25 min. The active fraction in this purification was by with a In some the pooled fractions from size exclusion were exchanged into 50 mm NaOAc, pH of these were as previously described Biochem. 1983; PubMed Scopus Google Scholar). The or were to a exchange column (Econo-Pac® 5-ml bed with a flow rate of 2 50 mm NaOAc, pH 5.0, and a from to 1 in 25 min. Western was performed as previously described with a and a Nature. PubMed Scopus Google Scholar). The was transferred to and a Western blot was performed to the using a of the The Western blot was by using an San of LTA4 stabilizing were performed by or antibodies or rabbit IgG at a of The was to with the protein at 50 of a 50% of Protein A San was and the mixture was to further for 2 at The mixture was at × to remove and the was for LTA4 stabilizing The protein A were in buffer for analysis by to and loss of LTA4 stabilizing The proteins were using LTA4 chemical LTA4 was to of buffer or protein and at were at various between 2 and 30 min and to of internal which was or LTA4. These were using an or a mass as previously described Murphy R.C. J. Am. 2002; PubMed Scopus (25) Google Scholar). In the of of its internal was and the half-life was using the of the of the stabilization of protein was used to protein For these LTA4 was to buffer or protein fraction was to at °C for 20 and was The was to HPLC conditions by the of of 10 mm and the from LTA4 and was determined at A column × 50 was used at a flow rate of with a using a A of 10 mm triethylamine at pH and a of 10 mm The at 30% for conditions and to in 5 min. of protein were as 10 the of after the of the same measured in buffer in the of factor in these was the of LTA4. of the LTA4 to the protein or buffer to the of the not this to the of the of stabilizing Protein tryptic were performed using a previously described J. J. P. Biochem. PubMed Scopus Google Scholar, U. C. J. Biochem. PubMed Scopus Google Scholar). peptide were by mass spectrometry after a and with an volume of acid. This was into an mass The were using a laser of and a of The mass was in the mass trypsin were used as internal for the this in mass of less than 10 peptide were also injected onto a internal column with a of 10 and using solvents of water and The at and to in min at a flow rate of with a from at ml/min. The was into a mass using a of The a of the was at and the was The were with a of 3 s and a mass of 2 the for these was at and the was from and were into the PubMed Scopus Google Scholar). studies were to critically the that cells in LTA4 synthesis proteins that stabilize this leukotriene and protect it from Cytosol obtained from RBL-1 cells was with LTA4, and the chemical half-life of LTA4 in this preparation was with the chemical half-life in The were performed at °C because the half-life of LTA4 been previously shown to be at (17Peters Jr., P. All About Albumin. Academic Press, New York1996: 76-132Google Scholar). shown in RBL-1 cytosol was able to increase the half-life of LTA4 to a increase buffer of stabilizing were used to the protein through purification sulfate precipitation of the cytosolic proteins that the between 30 and 75% The of total found in cytosol the protein was size exclusion chromatography as shown in 1 and However, the specific at this size exclusion chromatography. The active fraction from the size exclusion column at a volume to a molecular mass of the active cytosolic in the flow through from and anion exchange and with to a increase in specific the protein were to the active to the column and with mm with the same specific of RBL-1 cytosol by size exclusion chromatography and exchange to an of LTA4 after 20 min in to the protein as measured by the acid exclusion exchange to mm not exchange mm to an of LTA4 after 20 min in to the protein as measured by the acid not in a The molecular mass of the stabilizing protein together with its exchange and were with a known family of molecular mass proteins known as the fatty acid-binding proteins test for the found in the anion exchange flow this fraction was to an and the from the was tested for any LTA4 stabilizing of the anion exchange flow through with the to E-FABP, followed by precipitation of the with protein the stabilizing that was previously found in the anion exchange flow the from 5 to the proteins present in the anion exchange flow through fraction were with rabbit IgG or with an to the (21Hertzel A.V. Bennars-Eiden A. Bernlohr D.A. J. Lipid Res. 2002; 43: 2105-2111Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), the stabilizing in the the The protein A from the were with buffer and by This was and the were by tryptic digestion followed by to proteins to the E-FABP The tryptic obtained from at 25 and 50 and in 2 of the were found to derived from rabbit The protein identified from the other than rabbit and of was E-FABP The mass of in is shown in All with in were found to from E-FABP with an of These of the Other in the mass from the trypsin and with and tryptic of and A separate tryptic of the same was by ionization and the of the HPLC that to the at was as a activation of this generated a family of product with specific peptide to for a The product were at and and The in the was not the peptide was but this was in the of the peptide not for the tryptic The other major in the were also by and the from two of these are in product from activation of major from the tryptic of the protein with to the in product to the in in a A Western blot was used to the of E-FABP present in RBL-1 cytosol the cytosol of of total protein to ∼1–3 pmol/106 RBL-1 cells. For stabilization of LTA4 by E-FABP, the stabilizing of E-FABP were using LTA4 and the half-life of LTA4. a of LTA4 E-FABP its of at E-FABP The half-life of LTA4 μm) in the presence of E-FABP a half-life of min with 3 LTA4, but concentrations of LTA4 studies the ability of E-FABP to stabilize LTA4 to bovine serum albumin at and 37 °C The half-life of LTA4 in buffer at °C was at 37 the half-life of LTA4 in buffer was to a protein was found to have no stabilizing not bovine serum albumin μm) was tested for its ability to stabilize LTA4 at LTA4 a half-life of 1.5 and E-FABP μm) stabilized LTA4 to a half-life of min. 37 E-FABP stabilized LTA4 to a half-life of 7 bovine serum albumin the half-life of LTA4 to 5 min. Fatty acid-binding proteins are a family of molecular mass proteins found in the cytosol of cells J. 2000; PubMed Scopus Google Scholar). The of this family have between 20 and of 10 by hydrogen to form a J. Biol. PubMed Scopus Google Scholar). The fatty acid-binding protein family has been studied for its in of fatty acids as well as various in a of A.W. J. Biochem. Cell Biol. PubMed Scopus Google Scholar). This protein family has been as a soluble of insoluble found in the also have been to of into cells by of these in the the binding of these proteins with various have been extensively in is some as to physiological in P. S. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). studies with RBL-1 cell cytosol the presence of a factor that was able to stabilize LTA4 as measured by an half-life of the leukotriene in the with buffer The specific of this stabilization was by greater than using a of sulfate size and anion chromatography and using size exclusion chromatography in with exchange chromatography and The and molecular of this factor including its molecular mass and exchange chromatography after that it be a of the of RBL-1 cytosol with antibodies to E-FABP of the LTA4 stabilization in this fraction. In antibodies to were was stabilization of LTA4. E-FABP and acid and have fatty acid binding which suggest that E-FABP was active in LTA4 stabilization or more likely that any is in RBL-1 cells. Although the to with E-FABP protein the to E-FABP not any with any other of the of the proteins from the fraction by the E-FABP to the of rat E-FABP analysis of tryptic was first identified in and is to high in epithelial cells of the and and to in and binding studies have revealed high for and but or no for B. P. Bernlohr D.A. 1996; PubMed Scopus Google Scholar). acid binding to E-FABP has been by a of and high C. C. M. T. C. Biochem. J. 2002; PubMed Scopus Google Scholar, C. T. B. PubMed Scopus Google Scholar). The of the protein in to the of the fatty acid is and water. is the hydroxyl of that is from the that the of LTA4 is to that of acid, the be in of the epoxide is in a of including E-FABP was also found to to this fatty acid-binding protein play a in the of some chemically reactive lipid A. L. A.V. D.A. Bernlohr D.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). present an novel function for E-FABP as a binding protein that can stabilize the epoxide LTA4, by the nonenzymatic hydrolysis within the RBL of E-FABP in the leukotriene has not been previously but this protein play a critically important in the of the biologically active leukotrienes and studies of the of E-FABP LTA4 revealed a process in the of the conjugated triene present in The of E-FABP in the RBL-1 cells the half-life of this triene epoxide to min at 37 a it be for LTA4 to the RBL cell cytosol and LTC4 as well as cytosolic LTA4 Furthermore, this half-life be with the ability of LTA4 to participate in transcellular which the of intact LTA4 from the cell of synthesis to a cell LTA4 hydrolase or LTC4 Previous studies have the binding of eicosanoids with fatty acid-binding proteins. E-FABP has been shown to 5-lipoxygenase products and with high B. P. Bernlohr D.A. 1996; PubMed Scopus Google Scholar). products of metabolism of arachidonic acid, have also been found to be for other have shown that acids can be to and protect the acid from hydrolysis soluble epoxide hydrolase is to the buffer A.W. J. Spector 2003; Scopus Google Scholar). is a of to suggest that the fatty acid-binding proteins play an important in biosynthesis and In after purification and a cytosolic protein present in the RBL-1 cell that the half-life of LTA4 from less than 3 s in buffer at 37 °C to greater than 7 min was identified as the rat Although the of E-FABP in leukotriene biosynthesis has not been previously it is clear that it can play a in of the reactive chemical LTA4, into cellular compartments where chemical place to the biologically active LTC4 and is also that fatty acid-binding proteins are also critically important in the process transcellular biosynthesis where LTA4 is transferred from cell to the National Cell Culture Center for the RBL-1 cells. the for in the protein and peptide the of the Dr. and and Dr. of also for with Western blot

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