1-Oleoyl-2-acetylglycerol Stimulates 5-Lipoxygenase Activity via a Putative (Phospho)lipid Binding Site within the N-terminal C2-like Domain

Abstract

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2؉ ions and phospholipids that both act via the Nterminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca 2؉ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 ؋ g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca In the biosynthesis of leukotrienes (LTs) 1 , 5-lipoxygenase (5-LO) catalyzes the initial oxygenation of arachidonic acid (AA) leading to 5-HPETE, which is further metabolized by 5-LO to LTA 4 (for review, see Refs. 1 and 2). The mechanisms leading to activation of 5-LO in the cell are complex, and the enzymatic activity of 5-LO is tightly controlled. At its active site, 5-LO contains an essential nonheme-bound iron (3), which, in the resting state, is in the ferrous (Fe 2ϩ ) form and requires oxidation to the ferric (Fe 3ϩ ) state to enter the catalytic cycle (4, 5).In the absence of stimulating co-factors, 5-LO activity in vitro is low, and it has been found that Ca 2ϩ , ATP, and phospholipids are required for full enzyme activity (for review, see Refs. 1 and 2). Also, a threshold level of lipid hydroperoxides (LOOH) is necessary for initial enzyme activation, converting ferrous to ferric iron (4, 6, 7). For cellular 5-LO activation, elevated Ca 2ϩ levels (8), phosphorylation events by tyrosine kinases (9) and by members of the mitogen-activated protein kinase family (10, 11), an elevated peroxide tone (12-14), and nuclear membrane association (15, 16), including co-localization with the 5-LO-activating protein (17, 18), are determinants. Nevertheless, the precise pathway(s) and regulatory mechanisms of 5-LO activation in the cell are incompletely understood.Based on theoretical models of the tertiary structure, 5-LO consists of a catalytic and an N-terminal C2-like ␤-barrel domain (19 -22). The C2-like domain binds Ca 2ϩ and phosphatidylcholine (PC) (19, 21) and targets 5-LO to the nuclear membrane (20,21). Ca 2ϩ and/or phospholipids can strongly augment 5-LO activity in vitro but also may have no stimulatory effect, which depends on the assay conditions (see Refs. 1 and 23 and references therein). Ca 2ϩ has been shown to increase the affinity of 5-LO toward AA (24), to facilitate membrane association and binding to PC vesicles (21, 25), and appears to reduce the requirement of 5-LO for activating lipid hydroperoxides (26). For stimulation by phospholipids, apparently only the zwitterionic PC and a non-physiological cationic phospholipid (but not phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, or diacylglycerol (DAG)) stimulate 5-LO reactions (27,...

Lipoxin and resolvin biosynthesis is dependent on 5‐lipoxygenase activating protein

“…Moreover, when FLAP was coexpressed with the 5-LO 3W mutant, LXA 4 biosynthesis was impaired. 5-LO 3W is a mutant in which 3 tryptophan residues, which have been shown to play an important role in 5-LO phospholipid binding, are replaced by Ala (27,28). This attenuates the enzyme's membrane binding properties and thus its catalytic activity.…”

Lipoxygenases and their metabolites in formation of plant stress tolerance

“…Most of proteins, whose composition includes the С 2 -domain, are involved in the transduction of signals and membrane traffic including proteins that are implicated in the production of lipid secondary messengers (phospholipase А 2 , phospholipase С s , phosphatidilinositole-3-kіnase and in phosphorylation of other proteins (protein kinase С) [19][20][21]. That is the reason why LOX is referred to as "signal" enzyme that executes catalysis in the state associated with membrane structures [16,19,22]. It was established that the G. max section LOX-1 which is composed of 15 amino acid residues and localized between the last section of the β-folded structure in the N-domain and the first spiral of the catalytic C-domain may undergo proteolysis by trypsin involving a rupture of the connection between Lys-277 and Ser-278.…”

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain

“…Expression of recombinant GST-Dicer 1772-1912 (5LObd) fusion protein [3], GST alone [3], 5LO [28] and 5LO W13/75/102A [29] proteins have been described previously. For 5LO interaction studies, 5 μg of the GST-Dicer 5LObd fusion protein, or GST alone, coupled to glutathione (GSH)-Sepharose 4B beads (10 μl), was incubated with 0.5-10.0 μg 5LO or 0.5 μg 5LO W13/75/102A in buffer A (20 mM Tris•HCl, 100 mM KCl, 5 mM MgCl 2 , 1 mM dithiothreitol, 1 mM ATP, 0.5 mg/ml bovine serum albumin, 0.01% NP-40, pH 7.5), and the binding assay performed as described previously [24,25].…”

“…Ca 2+ has been shown to increase the hydrophobicity of 5LO as well as the affinity of the N-terminal β-sandwich for membrane phospholipids [28,35,36]. Three Trp ligands in the ligand binding loops of the 5LO N-terminal C2-like domain were shown to be important for binding of the isolated 5LO β-sandwich to PC [36] as well as for activation of 5LO by 1-oleoyl-2-acetylglycerol (OAG) [29]. Recently, the same three Trp residues (13, 75 and 102) were found to be required for binding of Coactosin-like Protein (CLP) to 5LO [23].…”