The C2-like β-Barrel Domain Mediates the Ca2+-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1

Abstract

Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca 2؉ -dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80 -100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca 2؉ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca 2؉ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca 2؉ . In summary, our data suggest that interaction of Ca 2؉ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca 2؉ , a low lipid hydroperoxide level is sufficient for 5-LO activation. 5-Lipoxygenase (5-LO)1 catalyzes the initial steps in the biosynthesis of leukotrienes (LTs) and 5(S)-hydro(pero)xyeicosatetraenoic acid (5(S)-H(P)ETE) from arachidonic acid (AA) (for review, see Ref. 1). Due to the pivotal biological functions of 5-LO metabolites (2, 3), the activity of 5-LO is tightly regulated. In intact cells, Ca 2ϩ and phosphorylation seem to be primary signals that activate 5-LO. Moreover, the membranebound 5-LO-activating protein (FLAP) (4) and the redox state (5, 6) have a strong impact on cellular 5-LO product formation.In cell-free systems Ca 2ϩ , ATP, phospholipids (membranes), lipid hydroperoxides (LOOH), and leukocyte-derived proteins have been shown to enhance 5-LO catalysis (reviewed in Ref. 1). However, the degree of stimulation by each of these components depends on the assay conditions, i.e. the source of 5-LO (isolated, in crude homogenates or cellular fractions), presence of other cofactors, the concentration of AA, etc. LOOH are of importance for the initial conversion of the active site iron from the ferrous (resting) to the ferric (active) state (7, 8). Accordingly, glutathione peroxidases (GPx) that reduce LOOH inhibit 5-LO product synthesis in vitro and in intact cells (5, 9 -15), and conditions that are associated with an increased peroxidetone promote 5-LO product formation (6,16,17). Two Ca 2ϩ ions bind to the N-terminal C2-like domain of 5-LO with a K d of 6 M (18, 19). Half-maximal activation of purified 5-LO was determined at 1-2 M Ca 2ϩ , whereas 4 -10 M Ca 2ϩ causes maximal activation of the enzyme (20,21). In intact cells lower concentrations of Ca 2ϩ (200 -300 nM) seem to be sufficient for 5-LO activation (22,23). It was shown that C...

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

“…1E). In our previous report (26) we suggest that stimulation of 5-LO by Ca 2ϩ is because of an increased affinity toward activating LOOH. In fact, the addition of 5-HPETE to PMNL-S100 concentration-dependently up-regulated the LTA 4 synthase activity of 5-LO in the absence of Ca 2ϩ or OAG (Fig.…”

“…OAG Renders 5-LO Activity Resistant against GPx-It was found that Ca 2ϩ renders 5-LO activity resistant against inhibition by GPx-1 (26). We attempted to investigate whether OAG could mimic the effect of Ca 2ϩ , rendering 5-LO activity GPx-resistant.…”

“…We attempted to investigate whether OAG could mimic the effect of Ca 2ϩ , rendering 5-LO activity GPx-resistant. To restore GPx activity in cell-free assays, PMNL-S100 were supplemented with defined amounts of exogenously added GPx-1 (isolated from bovine erythrocytes), where traces of endogenous thiols derived from the intracellular environment of the PMNL serve as the co-substrate for GPx-1 to suppress 5-LO activity (26). Alternatively, PMNL-S100 containing intrinsic GPx (ϳ2.8 milliunits GPx/10 6 cells (26)) were supplemented with 1 mM GSH to reconstitute the activity of endogenous GPx enzyme by elevating the co-substrate concentration (compare Ref.…”

“…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 (27,28).…”

Inhibition of 5-Lipoxygenase Product Synthesis by Natural Compounds of Plant Origin

“…Ca 2+ binds to the regulatory C2-like domain, mediates binding of 5-LO to PC-containing membranes or lipid vesicles, lowers the K m value for AA as substrate, and decreases the requirement of 5-LO for activating LOOH, which altogether potently enhances 5-LO catalysis in vitro [21], [24], [31]. In the cell, Ca 2+ facilitates 5-LO association with the nuclear membrane and protects 5-LO activity against GPx activity [48]. Hence, it is reasonable to assume that natural compounds that chelate Ca 2+ or interfere with Ca 2+ mobilization (i. e., by blocking channels, or interaction with signaling molecules such as PLC, IP3 receptors) prevent activation of 5-LO without inhibiting the enzyme directly.…”