Alkylglycerol monooxygenase as a potential modulator for PAF synthesis in macrophages
Graphical abstract
Proposed AGMO function in modulation of macrophage PAF levels. Left panel, in resting conditions, AGMO expression is high and lyso-PAF is effectively degraded by AGMO. Right panel, after stimulation with LPS, AGMO levels decrease. Consequently, degradation of lyso-PAF is attenuated and PAF production is enhanced in activated macrophages.
Introduction
Alkylglycerol monooxygenase (AGMO, glyceryl ether monooxygenase, EC 1.14.16.5) is the only enzyme, so far, known to catalyze the oxidative cleavage of O-alkyl bond of ether lipids. The enzymatic reaction was first described in 1964 as a tetrahydrobiopterin (BH4, (6R)-5,6,7,8-tetrahydro-l-biopterin)-requiring enzyme from rat liver homogenates [1], and later, several detailed studies reported tissue distribution, substrate specificity, and apparent kinetics with the BH4 cofactor [2], [3]. In 2010, using an in silico database search and the recombinant gene expression of selected candidates, Watschinger et al. [4] reported that a gene with an unassigned function, transmembrane protein 195 (TMEM195), encodes AGMO. Alkylglycerols and their various metabolites, including ether phospholipids, are believed to have important biological roles as membrane components and mediators of cell responses [5], [6], [7]. However, the molecular basis of the alkylglycerolipid function, as well as the importance of AGMO in regulating metabolite levels, remain unclear.
Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phosphatidylcholine (PC) with O-alkyl (typically O-hexadecyl) and acetyl groups at sn-1 and sn-2 positions, respectively. PAF is a potent phospholipid mediator that activates a G protein-coupled receptor (PAFR) and causes pleiotropic biological effects, including inflammation, platelet activation, airway constriction, hypotension, and hypersensitive reactions in allergy [8]. PAF is synthesized in tissues and cells via two distinct pathways, the de novo and remodeling pathways [9]. The de novo pathway is thought to maintain physiological levels of PAF for normal cellular functions, whereas the remodeling pathway is activated by inflammatory stimuli and is thought to be the primary source of PAF under pathological conditions. Induction of PAF synthesis by various extracellular stimuli has been reported in peritoneal and alveolar macrophages, polymorphonuclear neutrophils (PMN), vascular endothelial cells, basophils, and bone marrow-derived mast cells [8].
Watschinger et al. [4] reported that AGMO mRNA expression level and enzymatic activity are high in the murine macrophage cell line RAW264.7. Stimulation of RAW264.7 cells or mouse peritoneal macrophages with lipopolysaccharide (LPS) induces enhanced production of PAF in the remodeling pathway through two enzymes, lysophosphatidylcholine acyltransferase 2 (LPCAT2, EC 2.3.1.23 and EC 2.3.1.67) and phospholipases A2 (PLA2, EC 3.1.1.4) [10], [11]. Among PLA2s, cytosolic PLA2α (cPLA2α, group IVA PLA2) is a key enzyme for lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) production in inflammatory cells, the major source of PAF production [11], [12]. Lyso-PAF may either be converted to PAF or transformed back to PC by LPCAT2 [10]. PAF is then rapidly degraded to lyso-PAF by PAF acetylhydrolases (PAFAH) [13], [14]. AGMO is the only identified enzyme that can irreversibly cleave the O-alkyl bond of lyso-PAF to produce glycerophosphocholine (GPC) [2], [3], [4]. Thus, it is possible that this enzyme essentially eliminates lyso-PAF from the recycling system of lipid pool.
In this study, we investigated a possible role for AGMO in the modulation of macrophage PAF levels. The enzyme is primarily expressed in macrophages, and the expression as well as enzymatic activity is decreased by LPS stimulation, which coincides with increased cellular lyso-PAF and PAF levels. Exogenous expression of AGMO causes a reduction in cellular lyso-PAF and PAF levels. To our knowledge, this is the first report to demonstrate the biological importance of AGMO.
Section snippets
Reagents
PAF, lyso-PAF, PAF-d4 (1-O-hexadecyl-(7,7,8,8-d4)-2-acetyl-sn-glycero-3-phosphorylcholine), and lyso-PAF-d4 (1-O-hexadecyl-(7,7,8,8-d4)-2-lyso-sn-glycero-3-phosphorylcholine) were purchased from Cayman Chemical (Ann Arbor, MI). Oligonucleotide primers were from Sigma–Aldrich (St. Louis, MO) and Greiner Bio-One (Frickenhausen, Germany). LPS, from Salmonella minnesota, was purchased from Sigma–Aldrich. ODN1826 and poly(I:C)-LMW were from InvivoGen (San Diego, CA). Other reagents were from Wako
Downregulation of AGMO following LPS stimulation in mouse peritoneal macrophages and RAW264.7 cells
We first examined the effects of LPS treatment on the expression levels of AGMO in thioglycollate-induced peritoneal macrophages and RAW264.7 cells. LPS, a TLR4 ligand, is one of the major activators of macrophages and induces enhanced production of PAF in the remodeling pathway. We found that AGMO mRNA expression was reduced following LPS-stimulation in both peritoneal macrophages and RAW264.7 cells (Fig. 1A and B). We also examined the effect of the TLR3 ligand (poly(I:C)) and the TLR9 ligand
Discussion
In the present report, we demonstrated a possible role for AGMO in macrophage PAF production. BH4-dependent AGMO activity was first reported in 1964 using batyl alcohol (1-O-octadecylglycerol) as a substrate [1]. Later, substrate specificity of AGMO was investigated in vitro, and lyso-PAF was considered as a biological substrate candidate for AGMO [2], [3]. Although AGMO could cleave the O-alkyl bond of PAF itself in vitro, previous studies showed that BH4-dependent enzymatic activity was much
Acknowledgments
This work was supported by MEXT KAKENHI grant numbers 23790356, 23790324, and 24229003. We are grateful to Dr. H. Ichinose of the Tokyo Institute of Technology and Dr. T. Harayama of the University of Tokyo for valuable suggestions. We thank Dr. M. Yamada, Dr. W. Valentine and Ms. A. Kobayashi for assistance and Dr. J. Miyazaki (Osaka University) for supplying the expression vector, pCXN2.
References (28)
- et al.
A new pteridine-requiring enzyme system for the oxidation of glyceryl ethers
J. Biol. Chem.
(1964) - et al.
Substrate specificity in the biocleavage of the O-alkyl bond: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid) and its metabolites
Arch. Biochem. Biophys.
(1981) - et al.
Ether lipids
Chem. Phys. Lipids
(2011) - et al.
Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice
Prog. Lipid Res.
(2000) Platelet-activating-factor and its analogs - metabolic pathways and related intracellular processes
Biochim. Biophys. Acta
(1995)- et al.
A single enzyme catalyzes both platelet-activating factor production and membrane biogenesis of inflammatory cells. Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransferase
J. Biol. Chem.
(2007) - et al.
Roles of cytosolic phospholipase A2 and platelet-activating factor receptor in the Ca-induced biosynthesis of PAF
Biochem. Biophys. Res. Commun.
(2000) Platelet-activating factor acetylhydrolase
Prostaglandins Other Lipid Mediators
(2002)- et al.
Plasma platelet activating factor-acetylhydrolase (PAF-AH)
Prog. Lipid Res.
(2003) - et al.
Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay
J. Lipid Res.
(2007)
A multiplex quantitation method for eicosanoids and platelet-activating factor using column-switching reversed-phase liquid chromatography-tandem mass spectrometry
Anal. Biochem.
The p38 MAPK pathway mediates transcriptional activation of the plasma platelet-activating factor acetylhydrolase gene in macrophages stimulated with lipopolysaccharide
J. Biol. Chem.
Tetrahydropteridine-dependent cleavage enzyme for O-alkyl lipids: substrate specificity
Biochim. Biophys. Acta
The ether lipid-deficient mouse: tracking down plasmalogen functions
Biochim. Biophys. Acta
Cited by (20)
Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown
2022, Journal of Lipid ResearchMarginal BH4 deficiencies, iNOS, and self-perpetuating oxidative stress in post-acute sequelae of Covid-19
2022, Medical HypothesesCitation Excerpt :Other research has focused on platelet-activating factor (PAF), a powerful endogenous pro-inflammatory mediator [29]. Because PAF is an alkylglycerol, its regulation involves alkylglycerol monooxygenase (AGMO), the only enzyme capable of irreversibly cleaving the O-alkyl bond of PAF [30]. When macrophages are activated by inflammatory stimuli, they produce increased levels of PAF and decreased levels of AGMO.
Effect of acyl and alkyl analogs of platelet-activating factor on inflammatory signaling
2020, Prostaglandins and Other Lipid MediatorsCitation Excerpt :Although much is known about the pro-inflammatory actions of PAF including the crystal structure of PAF-R [105] and its catabolism, a dedicated PAF synthase that exclusively produces either alkyl-PAF or acyl-PAF has not yet been described [106]. Previous attempts to clone PAF-synthases ended up cloning enzymes that do not distinguish acylation of the acetyl group or the arachidonyl group at the sn-2 position of lyso PAF [107]. In addition, the recent identification of a nuclear PAF-R makes the intricate biology of PAF even more complex [108,109].
Plasmalogens, platelet-activating factor and beyond – Ether lipids in signaling and neurodegeneration
2020, Neurobiology of DiseaseCitation Excerpt :By the action of particular PLA2 enzymes named PAF-acetylhydrolases, of which several subtypes exist (McIntyre et al., 2009; Kono and Arai, 2019), PAF is hydrolyzed to lyso-PAF (Fig. 2E), which is thought to be biologically inactive (Marathe et al., 2001) and quickly metabolized (Snyder, 1994) either by further degradation or by reacetylation back to PAF. Interestingly, AGMO, an enzyme mainly associated with the degradation of alkylglycerols (cf. Section 2.6), has been suggested to cleave the O-alkyl bond of lyso-PAF, producing an aldehyde and glycerophosphocholine, and thus be an important player in the regulation of PAF levels (Tokuoka et al., 2013). The importance of PAF for the mammalian immune system is emphasized by the fact that mice with a genetic deficiency in the PAF receptor exhibit immunological hyporesponsiveness to a variety of stimuli, like allergens or viral infections (Ishii et al., 1998; Souza et al., 2009).
Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2
2014, Journal of Lipid ResearchCitation Excerpt :To analyze cellular PAF levels, lipids were extracted with 500 μl methanol containing d4-PAF and d4-lyso-PAF as internal standards. Each sample was submitted to solid phase extraction using an Oasis HLB 96-well cartridge cluster (Waters), and PAF and lyso-PAF were measured by LC-MS/MS as previously described (13). Chromatography was performed using an Acquity™ Ultra-performance LC system (Waters) coupled to a TSQ Vantage triple stage quadrupole mass spectrometer (Thermo Scientific) with a HESI-II electrospray ionization source (13).