Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP) Sawsan Amara a , Vincent Delorme a , Michel Record b , Frédéric Carrière a, ⁎ a CNRS-Aix-Marseille Université-Enzymologie Interfaciale et Physiologie de la Lipolyse-UMR 7282, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France b Cancer Research Center of Toulouse, INSERM UMR1037-CNRS ERL5294-Université Toulouse 3, Team Sterol Metabolism and Therapeutic Innovation in Oncology, Institut Claudius Regaud, 20-24 rue du Pont St Pierre, 31052 Toulouse cedex, France abstract article info Article history: Received 12 April 2012 Received in revised form 12 July 2012 Accepted 17 July 2012 Available online 24 July 2012 Keywords: Enzyme inhibition Galactolipase Lipase Phospholipase A1 Phosphonate PLRP2 Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the pres- ence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351 Da, suggesting a cova- lent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides > PC > MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Organophosphorus esters are well-known inhibitors of enzyme bearing a serine residue in their catalytic site [1]. Within this family of compounds, methyl arachidonyl fluorophosphonate (MAFP; Fig. 1), a phosphonate analog of arachidonic acid (AA), has been designed for the selective, active-site directed, inhibition of Group IVA calcium- dependent cytosolic phospholipase A2 (cPLA2) based on the substrate preference of this enzyme for AA [2–4]. This compound was shown to also inhibit the Group IV calcium-independent cytosolic phospholipase A2 (iPLA2) and further testing suggested that MAFP was not a general inhibitor of enzymes acting on arachidonoyl substrates [5]. Since secre- tory phospholipases A2 (sPLA2), such as Group IIA sPLA2, are not inhibited by MAFP, this inhibitor was considered as selective for cPLA2 and iPLA2 among phospholipases A2 and was used for distinguishing these enzymes [6]. MAFP was thus used in numerous studies with intact cells (human platelets, neutrophils) to specifically inhibit cPLA2 and iPLA2. A survey of recent cell biology articles reveals that the blockade of AA release by MAFP is still associated in most arti- cles with the involvement of cPLA2. There is now evidence that MAFP can inhibit other enzymes. A PLA2 isolated from macrophages acting on phosphatidylglycerol and which also exhibits some phospholipase A1 (PLA1) activity has been shown to be strongly inhibited by this compound [7]. MAFP inhibits a human brain 25 kDa lysophospholipid-speci fic lysophospholipase [8]. MAFP is also a potent irreversible inhibitor of anandamide amidase (also named fatty acid amide hydrolase) [9] and human monoacylglycerol lipase (MAGL) [10], two enzymes involved in the degradation of endocannabinoids, anandamide and 2-monocylgycerol, respectively. As such, MAFP has been used for investigating the role of these enzymes in the control of endocannabinoid levels. Since MAGL is also involved in the release of AA from the endocannabinoid 2-arachidonoylglycerol [11], the blockade of AA release by MAFP in cell cultures could therefore partly result from the inhibition of MAGL and alternative pathways of AA production. In the present study, we identified human pancreatic lipase-related protein 2 (HPLRP2) as a novel potential target for MAFP. HPLRP2 is a serine enzyme exhibiting lipase, PLA1 and galactolipase activities [12]. Biochimica et Biophysica Acta 1821 (2012) 1379–1385 ⁎ Corresponding author. Tel.: +33 4 91 16 41 34; fax: +33 4 91 71 58 57. E-mail addresses: michel.record@inserm.f (M. Record), carriere@imm.cnrs.fr (F. Carrière). 1388-1981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbalip.2012.07.014 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip