A Lipidomic Perspective of the Action of Group IIA Secreted Phospholipase A2 on Human Monocytes: Lipid Droplet Biogenesis and Activation of Cytosolic Phospholipase A2α

Abstract

Phospholipase A2s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca2+-dependent secreted phospholipase A2 (sPLA2) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A2s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A2α. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A2, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response.

A Representative GIIA Phospholipase A2 Activates Preadipocytes to Produce Inflammatory Mediators Implicated in Obesity Development

“…In this context, our finding that inhibition of the cytosolic (cPLA 2 )-α by compound Pyr-2 abrogated the release of PGE 2 induced by MT-III indicates that the cPLA 2 -α is a crucial partner for the effect triggered by MT-III in preadipocytes. This finding is in line with our previous data showing that MT-III increased phosphorylation of cPLA 2 -α at Ser505, a hallmark of cPLA 2 -alpha activation, in human monocytes [63]. Furthermore, although MT-III has the ability to release arachidonic acid from membrane phosphatidylcholine [63], our results evidence that the catalytic activity of MT-III does not play a role in production of PGE 2 in preadipocytes.…”

“…This finding is in line with our previous data showing that MT-III increased phosphorylation of cPLA 2 -α at Ser505, a hallmark of cPLA 2 -alpha activation, in human monocytes [63]. Furthermore, although MT-III has the ability to release arachidonic acid from membrane phosphatidylcholine [63], our results evidence that the catalytic activity of MT-III does not play a role in production of PGE 2 in preadipocytes. A similar mechanism is widely accepted for mammalian GIIA sPLA2s [57].…”

Phospholipid Arachidonic Acid Remodeling During Phagocytosis in Mouse Peritoneal Macrophages

“…Analysis of eicosanoids by LC/MS was carried out exactly as described elsewhere [15,16,49,50], using an Agilent 1260 Infinity high-performance liquid chromatograph equipped with an Agilent G1311C quaternary pump and an Agilent G1329B Autosampler, coupled to an API2000 triple quadrupole mass spectrometer (Applied Biosystems, Carlsbad, CA, USA). Quantification was carried out by integrating the chromatographic peaks of each species and comparing with an external calibration curve made with analytical standards [15,18,49,50].…”

Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2

“…sPLA2s oligomers participate in functional molecular condensates on the PM with other peptides and proteins, such as melittin, HSP70, and nucleolin (NCL), which can modulate the enzymatic activity of sPLA2s or even regulate the activity of other enzymes [26,48,50]. For example, sPLA2s can trigger the activity of cytosolic phospholipases and of enzymes that produce oxygenated fatty acid derivatives [52][53][54]. A direct physical interaction among sPLA2s and other PM proteins or enzymes could occur when sPLA2s are internalized.…”