Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A 2 W. Pruzanski a , G. Lambeau b , M. Lazdunski b , W. Cho c , J. Kopilov a , A. Kuksis d, ⁎ a Inflammation Research group, University of Toronto, Toronto, Canada b Institut de Pharmacologie Moleculaire et Cellulaire, Sophia Antipolis, Valbonne, France c Department of Chemistry, University of Illinois, IL 60607-7061, USA d Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada M5G 1L6 Received 10 January 2006; received in revised form 22 November 2006; accepted 28 November 2006 Available online 5 December 2006 Abstract We investigated the hydrolysis of the minor glycerophospholipids of human HDL 3 , total HDL and LDL using human group IIA, V and X secretory phospholipases A 2 (sPLA 2 s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA 2 s, and to a lesser extent by group IIA sPLA 2 , the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA 2 s. Thus, when PtdCho was hydrolyzed about 80%, the ethanolamine and inositol glycerophospholipids reached a maximum of 40% hydrolysis. The hydrolysis of phosphatidylserine (PtdSer), which was examined to a more limited extent, showed similar resistance to group IIA, V and X sPLA 2 s, although the group V sPLA 2 attacked it more readily than group X sPLA 2 (52% versus 39% hydrolysis, respectively). Surprisingly, the group IIA sPLA 2 hydrolysis remained minimal at 10– 15% for all minor glycerophospholipids, and was of the order seen for the PtdCho hydrolysis by group IIA sPLA 2 at the 4-h digestion time. All three enzymes attacked the oligo- and polyenoic species in proportion to their mole percentage in the lipoproteins, although there were exceptions. There was evidence of a more rapid destruction of the palmitoyl compared to the stearoyl arachidonoyl glycerophospholipids. Overall, the characteristics of hydrolysis of the molecular species of the lipoprotein-bound diradyl GroPEtn, GroPIns and GroPSer by group V and X sPLA 2 s differed significantly from those observed with lipoprotein-bound PtdCho. As a result, the acidic inositol and serine glycerophospholipids accumulated in the digestion residues of both LDL and HDL, and presumably increased the acidity of the residual particles. An accumulation of the ethanolamine glycerophospholipids in the sPLA 2 digestion residues also had not been previously reported. These results further emphasize the diversity in the enzymatic activity of the group IIA, V and X sPLA 2 s. Since these sPLA 2 s possess comparable tissue distribution, their combined activity may exacerbate their known proinflammatory and proatherosclerotic function. © 2007 Elsevier B.V. All rights reserved. Keywords: Ethanolamine, inositol and serine glycerophospholipids; High density lipoprotein; Low density lipoprotein; In vitro incubation; Human group IIA; Group V and group X secretory phospholipases A 2 ; Liquid chromatography/on-line mass spectrometry 1. Introduction We have recently compared the fatty acid specificity of hydrolysis of PtdCho of plasma lipoproteins by group IIA, V and X secretory phospholipases A 2 (sPLA 2 s) [1]. It was shown that the group V and X sPLA 2 s possessed significantly higher activity than the group IIA sPLA 2 , when compared per mg protein of purified enzyme. Furthermore, the group V sPLA 2 attacked the oligoenoic (one and two double bonds) in preference to polyenoic (three or more double bonds) species, Biochimica et Biophysica Acta 1771 (2007) 5 – 19 www.elsevier.com/locate/bbalip Abbreviations: BSA, bovine serum albumin; CapEx, capillary exit voltage; CID, collision induced dissociation; CL, cardiolipin; GLC, gas–liquid chromatography; GroPCho, glycerophosphocholine; GroPEtn, glyceropho- sphoethanolamine; GroPGro, glycerophosphoglycerol; GroPSer, glyceropho- sphoserine; HDL, high density lipoprotein; HPLC, high performance liquid chromatography; LC/ESI-MS, liquid chromatography with on-line electrospray mass spectrometry; LDL, low density lipoprotein; LysoPtdCho, lysopho- sphatidylcholine; PLA 2 , phospholipase A 2 ; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdGro, phos- phatidylglycerol; PtdIns, phosphatidylinositol; sPLA 2 , secretory PLA 2 ⁎ Corresponding author. E-mail address: arnis.kuksis@utoronto.ca (A. Kuksis). 1388-1981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2006.11.008