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Mature endothelial lipase (EL) is a 68 kDa glycoprotein. In HepG2 cells infected with adenovirus encoding human EL, the mature EL was detectable in the cell lysates and heparin-releasable fractions. In contrast, cell media of these cells contained two EL fragments: an N-terminal 40 kDa fragment and a C-terminal 28 kDa fragment. N-terminal protein sequencing of the His-tagged 28 kDa fragment revealed that EL is cleaved on the C terminus of the sequence RNKR330↓, the consensus cleavage sequence for mammalian proprotein convertases (pPCs). Replacement of Arg-330 with Ser by site-directed mutagenesis totally abolished EL processing. EL processing could efficiently be attenuated by specific inhibitors of pPCs, α1-antitrypsin Portland (α1-PDX) and α1-antitrypsin variant AVRR. Coexpression of the pPCs furin, PC6A, and PACE4 with EL resulted in a complete conversion of the full-length EL to a truncated 40 kDa fragment. Exogenously added EL was also processed by cells, and the processing could be attenuated by α1-PDX. The expressed N-terminal 40 kDa fragment of EL (EL-40) harboring the catalytic site failed to hydrolyze [14C]NEFA from [14C]dipalmitoyl-PC-labeled HDL. EL-40 was incapable of bridging 125I-labeled HDL to the cells and had no impact on plasma lipid concentration when overexpressed in mice.Thus, our results demonstrate that pPCs are involved in the inactivation process of EL.

GM2 activator protein (GM2AP) is a specific protein cofactor that stimulates the enzymatic hydrolysis of the GalNAc from GM2, a sialic acid containing glycosphingolipid, both in vitro and in lysosomes. While phospholipids together with glycosphingolipids are important membrane constituents, little is known about the possible effect of GM2AP on the hydrolysis of phospholipids. Several recent reports suggest that GM2AP might have functions other than stimulating the conversion of GM2 into GM3 by β-hexosaminidase A, such as inhibiting the activity of platelet activating factor and enhancing the degradation of phosphatidylcholine by phospholipase D (PLD). We therefore examined the effect of GM2AP on the in vitro hydrolyses of a number of phospholipids and sphingomyelin by microbial (Streptomyces chromofuscus) and plant (cabbage) PLD. GM2AP, at the concentration as low as 1.08 μM (1 μg/50 μl) was found to inhibit about 70% of the hydrolyses of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol by PLD, whereas the same concentration of GM2AP only inhibited about 20–25% of the hydrolysis of sphingomyelin by sphingomyelinase and had no effect on the hydrolysis of sphingosylphosphorylcholine by PLD.Thus, GM2AP exerts strong and broad inhibitory effects on the hydrolysis of phospholipids carried out by plant and microbial PLDs. High ammonium sulfate concentration (1.6 M or 21.1%) masks this inhibitory effect, possibly due to the alteration of the ionic property of GM2AP.

Classical lipases are well known for being enzymes hydrolysing triacylglycérols as substrate, except the porcine pancreatic lipase (PPL) which was able to hydrolyze phosphatidylcholine. Amino acid sequence alignments revealed that Valine 260 residue in PPL lid, postulated to be responsible for the PPL phospholipase activity, was present in the Turkey pancreatic lipase (TPL). The importance of Val 260 in the phospholipase activities expression has been reported. To confirm this fact, Val 260 was mutated to Alanine in the TPL lid. Mutated protein has conserved its phospholipase activity as well as the non mutated TPL. Therefore, Valine 260 residue in the lid is not involved in the pancreatic lipases phospholipase activity. The rTPL phospholipase activity was also studied using monolayer technique. This avian pancreatic lipase has shown phospholipase activity toward differently charged phospholipids. The highest phospholipase activity was found on phosphatidylglycerol (negatively charged substrate) at a surface pressure of 20mN/m, but when a zwitterionic substrate was used (DLPC), a lower activity was found at a surface pressure of 10mN/m. However, it is worth noticing that the TPL phospholipase activity is about 100 fold lower than its lipase activity. GC chromatography analyses of the released fatty acids from the hydrolysis of 1,2-POPC have shown that rTPL hydrolyses esters bonds at the sn-1 as well as the sn-2 position of phospholipids. Hence, rTPL shows a low phospholipase activity in comparison to its activity toward triacylglycerols., (Copyright © 2015 Elsevier B.V. All rights reserved.)