Lipoprotein-Associated Phospholipase A₂ Interacts with Phospholipid Vesicles via a Surface-Disposed Hydrophobic α-Helix

Abstract

Lipoprotein-associated phospholipase A2 (Lp-PLA2) plays important roles in both the inhibition and promotion of inflammation in human disease. It catalyzes the hydrolytic inactivation of plasma platelet activating factor (PAF) and is also known as PAF acetylhydrolase. High levels of PAF are implicated in a variety of inflammatory diseases such as asthma, necrotizing enterocolitis and sepsis. Lp-PLA2 also associates with lipoproteins in human plasma where it hydrolyzes oxidized phospholipids to produce pro-inflammatory lipid mediators that can promote inflammation and the development of atherosclerosis. Lp-PLA2 plasma levels have recently been identified as a biomarker of vascular inflammation, atherosclerotic vulnerability, and future cardiovascular events. The enzyme is thus a prominent target for the development of inflammation and atherosclerosis-modulating therapeutics. While the crystallographically-determined structure of the enzyme is known, the enzyme's mechanism of interaction with PAF and the function-modulating lipids in lipoproteins is unknown. We have employed peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to characterize the association of Lp-PLA2 with dimyristoyl phosphatidylcholine (DMPC) vesicles, and found that specific residues 113-120 in one of the enzyme's surface-disposed hydrophobic α-helices likely mediate liposome binding.

Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry

“…The recombinant human Lp-PLA 2 protein including residues 47-441 with an additional alanine residue at the N terminus was prepared as described previously ( 25 ). Purifi ed Lp-PLA 2 was concentrated to 2.5 mg/ml in protein buffer (20 mM Tris, 150 mM NaCl, 10% glycerol, pH 7.5, 1 mM DTT) with an Amicon Ultra-15 (Millipore) with the protein concentration determined by bicinchoninic acid protein assay.…”

“…All steps were performed at 0°C as described previously ( 25 ). Data processing of DXMS experiments utilized specialized software as previously described (DXMS Explorer, Sierra Analytics Inc.) ( 25 ).…”

“…The Lp-PLA 2 protein peptide digestion was optimized as described previously and gave a 94% coverage of recombinant protein ( 25 ).…”

“…Previously, we have used peptide amide deuterium exchange mass spectrometry (DXMS) to investigate Lp-PLA 2 /liposome interactions ( 25 ). DXMS has been widely used to analyze protein-protein, protein-substrate, and protein-inhibitor interactions, as well as protein dynamics and protein conformational changes (26)(27)(28).…”

Liberating Chiral Lipid Mediators, Inflammatory Enzymes, and LIPID MAPS from Biological Grease

“…This approach has traditionally been used to study protein-protein and protein-ligand interactions, but we applied it to look at the interaction of water-soluble proteins with membranes and demonstrated this approach with PLA 2 (136 -138). We have now studied four of the major types of PLA 2 , including the secreted (139), cytosolic (140,141), and Ca 2ϩ -independent forms (142), as well as lipoprotein-associated/platelet-activating factor (PAF) acetyl hydrolase (143) and the interactions of the latter with lipoproteins such as HDL (144). These DXMS methods also led to a better understanding of the catalytic action of an unusual form with a Cys in the catalytic site (145).…”