Investigation of Molecular Interactions Between Lysophospholipase A2 and its Diverse Bioactive Lipid Substrates

Abstract

Lysophospholipase A2 (LYPLA2) is a serine hydrolase responsible for the hydrolysis of lysophospholipids, including lysophosphatidic acids, lysophosphatidylcholines and lysophosphatidylserines. These lysophospholipids are known to elicit a variety of physiological effects, such as the activation and recruitment of phagocytes, induction of cytokine expression, aggregation of platelets, calcium mobilization, and proliferation of vascular smooth muscle cells. We have also recently identified LYPLA2 as the major prostaglandin glyceryl ester (PGG) hydrolase in human cancer cells. These metabolites of the endocannabinoid species, 2‐arachidonoylglycerol (2‐AG), are precursors to the bioactive prostaglandin species, which also elicit a variety of effects, including vasoregulation, hyperalgesia, and inflammatory responses.With purified recombinant LYPLA2, we have used mass spectrometric approaches to evaluate and compare the kinetic rates of hydrolysis of its various substrates, including PGGs, lysophospholipids, and the prostanoid precursors, 2‐AG and arachidonoylethanolamide. With these data, we have shown that LYPLA2 preferentially hydrolyzes 1‐glyceryl esters of esterified lipids, and selectively metabolizes PGGs over their endocannabinoid precursor, 2‐AG. We have recently solved the first crystal structure of LYPLA2 and used this data to develop site‐directed mutants, providing new insights into the binding site of this enzyme. Structural investigations of LYPLA2 reveal a large channel at the surface of the protein, as well as a mobile loop near its catalytic triad. To further investigate the binding site interactions of LYPLA2 with its structurally diverse substrates, site‐directed mutagenesis was employed to express, purify and assess the hydrolytic activity of mutants at these hypothesized binding sites. Mutations of amino acids located in the surface channel were mutated to large tryptophan residues to sterically hinder binding of lipid substrates in the channel, and have shown to significantly inhibit the enzyme's hydrolytic activity. Here we describe the first kinetic analyses of this enzyme's ability to hydrolyze a wide range of PGG, endocannabinoid, and lysophospholipid substrates. Collectively, these data provide the first structural interpretation of the interactions of LYPLA2 with its bioactive lipid substrates.Support or Funding InformationAuthors would like to acknowledge sources of funding for this research including:Center in Molecular Toxicology Predoctoral Training Grant (NIEHS T32 ES007028)National Institute of Health Research Grant (GM15431)