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J Lipid Res. 2018 Jul;59(7):1205-1218. doi: 10.1194/jlr.M084012. Epub 2018 May 3.

Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A2.

Author information

1
Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, MI.
2
Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, MI.
3
Department of Pharmaceutical Science, Biointerfaces Institute, University of Michigan, Ann Arbor, MI.
4
Department of Biological Sciences, Purdue University, West Lafayette, IN.
5
Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, MI jshayman@umich.edu.

Abstract

Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.

KEYWORDS:

1-O-acylceramide; acyltransferase; ceramide; cholesterol; crystallography; lysosome; pH dependence; phospholipase A2 group XV; selectivity

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