Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids

Abstract

All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein α/β-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.

Fig. 1

Metabolomic profiling of an enzyme library identifies metabolites altered by ABHD3 overexpression. (a) Representative overlaid extracted ion chromatograms at m/z = 523.5–524.5 from HEK293T cells transfected with ABHD3 (red trace) versus other enzymes (blue traces). Insert: Activity-based labeling using the serine hydrolase-directed probe fluorophosphonate rhodamine (FP-Rh) in mock versus ABHD3-transfected cells. Arrowhead designates FP-Rh-labeled ABHD3 protein. (b) Representative MS/MS fragmentation of synthetic C18-LPC (top, in green) and endogenous m/z = 524 (bottom, in brown) gave identical daughter ions of 104.1 (choline), 184.1 (phosphocholine), and 506.1 (dehydro-C18-LPC). (c) Targeted MRM measurements of phosphocholines (PCs) from C8161 cells stably overexpressing epitope-tagged ABHD3 (dark grey), the catalytically dead ABHD3-S220A mutant (black), or GFP (light grey). PC species are indicated by C#/##, where the # indicates the sn-1 acyl chain and ## indicates the sn-2 acyl chain. Top insert: C16-containing PC species; bottom insert: an enlarged graph showing C14/20:4-PC. (d) PLA1 and PLA2 hydrolysis activities for ABHD3 using C14/18:2-PC as a substrate and lysates from stably transfected C8161 cells as the protein source. Data are presented as mean ± standard error; n = 4/group; * P < 0.05, ** P < 0.01, *** P < 0.001 for ABHD3 versus ABHD3-S220A groups.

Fig. 2

Abhd3^−/− mice possess elevated C14-PCs. (a) Untargeted LC-MS profiling of kidney metabolomes from Abhd3^+/+ or Abhd3^−/− mice (plot is shown in the positive ionization mode). Only those metabolites with P < 0.05 and fold change > 1.5 are shown. Data are presented as mean fold changes; n = 6/group. (b) Targeted MRM measurements of PCs from kidney tissue of Abhd3^+/+ (black bars) or Abhd3^−/− (grey bars) mice. Similar changes in PCs were observed in brain, liver, and plasma from Abhd3^−/− mice (see Supplementary Table 3). (c) Fold change of PC species from kidney tissue of Abhd3^−/− versus Abhd3^+/+ mice. The PC acyl chains for a given species are indicated by the top and left axis. Lighty grey indicates no change between genotypes whereas the darkest blue indicates a 8.5-fold change. Data are presented as means; n = 4/group. (d) Relative levels of azPAF in C8161 cells following live-cell incubation with azPAF (10 μM, 4 h, 37°C). For (b) and (d), data are presented as mean ± standard error; n = 3–4/group; * P < 0.05, ** P < 0.01, *** P < 0.001 for Abhd3^+/+ versus Abhd3^−/− or ABHD3 versus ABHD3-S220A.