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. 2011 Oct 28;286(43):37085-93.
doi: 10.1074/jbc.M111.290114. Epub 2011 Aug 30.

Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease

Yongcheng Huang  1 , Jonathan C Cohen, Helen H Hobbs
Affiliations

Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease

Yongcheng Huang et al. J Biol Chem. .

Abstract

A genetic variant of PNPLA3 (patatin-like phospholipase domain-containing 3; PNPLA3-I148M), a serine protease of unknown function, is associated with accumulation of triacylglycerol (TAG) in the liver. To determine the biological substrates of PNPLA3 and the effect of the I148M substitution on enzymatic activity and substrate specificity, we purified and characterized recombinant human PNPLA3 and PNPLA3-I148M. Maximal hydrolytic activity of PNPLA3 was observed against the three major glycerolipids, TAG, diacylglycerol, and monoacylglycerol, with a strong preference for oleic acid as the acyl moiety. Substitution of methionine for isoleucine at position 148 markedly decreased the V(max) of the enzyme for glycerolipids but had only a modest effect on the K(m). Purified PNPLA3 also catalyzed the hydrolysis of oleoyl-CoA, but the V(max) was 100-fold lower for oleoyl-CoA than for triolein. The thioesterase activity required the catalytic serine but was only modestly decreased by the I148M substitution. The enzyme had little or no hydrolytic activity against the other lipid substrates tested, including phospholipids, cholesteryl ester, and retinyl esters. Neither the wild-type nor mutant enzyme catalyzed transfer of oleic acid from oleoyl-CoA to glycerophosphate, lysophosphatidic acid, or diacylglycerol, suggesting that the enzyme does not promote de novo TAG synthesis. Taken together, our results are consistent with the notion that PNPLA3 plays a role in the hydrolysis of glycerolipids and that the I148M substitution causes a loss of function, although we cannot exclude the possibility that the enzyme has additional substrates or activities.

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Figures

FIGURE 1.
FIGURE 1.
Triglyceride (TAG) lipase activity of PNPLA3. A, recombinant human PNPLA3-WT, PNPLA3-I148M, and PNPLA3-S47A were purified from insect (Sf9) cells. A total of 1.8 μg of protein was size-fractionated on an 8% SDS-polyacrylamide gel, and the gel was stained with Coomassie Blue. B, wild-type or mutant PNPLA3 (2 μg) was incubated with [3H]triolein liposome substrate for 20 min at 37 °C as described under “Experimental Procedures.” Lipids were extracted and then fractionated by TLC using chloroform/methanol/ammonium hydroxide (65:25:5, v/v/v). Free oleate was isolated, and the radioactivity quantitated by scintillation counting. Values were normalized to the levels in the buffer-only control sample and fitted to Michaelis-Menten kinetic curves. The experiments were performed four times, and the results from a representative experiment are shown.
FIGURE 2.
FIGURE 2.
PNPLA3 hydrolysis of TAG. A total of 100 μm [14C]triolein that was uniformly labeled on glycerol was prepared in liposomes. Substrate (100 μl) was incubated with 5 μg of wild-type or mutant PNPLA3 in a 200-μl reaction volume for 60 min at 37 °C. The extracted lipids were spotted onto a TLC plate that was flushed by 2.3% boric acid in ethanol and dried at 100 °C. The plate was developed in chloroform/acetone (96:4, v/v) and subjected to phosphorimaging analysis. Positions of TAG, 1,3-DAG, 1,2-DAG, and MAG standards were determined. The experiment was performed twice, and the results were similar.
FIGURE 3.
FIGURE 3.
PNPLA3 hydrolyzes DAG and MAG. A, PNPLA3-WT, PNPLA3-I148M, or PNPLA3-S47A (2 μg) was incubated for 20 min at 37 °C with [14C]diolein (A) or [14C]monoolein (B) prepared in liposomes as described under “Experimental Procedures.” Total lipids were extracted, and free oleate was measured as described in the legend to Fig. 1. The values were normalized to the levels obtained when buffer alone was added to the reaction. The experiments were performed three times for either substrate, and the results from one representative experiment are shown.
FIGURE 4.
FIGURE 4.
CGI-58 does not activate PNPLA3. COS-7 cells were transfected with the vector control (Vec.), PNPLA3-WT, PNPLA3-I148M, PNPLA3-S47A, ATGL-WT, ATGL-I148M, or ATGL-S47A. Cells were disrupted by sonication, and 100 mg of cell lysates was used for TAG hydrolase assay in the presence or absence of 200 ng of bacterially purified GST-CGI-58. The released free oleate was extracted and measured by scintillation counting. Expression of PNPLA3, PNPLA3 mutants, ATGL, ATGL mutants, and calnexin was determined by Western blotting using antibodies against the V5 tag and calnexin. This experiment was performed twice, and the results were similar.
FIGURE 5.
FIGURE 5.
Purified PNPLA3 has no detectable GPAT, LPAAT, or DGAT activity. A schematic of the Kennedy pathway of TAG synthesis (upper left) and Coomassie Blue staining of PNPLA3-WT, PNPLA3-I148M, PNPLA3-S47A, and GST-CGI-58 used in the experiments (upper right) are shown. A total of 4 μg of PNPLA3-WT, PNPLA3-I148M, or PNPLA3-S47A in a volume of 100 μl was mixed with 100 μl of (A) 200 μm glycerol 3-phosphate and 20 μm [14C]oleoyl-CoA (B), 200 μm LPA and 20 μm [14C]oleoyl-CoA (C), or 200 μm DAG and 20 μm [14C]oleoyl-CoA and incubated at 37 °C for 15 min. Liver lysate (100 μg) was used as a positive control. For the LPAAT assay, recombinant GST-CGI-58 (4 μg) was also used as a positive control. Total lipids were extracted and resolved as described under “Experimental Procedures.” The positions of oleoyl-CoA and of the reaction end products are indicated. Each of these experiments was repeated at least three times, and the results were similar. PAP, phosphatidic acid phosphohydrolase; FA, fatty acid; C, control. *, unidentified lipid.
FIGURE 6.
FIGURE 6.
Transacylation activity of PNPLA3. WT and mutant PNPLA3 (5 μg) were incubated with 20 μm [14C]diolein (A) or [14C]oleate (B) for 20 min at 37 °C. Total lipids were extracted and loaded onto a TLC plate pretreated with 2.3% boric acid in ethanol. The plate was developed in chloroform/acetone (96:4, v/v) and subjected to phosphorimaging analysis. The positions of TAG, DAG, MAG, and free fatty acid (FFA) are indicated. The experiment was repeated twice, and the results were similar.
FIGURE 7.
FIGURE 7.
PNPLA3 has thioesterase activity. PNPLA3-WT, PNPLA3-I148M, or PNPLA3-S47A was incubated for 20 min at 37 °C with [14C]oleoyl-CoA (A), [14C]palmitoyl-CoA (B), or [14C]stearoyl-CoA (C). Lipids were extracted, applied to TLC plates, and resolved in chloroform/methanol/ammonium hydroxide (65:25:5, v/v/v). The activity of the released free oleate was analyzed by scintillation counting and calculated after normalization to the levels obtained using buffer alone. The final values obtained were plotted using GraphPad Prism 5 software. The experiments were performed three times for each substrate, and the results from one representative experiment are shown.
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