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Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function
Piero Pingitore1, Carlo Pirazzi2, Rosellina M Mancina3
1Department BEST (Biologia, Ecologia, Scienze della Terra), Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende, Italy; Department of Chemistry and Molecular Biology, University of Gothenburg, PO Box 462, SE-405 30 Göteborg, Sweden.
Abstract:
The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function.
Insights
The patatin-like phospholipase domain containing 3 (PNPLA3) protein, crucial in liver health, exhibits lipase activity. A common genetic variant (I148M) impairs this function, contributing to liver disease progression.
Area of Science:
- Biochemistry
- Molecular Biology
- Hepatology
Background:
- Patatin-like phospholipase domain containing 3 (PNPLA3), also known as adiponutrin (ADPN), is a liver-expressed membrane protein.
- The PNPLA3 I148M genetic variant (rs738409) is linked to the advancement of chronic liver disease.
Purpose of the Study:
- To develop a yeast-based protein purification protocol for human PNPLA3.
- To investigate the enzymatic activity, substrate specificity, and functional impact of the PNPLA3 I148M mutation.
Main Methods:
- Cloning of wild-type (148I) and mutant (148M) hPNPLA3 cDNA into Pichia pastoris expression vectors.
- Protein expression in 3L fermentors and purification from membrane fractions using Ni-affinity chromatography.
- Enzymatic activity assays utilizing radiolabeled substrates to assess lipase and lysophosphatidic acid acyl transferase (LPAAT) activities.
Main Results:
- Both wild-type and I148M mutant PNPLA3 proteins were successfully localized to the cell membrane.
- Wild-type PNPLA3 demonstrated predominant lipase activity with secondary mild LPAAT activity.
- The I148M mutation resulted in a significant loss of both lipase and LPAAT activities.
Conclusions:
- Human PNPLA3 possesses primary lipase activity.
- The I148M mutation leads to a loss-of-function phenotype, implicating PNPLA3 dysfunction in liver disease pathogenesis.
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