title: Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function.
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.
authors.affiliation: Department 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.
authors.forename: Piero
authors.initials: P
authors.lastname: Pingitore
authors.affiliation: Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, University of Gothenburg, Bruna Stråket, 16 SE-413 45 Göteborg, Sweden.
authors.forename: Carlo
authors.initials: C
authors.lastname: Pirazzi
authors.affiliation: Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, University of Gothenburg, Bruna Stråket, 16 SE-413 45 Göteborg, Sweden; Department of Medical and Surgical Sciences, Clinical Nutrition Unit, University Magna Graecia of Catanzaro, Viale Europa, Localitá Germaneto, 88100 Catanzaro, Italy.
authors.forename: Rosellina M
authors.initials: RM
authors.lastname: Mancina
authors.affiliation: Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, University of Gothenburg, Bruna Stråket, 16 SE-413 45 Göteborg, Sweden; Department of Pathophysiology and Transplantation, University of Milan, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Via Francesco Sforza 35, 20122 Milan, Italy.
authors.forename: Benedetta M
authors.initials: BM
authors.lastname: Motta
authors.affiliation: Department BEST (Biologia, Ecologia, Scienze della Terra), Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende, Italy.
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
authors.affiliation: Department of Medical and Surgical Sciences, Clinical Nutrition Unit, University Magna Graecia of Catanzaro, Viale Europa, Localitá Germaneto, 88100 Catanzaro, Italy.
authors.forename: Arturo
authors.initials: A
authors.lastname: Pujia
authors.affiliation: Department of Medical and Surgical Sciences, Clinical Nutrition Unit, University Magna Graecia of Catanzaro, Viale Europa, Localitá Germaneto, 88100 Catanzaro, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Montalcini
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, PO Box 462, SE-405 30 Göteborg, Sweden. Electronic address: kristina.hedfalk@chem.gu.se.
authors.forename: Kristina
authors.initials: K
authors.lastname: Hedfalk
authors.affiliation: Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, University of Gothenburg, Bruna Stråket, 16 SE-413 45 Göteborg, Sweden; Department of Medical and Surgical Sciences, Clinical Nutrition Unit, University Magna Graecia of Catanzaro, Viale Europa, Localitá Germaneto, 88100 Catanzaro, Italy. Electronic address: stefano.romeo@wlab.gu.se.
authors.forename: Stefano
authors.initials: S
authors.lastname: Romeo
mesh_terms: D003001:Cloning, Molecular; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology / Q000473:pathology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014280:Triglycerides / Q000378:metabolism; D000217:Acyltransferases; D054512:Phospholipases A2, Calcium-Independent
chemical_list: D006867:Hydrolases; D008049:Lipase; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014280:Triglycerides; C533682:PNPLA3 protein, human; D000217:Acyltransferases; D054512:Phospholipases A2, Calcium-Independent
keywords: Adiponutrin (ADPN); Lysophosphatidic acid acyltransferase activity; Patatin-like phospholipase domain containing 3 (PNPLA3); Pichia pastoris; Triglyceride hydrolase activity; rs738409 (I148M)