Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function

Piero Pingitore, Carlo Pirazzi, Rosellina M. Mancina, Benedetta M. Motta, Cesare Indiveri, Arturo Pujia, Tiziana Montalcini, Kristina Hedfalk, Stefano Romeo

Research output: Contribution to journalArticle

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 3 L 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.

Original languageEnglish
Pages (from-to)574-580
Number of pages7
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1841
Issue number4
DOIs
Publication statusPublished - Apr 2014

Keywords

  • Adiponutrin (ADPN)
  • Lysophosphatidic acid acyltransferase activity
  • Patatin-like phospholipase domain containing 3 (PNPLA3)
  • Pichia pastoris
  • rs738409 (I148M)
  • Triglyceride hydrolase activity

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Medicine(all)

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    Pingitore, P., Pirazzi, C., Mancina, R. M., Motta, B. M., Indiveri, C., Pujia, A., Montalcini, T., Hedfalk, K., & Romeo, S. (2014). Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1841(4), 574-580. https://doi.org/10.1016/j.bbalip.2013.12.006