TY - JOUR
T1 - Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function
AU - Pingitore, Piero
AU - Pirazzi, Carlo
AU - Mancina, Rosellina M.
AU - Motta, Benedetta M.
AU - Indiveri, Cesare
AU - Pujia, Arturo
AU - Montalcini, Tiziana
AU - Hedfalk, Kristina
AU - Romeo, Stefano
PY - 2014/4
Y1 - 2014/4
N2 - 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.
AB - 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.
KW - Adiponutrin (ADPN)
KW - Lysophosphatidic acid acyltransferase activity
KW - Patatin-like phospholipase domain containing 3 (PNPLA3)
KW - Pichia pastoris
KW - rs738409 (I148M)
KW - Triglyceride hydrolase activity
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U2 - 10.1016/j.bbalip.2013.12.006
DO - 10.1016/j.bbalip.2013.12.006
M3 - Article
C2 - 24369119
AN - SCOPUS:84896895656
VL - 1841
SP - 574
EP - 580
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
SN - 1388-1981
IS - 4
ER -