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.

Association of PNPLA3 Polymorphism with Hepatocellular Carcinoma Development and Prognosis in Viral and Non-Viral Chronic Liver Diseases

“…Although the function of PNPLA3 in vivo is currently unknown, in vitro studies have demonstrated that PNPLA3 is able to act as a transacylase that synthesizes intracellular triglycerides by transferring acyl groups (Jenkins et al, 2004). Recent data have also showed that wild type PNPLA3 has a lipase activity and Ile148Met substitution causes a loss of enzyme activity (Pingitore et al, 2014). In animal model, recent evidence from a knock-in mice study has demonstrated that the Ile148Met variant of adiponutrin directly results in the accumulation of hepatic steatosis when fed a high-sucrose diet (Smagris et al, 2015).…”

PNPLA3 gene in liver diseases

“…In mice, He et al showed that the I148M substitution seems to abolish hydrolase activity by reducing substrate access to the enzyme's active site and so promotes TG accumulation suggesting a loss of function [76]. Using recombinant human PNPLA3 and PNPLA3-I148M proteins, studies showed that fatty acid release increased linearly with increased TG and was reduced by the I148M substitution, indicating a loss of function [78,82]. Nevertheless, He et al also observed that an overexpression of wild-type (WT) PNPLA3 in mouse liver created no obvious phenotype and, more specifically, did not reduce hepatic TG content [76].…”

Treating liver fat and serum triglyceride levels in NAFLD, effects of PNPLA3 and TM6SF2 genotypes: Results from the WELCOME trial

“…regulates the efflux and remodelling, but not the influx, of lipid into hepatic lipid droplets (17,(20)(21)(22). In mice, Peter et al (23) have shown that PNPLA3I148M GG genotype is characterised by a decrease in lipolytic activity and lysophosphatidic acid acyl-CoA transferase activity, with a greater than twofold preference for PUFAs.…”