Abstract
Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.
Highlights
Bacterial PC-phospholipase C (PLC) was cloned previously [5], the gene, which is responsible for mammalian PC-PLC, is still unknown so far
From sphingomyelin synthase (SMS) catalytic activity, we noticed that SMS1- or sphingomyelin synthase and 2 (SMS2)-mediated SM formation can be separated into two steps: [1] PC hydrolysis step, where PC is hydrolyzed into P-choline and DAG and [2] SM formation step, where P-choline is added onto ceramide
To avoid the noise from endogenous SMS activity, we immunoprecipitated each flagged SMS using a flag antibody and utilizing the precipitate to perform SMS activity measurement, using PC and nitrobenz-diazol (NBD)-ceramide as two substrates. We found that both SMS1-flag and SMS2-flag but not SMS-related protein (SMSr)-flag and empty vector had SM synthase activity (Fig. 1A)
Summary
Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity. Yeun-po Chiang , Zhiqiang Li1,2 , Yang Chen, Yu Cao3,* , and Xian-Cheng Jiang1,2,* From the 1Department of Cell Biology, SUNY Downstate Health Sciences University, Brooklyn, New York, USA; 2Molecular and Cellular Cardiology Program, VA New York Harbor Healthcare System, Brooklyn, New York, USA; 3Institute of Precision Medicine, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
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