Biochimica et Biophysica Acta 1781(2008)610-E Contents lists available at science Direct Biochimica et Biophysica Acta ELSEVIER journalhomepagewww.elsevier.com/locate/bbalip The domain responsible for sphingomyelin synthase(SMS) activity Calvin Yeang Shweta Varshney a, Renxiao Wang Ya Zhang Deyong Ye b. Xian-Cheng Jiang , Department of Anatomy and Cell Biology, SUNY Downstate Medical Center, USA b School of pharmacy, Fudan University ghai, The People's Republic of china ARTICLE INFO A BSTRACT a间2 Sphingomyelin synthase(SMS)sits at the crossroads of sphingomyelin(SM), ceramide, diacylglycerol (DAG) 2008 metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby form 2 July 2008 regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the Available online 23 July 2008 enzyme, SMSI and SMS2 Both SMSI and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this study, we systematically Keywords: mutated these amino acids using site-directed mutagenesis and found that each point mutation abolished phingomyelin synthase 1 and 2 SMS activity without altering cellular distribution. We also explored the domains which are responsible for oint mutagenesis ellular distribution of both enzymes. Given their role as a potential regulator of diseases, these findings, pled with homology modeling of SMSI and SMS2, will be useful for drug development targeting SMS. o 2008 Elsevier B.V. All rights reserved. 1 Introduction (underlined), are similar to the C2 and C3 motifs in lipid phosphate phosphatase(LPPs) which form a catalytic triad mediating the The de novo synthesis of SM requires the action of nucleophilic attack on the lipid phosphate ester bond [17, 19(Fig. 1). palmitoyl-CoA transferase (SPT). 3-ketosphinganine reduc In this study, using site-directed mutagenesis, we systematically amide synthase, dihydroceramide desaturase, and SMs [1 changed the HHD motif and found that each point mutation last enzyme for SM biosynthesis. It utilizes ceramide and pc as completely abolished SMs activity without altering cellular distribu- substrates to produce SM and DAG 1 and its activity thus directly tion. These results provide a molecular basis for designing SMs influences SM, PC, and ceramide, as well as DAG levels. SMs activity inhibitors. Furthermore, we analyzed the contribution of domains directly links to plasma membrane structures and cell functions which unique to each isoform on the differential localization pattern for y well have impact on disease development [2-7. Many studies SMSI and SMsz ave indicated that SMs is located mainly in the cis-, medial-Golgi 8,91. and plasma membranes [10-12]. There has been evidence of a 2. Methods and materials form of SMs in the trans-Golgi network [13 and at the nuclear level [14]. In addition, SMS activity has been found in chromatin, and 2. 1. Reagents chromatin-associated SMS modifies the SM content [14-16. Of the two mammalian SMS gene isoforms, SMSI is located on cis, medial-Golgi, Bovine brainL-a-phosphatidylcholine(PC)and NBD-C6-ceramide while SMS2 is found in plasma membranes [17). However, a recent were purchased from Sigma. report showed that SMS1 localized to the trans-Golgi network [18] Hydrophobicity analysis has postulated that both SMS1 and SMS2 2.2. Plasmids contain six membrane-spanning alpha helices connected by hydr hilic regions that would form extramembrane loops [16 SMSI and Expression vectors containing SMS1(BC117782)and SMS2 SMS2 contain four highly conserved sequence motifs, designated D1, (BC042899)were purchased from Open Biosystems SMSI and SMS2 D2, D3, and D4(Fig. 1)(17). Motifs D3(G-G-D-X3-S-G-H-T)and D4(H- were subcloned into pCMV-3xFlag14 (Sigma). All point mutations Y-T-X-D-V-X3-Y-X6-F-X2-Y-H), containing conserved amino acids HHd were introduced using the QuikChange Site-Directed Mutagenesis Kit (Strategene). N-terminal SAM domain deletion from SMS1 was Abbreviations: SM, sphingomyelin: SMS, sphingomyelin synthase: LPPs, lipid performed by PCR amplification of base pairs 181-1239 using a s Corresponding authors. D. Ye is to be contacted at Department of Organic BamHl cutting site. N-terminal SAM addition to SMS2 was performed School of Pharmacy Fudan University, 138 Yi Xue Yuan Road, Shanghai by ligation of a PCR amplicon of base pairs 1-184 from SMS1 (using a ina X-C Jiang, Department of Anatomy and Cell Biology, SUNY Downsta Center, 450 Clarkson Ave Box 5, Brooklyn, NY 11203, USA Tel: +1 718 270 3' primer containing an Xbal cutting site)to a PCR amplicon of base 6701;fax:+17182703732 pairs 4-1095 from SMS2 (using a 5 primer containing an Xbal cutting E-mail addresses: dyye@shmuedu cn(D Ye). xiang@downstate edu(X-C. Jiang). site). All constructs were verified by dNA sequencing 1388-19 ee front matter 2008 Elsevier B V. All rights reserved.The domain responsible for sphingomyelin synthase (SMS) activity Calvin Yeang a , Shweta Varshney a , Renxiao Wang b , Ya Zhang b , Deyong Ye b, ⁎, Xian-Cheng Jiang a, ⁎ a Department of Anatomy and Cell Biology, SUNY Downstate Medical Center, USA b School of Pharmacy, Fudan University, Shanghai, The People's Republic of China article info abstract Article history: Received 27 April 2008 Received in revised form 2 July 2008 Accepted 4 July 2008 Available online 23 July 2008 Keywords: Sphingomyelin synthase 1 and 2 Point mutagenesis Lipid phosphate phosphatase Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this study, we systematically mutated these amino acids using site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. We also explored the domains which are responsible for cellular distribution of both enzymes. Given their role as a potential regulator of diseases, these findings, coupled with homology modeling of SMS1 and SMS2, will be useful for drug development targeting SMS. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The de novo synthesis of SM requires the action of a serine palmitoyl-CoA transferase (SPT), 3-ketosphinganine reductase, cer￾amide synthase, dihydroceramide desaturase, and SMS [1]. SMS is the last enzyme for SM biosynthesis. It utilizes ceramide and PC as substrates to produce SM and DAG [1], and its activity thus directly influences SM, PC, and ceramide, as well as DAG levels. SMS activity directly links to plasma membrane structures and cell functions which may well have impact on disease development [2–7]. Many studies have indicated that SMS is located mainly in the cis-, medial-Golgi [8,9], and plasma membranes [10-12]. There has been evidence of a form of SMS in the trans-Golgi network [13] and at the nuclear level [14]. In addition, SMS activity has been found in chromatin, and chromatin-associated SMS modifies the SM content[14-16]. Of the two mammalian SMS gene isoforms, SMS1 is located on cis-, medial-Golgi, while SMS2 is found in plasma membranes [17]. However, a recent report showed that SMS1 localized to the trans-Golgi network [18]. Hydrophobicity analysis has postulated that both SMS1 and SMS2 contain six membrane-spanning alpha helices connected by hydro￾philic regions that would form extramembrane loops [16]. SMS1 and SMS2 contain four highly conserved sequence motifs, designated D1, D2, D3, and D4 (Fig. 1) [17]. Motifs D3 (C-G-D-X3-S-G-H-T) and D4 (H￾Y-T-X-D-V-X3-Y-X6-F-X2-Y-H), containing conserved amino acids HHD (underlined), are similar to the C2 and C3 motifs in lipid phosphate phosphatase (LPPs) which form a catalytic triad mediating the nucleophilic attack on the lipid phosphate ester bond [17,19] (Fig. 1). In this study, using site-directed mutagenesis, we systematically changed the HHD motif and found that each point mutation completely abolished SMS activity without altering cellular distribu￾tion. These results provide a molecular basis for designing SMS inhibitors. Furthermore, we analyzed the contribution of domains unique to each isoform on the differential localization pattern for SMS1 and SMS2. 2. Methods and materials 2.1. Reagents Bovine brainL-α-phosphatidylcholine (PC) and NBD-C6-ceramide were purchased from Sigma. 2.2. Plasmids Expression vectors containing SMS1 (BC117782) and SMS2 (BC042899) were purchased from Open Biosystems. SMS1 and SMS2 were subcloned into pCMV-3 × Flag14 (Sigma). All point mutations were introduced using the QuikChange Site-Directed Mutagenesis Kit (Strategene). N-terminal SAM domain deletion from SMS1 was performed by PCR amplification of base pairs 181–1239 using a 5′ primer containing an EcoR1 cutting site and a 3′ primer containing a BamH1 cutting site. N-terminal SAM addition to SMS2 was performed by ligation of a PCR amplicon of base pairs 1–184 from SMS1 (using a 3′ primer containing an Xba1 cutting site) to a PCR amplicon of base pairs 4–1095 from SMS2 (using a 5′ primer containing an Xba1 cutting site). All constructs were verified by DNA sequencing. Biochimica et Biophysica Acta 1781 (2008) 610–617 Abbreviations: SM, sphingomyelin; SMS, sphingomyelin synthase; LPPs, lipid phosphate phosphatase; SAM, Sterile Alpha Motif ⁎ Corresponding authors. D. Ye is to be contacted at Department of Organic Chemistry, School of Pharmacy, Fudan University, 138 Yi Xue Yuan Road, Shanghai 100003, China. X.-C. Jiang, Department of Anatomy and Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Ave., Box 5, Brooklyn, NY 11203, USA. Tel.: +1 718 270 6701; fax: +1 718 270 3732. E-mail addresses: dyye@shmu.edu.cn (D. Ye), xjiang@downstate.edu (X.-C. Jiang). 1388-1981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2008.07.002 Contents lists available at ScienceDirect Biochimica et Biophysica Acta j o u r n a l h om e p a g e : www. e l s ev i e r. c om / l o c a t e / b b a l i p