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, ceramide 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 hydrophilic 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 (HY-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 distribution. 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
C. Yeang et aL/ Biochimica et Biophysica Acta 1781(2008)610-617 SMS1 168 PLPD 213 RRFFCIVGTLYLYRCITMYVT 277 CGDYLYSGHT 328 HYTVDVVVAYYITTRLFWWYH SMS2 112 PLPD 157 RRFCFILGTLYLYRCITMYVT 221 CGDFLFSGHT 272 HYTIDVILAYYITTRLFWWYH KYSIGRLRPHFLD KYMIGRLRPNFLA KVSIGRLRPHGLS RLSFYSGHS 2241 HHWSDVLTGLIQGAL HHWSDVLVGLLQGAL HHPSDVLAGFAQGAL Fig 1. Alignment of conserved regions between SMS and LPPs. Evolutionary conserved domains(D1-D4)in human SMSI and human SMS2 are aligned with conserved domains (C1-C3)in three human LPPs: LPPL, LPP2, and LPP3 Highlighted amino acids represent residues responsible for catalytic activity in LPPs 2.3. Cell culture and transfection eluted proteins were immunoblotted with an anti-Flag HRP(Sigma) (1: 2000)for 1 h. Following three washes, protein ns were detected by Hela and HEK-293 cells were grown in monolayers at 37C in 5% the chemiluminescence method(Pierce). CO2. All cells were cultured in DMEM containing 10%(v/v ) FBS, 100 U ml penicillin and streptomycin, and 2 mM glutamine Plasmids were 2.5. Immunohistochemistry transfected using Lipofectamine 2000(Invitrogen). Cells were grown, transfected with plasmid, and prepared for 2.4. Immunoprecipitation and immunoblotting microscopy directly on an eight-well chamber slide( Nunc) for 48 h. s. Cells were lysed in 200 mM NaCl, 50 mM Tris(pH 7.5). 1 mM EDTA, in 4 formaldehyde in PBS for 10 min Cells were washed with PBS and nd 1%(v/v) protease inhibitor cocktail(Sigma). Cell debris were permeabilized with PBS containing 0.1% Triton X-100, washed, and cleared by centrifugation at 8200 g for 10 min For immunoprecipita- then blocked in 3% BSA in PBS for 1 h Cells were then incubated with tion, lysates were incubated with Flag antibody conjugated Nickel anti-Flag Cy3 1: 250(Sigma) and anti-panCadherin FITC 1: 250 Affinity Gel (Sigma) for 2 h. The gel was then washed in lysis buffer.(Abcam) or anti-Flag Cy3 1: 250 and anti-cMannosidase Il 1: 25 bound proteins were eluted by adding SDS PAGE loading buffer. The (USBiological) diluted in blocking buffer. Where applicable, cells H+NBD-SM 器二=二==98H Cadherin Flag TOPRO-3 Merge SMS1-Flag TOPAO-3 a Mannosidase ll Flag TOPRO-3 erge abolishes SMSI activity. (A) SMS activity was performed on wild type(wt) or mutant SMSI munoprecipitation from Hela cells transiently expressing the respective enzyme. The protein level of each targeted protein. Both cadherin( top)and c Mannosidase ll (bottom) are depicted in green, while SMSl-Flag is depicted in red. TOPRO-3, a DNA dye is in blue. (C)Each SMS (S283A, H285A, H328A, H332A, S273A)exhibits a wTlocalization pattern a Mannosidase ll is depicted in green. Flag-tagged SMSI and mutants are depicted in red. TOPRO-3
2.3. Cell culture and transfection Hela and HEK-293 cells were grown in monolayers at 37 °C in 5% CO2. All cells were cultured in DMEM containing 10% (v/v) FBS, 100 U/ ml penicillin and streptomycin, and 2 mM glutamine. Plasmids were transfected using Lipofectamine 2000 (Invitrogen). 2.4. Imunoprecipitation and immunoblotting Cells were lysed in 200 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, and 1% (v/v) protease inhibitor cocktail (Sigma). Cell debris were cleared by centrifugation at 8200 g for 10 min. For immunoprecipitation, lysates were incubated with Flag antibody conjugated Nickel Affinity Gel (Sigma) for 2 h. The gel was then washed in lysis buffer, bound proteins were eluted by adding SDS PAGE loading buffer. The eluted proteins were immunoblotted with an anti-Flag HRP (Sigma) (1:2000) for 1 h. Following three washes, proteins were detected by the chemiluminescence method (Pierce). 2.5. Immunohistochemistry Cells were grown, transfected with plasmid, and prepared for microscopy directly on an eight-well chamber slide (Nunc) for 48 h. Subconfluent cells were washed three times with PBS and then fixed in 4% formaldehyde in PBS for 10 min. Cells were washed with PBS and permeabilized with PBS containing 0.1% Triton X-100, washed, and then blocked in 3% BSA in PBS for 1 h. Cells were then incubated with anti-Flag Cy3 1:250 (Sigma) and anti-panCadherin FITC 1:250 (Abcam) or anti-Flag Cy3 1:250 and anti-αMannosidase II 1:25 (USBiological) diluted in blocking buffer. Where applicable, cells Fig. 1. Alignment of conserved regions between SMS and LPPs. Evolutionary conserved domains (D1–D4) in human SMS1 and human SMS2 are aligned with conserved domains (C1–C3) in three human LPPs: LPP1, LPP2, and LPP3. Highlighted amino acids represent residues responsible for catalytic activity in LPPs. Fig. 2. Point mutation of individual conserved residues within D3 and D4 abolishes SMS1 activity. (A) SMS activity was performed on wild type (WT) or mutant SMS1 following immunoprecipitation from Hela cells transiently expressing the respective enzyme. The protein level of each enzyme is shown in a western blot below. (B) Wild type SMS1 is a Golgi targeted protein. Both cadherin (top) and α Mannosidase II (bottom) are depicted in green, while SMS1-Flag is depicted in red. TOPRO-3, a DNA dye is in blue. (C) Each SMS1 mutant (S283A, H285A, H328A, H332A, S273A) exhibits a WT localization pattern. α Mannosidase II is depicted in green. Flag-tagged SMS1 and mutants are depicted in red. TOPRO-3 is in blue. C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617 611
612 a Mannosidase‖l Fla TOPRO-3 S283A H285A H328A H332A S733A were incubated with anti-rabbit FITC 1: 1000(Vector Labs ). After three (v/v) protease inhibitor cocktail (Sigma). Cell debris were cleared washes, TOPRO-3(Invitrogen), diluted 1: 1000 in blocking buffer, was by centrifugation at 8200 g for Immunoprecipitation was added to cells to stain the nuclei. Slides were mounted in Vectashield performed by incubating lysate 30 Al of an anti-Flag M2 Vector Labs) and analyzed on a confocal microscope(Bio-Rad monoclonal antibody covalently bound to agarose beads(Sigma) Radiance 2000)using 488 nm, 543 nm, and 638 nm excitation and a for 2 h Immune complexes were washed three times with 50 mM 40x objective lens. Images were processed with Photoshop(Adobe). Tris(pH 7.5), 5% sucrose, 1 mM EDTA SMSI or SMS2 activity assay was then performed as previously described [2 Briefly, 50 mM 2.6. SMSI, SMS2-specific activity assay Tris-HCl(pH 7.4). 25 mM KCl, C6-NBD-ceramide(o1 ug/ul), and phosphatidylcholine(0.01 ug/y) was incubated with the immuno- Cells, transiently expressing Flag-tagged SMS1 or SMS2, were precipitate at 37 C for 45 min. Lipids were extracted in chloro- lysed in 200 mM NaCl, 50 mM Tris(pH 7.5). 1 mM EDTA, and 1% form: methanol (2: 1), dried under N2 gas, and separated by thin
were incubated with anti-rabbit FITC 1:1000 (Vector Labs). After three washes, TOPRO-3 (Invitrogen), diluted 1:1000 in blocking buffer, was added to cells to stain the nuclei. Slides were mounted in VectaShield (Vector Labs) and analyzed on a confocal microscope (Bio-Rad Radiance 2000) using 488 nm, 543 nm, and 638 nm excitation and a 40× objective lens. Images were processed with Photoshop (Adobe). 2.6. SMS1-, SMS2-specific activity assay Cells, transiently expressing Flag-tagged SMS1 or SMS2, were lysed in 200 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, and 1% (v/v) protease inhibitor cocktail (Sigma). Cell debris were cleared by centrifugation at 8200 g for 10 min. Immunoprecipitation was performed by incubating lysate with 30 μl of an anti-Flag M2 monoclonal antibody covalently bound to agarose beads (Sigma) for 2 h. Immune complexes were washed three times with 50 mM Tris (pH 7.5), 5% sucrose, 1 mM EDTA. SMS1 or SMS2 activity assay was then performed as previously described [2]. Briefly, 50 mM Tris–HCl (pH 7.4), 25 mM KCl, C6-NBD-ceramide (0.1 μg/μl), and phosphatidylcholine (0.01 μg/μl) was incubated with the immunoprecipitate at 37 °C for 45 min. Lipids were extracted in chloroform: methanol (2:1), dried under N2 gas, and separated by thin Fig. 2 (continued). 612 C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617
C. Yeang et aL/ Biochimica et Biophysica Acta 1781(2008)610-617 layer chromatography (TLC) using Chloroform: MeOH: NH4OH catalytic acid-base residue in LPPs (19. H328 and D332, meanwhile, act coordinately to mediate a nucleophilic attack on the phosphomo- nester bond. Serine 283 is evolutionarily conserved between animal 3. Results and discussion SMSs 17 as well as within human LPPs[ 20). This serine in LPPs has been implicated in hydrogen binding to the phosphate 3. 1. Domains responsible for SMS activity Serine 273 mutant served as a control. since $273 is outside of D3 and is not in the putative active site. In order to contr From ceramide and phosphatidylcholine, SMs generates SM, expression levels, we added a Flag tag at the c terminus of each and DAG as a side product. In this process the phosphomonoester construct To accurately compare the specific activity of each mutant bond between phosphocholine and DAG of phosphatidylcholine with wild type SMSI without noise from endogenous SMSI and SMS2 must be broken, allowing the transfer of phosphocholine to(both are expressed in all tissues which were tested ) SMS activity ceramide. Due to similarities between SMs and LPPs, it has been assay was performed following Flag affinity immunopurification. speculated that they share a common catalytic mechanism, indicated in Fig. 2A, SMS1-Flag(WT) and S273A have comparable ever, this concept has not been proven. Domains designated specific SMS activity, but H285A, H328A, D332A, and S283A have no D3 and D4 from both SMSI and SMS2 share key sequence ho- detectable activity. Therefore, each amino acid of the hhd triad in with the active site of LPPs. Within the C2 and C3 SMSl, as well as the conserved serine 283 is necessary for SMS1 of LPPs, two histidines and one aspartic acid form a activity triad mediating phosphomonoester hydrolysis [17, 19 To rule out the possibility that any of these point mutations yielded (Fig. 1). an inactive enzyme secondary to causing a global change in protein To demonstrate that the analogous His. His, Asp(HHD) triad in structure, we confirmed that the subcellular localization of each SMSI is responsible for SMS activity, we utilized site-directed mutant remained identical to the wild type enzyme. As expected mutagenesis to replace each amino acid with alanine and prepared confocal analysis showed that SMsl-Flag(wt) are located on golgi five unique SMSI mutants(S273A, S283A, H285A, H328A, and D332A) complex, since it was co-localized with a Mannosidase Il, a well- along with WT SMSI(Fig. 2A). In SMS1, H285 corresponds to the known Golgi marker(Fig 2B). There is no detectable signal on plasma HNBD-SM 40 kD < - SMS2-Flag Cadherin Flag TOPRO-3 Cadherin TOPRO TOPRO-3 a Mannosidase ll TOPRO-3 Fig. 3. Point mutation of individual conserved residues within D3 and D4 abolishes SMS2 activity with the exception of $227(A)SMs activity was performed on wild type (wt)or autant SMSI following immunoprecipitation fro la cells transiently expressing the respective enzyme. The protein levels of each enzyme is shown below. (B)Wild type SMS2 ocalizes to both the plasma membrane and Golgi. Both cadherin(top)and a Mannosidase ll(bottom)are depicted in green, while SMS2-Flag is depicted in red. TOPRO-3 is in blue. (C) Each SMSI mutant(S2227A, H229A, H272A H276A, S217A)exhibits a WT localization pattern. Cadherin is depicted in green. Flag-tagged SMSI and mutants are depicted in red. TOPRo-3 is in blue
layer chromatography (TLC) using Chloroform:MeOH:NH4OH (14:6:1). 3. Results and discussion 3.1. Domains responsible for SMS activity From ceramide and phosphatidylcholine, SMS generates SM, and DAG as a side product. In this process the phosphomonoester bond between phosphocholine and DAG of phosphatidylcholine must be broken, allowing the transfer of phosphocholine to ceramide. Due to similarities between SMS and LPPs, it has been speculated that they share a common catalytic mechanism, however, this concept has not been proven. Domains designated D3 and D4 from both SMS1 and SMS2 share key sequence homologies with the active site of LPPs. Within the C2 and C3 domains of LPPs, two histidines and one aspartic acid form a catalytic triad mediating phosphomonoester hydrolysis [17,19] (Fig. 1). To demonstrate that the analogous His, His, Asp (HHD) triad in SMS1 is responsible for SMS activity, we utilized site-directed mutagenesis to replace each amino acid with alanine and prepared five unique SMS1 mutants (S273A, S283A, H285A, H328A, and D332A) along with WT SMS1 (Fig. 2A). In SMS1, H285 corresponds to the catalytic acid–base residue in LPPs [19]. H328 and D332, meanwhile, act coordinately to mediate a nucleophilic attack on the phosphomonoester bond. Serine 283 is evolutionarily conserved between animal SMSs [17] as well as within human LPPs [20]. This serine in LPPs has been implicated in hydrogen binding to the phosphate group [20]. Serine 273 mutant served as a control, since S273 is outside of D3, and therefore is not in the putative active site. In order to control for expression levels, we added a Flag tag at the C terminus of each construct. To accurately compare the specific activity of each mutant with wild type SMS1 without noise from endogenous SMS1 and SMS2 (both are expressed in all tissues which were tested), SMS activity assay was performed following Flag affinity immunopurification. As indicated in Fig. 2A, SMS1-Flag (WT) and S273A have comparable specific SMS activity, but H285A, H328A, D332A, and S283A have no detectable activity. Therefore, each amino acid of the HHD triad in SMS1, as well as the conserved serine 283 is necessary for SMS1 activity. To rule out the possibility that any of these point mutations yielded an inactive enzyme secondary to causing a global change in protein structure, we confirmed that the subcellular localization of each mutant remained identical to the wild type enzyme. As expected, confocal analysis showed that SMS1-Flag (WT) are located on Golgi complex, since it was co-localized with α Mannosidase II, a wellknown Golgi marker (Fig. 2B). There is no detectable signal on plasma Fig. 3. Point mutation of individual conserved residues within D3 and D4 abolishes SMS2 activity with the exception of S227. (A) SMS activity was performed on wild type (WT) or mutant SMS1 following immunoprecipitation from Hela cells transiently expressing the respective enzyme. The protein levels of each enzyme is shown below. (B) Wild type SMS2 localizes to both the plasma membrane and Golgi. Both cadherin (top) and α Mannosidase II (bottom) are depicted in green, while SMS2-Flag is depicted in red. TOPRO-3 is in blue. (C) Each SMS1 mutant (S2227A, H229A, H272A, H276A, S217A) exhibits a WT localization pattern. Cadherin is depicted in green. Flag-tagged SMS1 and mutants are depicted in red. TOPRO-3 is in blue. C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617 613
c Cadherin Fla TOPRO-3 Merge S227A H229A H272A D276A s217A membrane(Fig. 2B). This result confirmed a previous report (17. approximately 30% of a wild type specific activity, suggesting that Moreover, all mutants have a normal cellular distribution, compared although both serines are located next to HHd motifs(Fig 1A), they with WT( Fig 2B and C), indicating the normal enzyme topology is still have a different impact on catalytic domain formation. It has been maintained in the"dead"enzyme suggested that S181 in LPPl, analogous to S283 in SMSI and S227 in We also prepared Flag-tagged wild type SMS2 as well as mutants SMSZ, can form a hydrogen bond with the phosphate group while it analogous to those described above for SMS1( Fig. 3). Similarly, is in the active site instead of directly participating in catalysis [ 19]. individual mutations of the SMSz HHD triad all abolished SMS2 Therefore, it is not surprising that $227A is not completely activity as opposed to wild type SMS2 and the control(S217A) necessary for SMs activity. there is likely a neighboring residue nutant(Fig 3A). Interestingly, one difference between SMSI and within the tertiary structure of SMS2 which is functionally equi SMS2 is that SMS1-S283A has no activity, while SMS2-S227A retains valent to $227
membrane (Fig. 2B). This result confirmed a previous report [17]. Moreover, all mutants have a normal cellular distribution, compared with WT (Fig. 2B and C), indicating the normal enzyme topology is still maintained in the “dead” enzyme. We also prepared Flag-tagged wild type SMS2 as well as mutants analogous to those described above for SMS1 (Fig. 3). Similarly, individual mutations of the SMS2 HHD triad all abolished SMS2 activity as opposed to wild type SMS2 and the control (S217A) mutant (Fig. 3A). Interestingly, one difference between SMS1 and SMS2 is that SMS1-S283A has no activity, while SMS2-S227A retains approximately 30% of a wild type specific activity, suggesting that although both serines are located next to HHD motifs (Fig. 1A), they have a different impact on catalytic domain formation. It has been suggested that S181 in LPP1, analogous to S283 in SMS1 and S227 in SMS2, can form a hydrogen bond with the phosphate group while it is in the active site instead of directly participating in catalysis [19]. Therefore, it is not surprising that S227A is not completely necessary for SMS activity. There is likely a neighboring residue within the tertiary structure of SMS2 which is functionally equivalent to S227. Fig. 3 (continued). 614 C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617
C. Yeang et aL/ Biochimica et Biophysica Acta 1781(2008)610-617 615 As shown in Fig. 3B, a portion of SMS2-Flag(WT)was located on 3. 2. Analysis of a domain unique to SMSI on subcellula plasma membrane where it co-localized with cadherin(a well-know plasma membrane marker), and a portion was found in the peri- ASl is a Golgi protein which has been co-localized with the golgi nuclear region where it co-localized with Golgi marker a Mannosi- marker Mannosidase ll [17](Fig 2B). SMS2 on the other hand localizes dase ll. This result also confirmed a previous report[17. Furthermore, to both the plasma membrane as well as the Golgi [17 ( Fig. 3B). There all mutants have an identical cellular distribution as WT (Fig. 3B and are no known or predicted targeting signals in either protein. C), suggesting that these point mutations only influenced SMS2 Although the genes encoding these two isoforms are located on catalytic activity but not the enzyme topology distinct chromosomes, SMSI and SMS2 are 51.5% identical in protein So far, no experimental studies identifying the locale of an SMs sequence active site have been reported. In this study, we utilized site- Sequence-wise, the most striking difference between the two directed mutagenesis to elucidate the catalytic structure for both isoforms is that SMSI, but not SMS2 has an N-terminal Sterile alpha SMSI and SMSZ SMS activity is closely related with four important Motif (SAM) domain. We explored the possibility that this SMs1 lipids, ceramide, DAG, SM, and phosphatidylcholine, which are unique sequence may play a role in the differential subcellular involved in plasma membrane formation, signal transduction, and localization of SMSI and SMS2 lipoprotein metabolism. The catalytically inactive enzymes created Truncation of 61 amino acids from the sms1 sam domain resulted by this study will be ful in the future studies in a mutant with an identical distribution pattern as the wild type SMS is a therapeutie for the treatment of disease enzyme( Fig. 5C). Furthermore, addition of these 61 amino acids to the cancer and atheros and elucidating the amino N-terminus of SMS2 did not alter localization of the protein( Fig. 5D). form an active site will provide a molecular basis for de- This was surprising because trans-Golgi resident proteins have been signing the drugs known to target to the Golgi through aggregation and subsequent Based on the LPP structure [18 and through homology modeling, exclusion from transport vesicles. SAM, conventionally thought of as a we have created a tertiary structure model of SMS1, which shows a protein interaction domain, has also been shown to mediate homo- favorable distance of 4.27 A for the interaction between H328 and oligomerization [21 Although the results are not expected, we D332, as well as an 11.90 A distance between H328 and H283 which conclude that the SMS1 SAM domain is neither necessary nor can accommodate the phosphate group from phosphatidylcholine sufficient for Golgi targeting of SMS. The domains, responsible to (Fig 4). This model is also applicable to SMSZ From this model, we can subcellular distribution of both enzymes, should be located within the clearly see that the replacement of any one of these three amino acids region where SMSI and SMS2 share the similarity. Furthermore, the abolishes SMSI or SMS2 activity in the cells. SAM domain does not influence SMS activity As shown in Fig 5B, 1190A otein(PDB entry 2IC8)and PhoN protein(PDB entry 2AKC), with known structures, were used as templates. Multiple sequence alignments and construction of homology models the respective distances between residues are depicted
As shown in Fig. 3B, a portion of SMS2-Flag (WT) was located on plasma membrane where it co-localized with cadherin (a well-known plasma membrane marker), and a portion was found in the perinuclear region where it co-localized with Golgi marker α Mannosidase II. This result also confirmed a previous report [17]. Furthermore, all mutants have an identical cellular distribution as WT (Fig. 3B and C), suggesting that these point mutations only influenced SMS2 catalytic activity but not the enzyme topology. So far, no experimental studies identifying the locale of an SMS active site have been reported. In this study, we utilized sitedirected mutagenesis to elucidate the catalytic structure for both SMS1 and SMS2. SMS activity is closely related with four important lipids, ceramide, DAG, SM, and phosphatidylcholine, which are involved in plasma membrane formation, signal transduction, and lipoprotein metabolism. The catalytically inactive enzymes created by this study will be very useful in the future studies. Potentially, SMS is a therapeutic target for the treatment of diseases, such as cancer and atherosclerosis, and elucidating the amino acids that form an active site for SMS will provide a molecular basis for designing the drugs. Based on the LPP structure [18] and through homology modeling, we have created a tertiary structure model of SMS1, which shows a favorable distance of 4.27 Å for the interaction between H328 and D332, as well as an 11.90 Å distance between H328 and H283 which can accommodate the phosphate group from phosphatidylcholine (Fig. 4). This model is also applicable to SMS2. From this model, we can clearly see that the replacement of any one of these three amino acids abolishes SMS1 or SMS2 activity in the cells. 3.2. Analysis of a domain unique to SMS1 on subcellular localization SMS1 is a Golgi protein which has been co-localized with the Golgi marker Mannosidase II [17] (Fig. 2B). SMS2 on the other hand localizes to both the plasma membrane as well as the Golgi [17] (Fig. 3B). There are no known or predicted targeting signals in either protein. Although the genes encoding these two isoforms are located on distinct chromosomes, SMS1 and SMS2 are 51.5% identical in protein sequence. Sequence-wise, the most striking difference between the two isoforms is that SMS1, but not SMS2 has an N-terminal Sterile Alpha Motif (SAM) domain. We explored the possibility that this SMS1 unique sequence may play a role in the differential subcellular localization of SMS1 and SMS2. Truncation of 61 amino acids from the SMS1 SAM domain resulted in a mutant with an identical distribution pattern as the wild type enzyme (Fig. 5C). Furthermore, addition of these 61 amino acids to the N-terminus of SMS2 did not alter localization of the protein (Fig. 5D). This was surprising because trans-Golgi resident proteins have been known to target to the Golgi through aggregation and subsequent exclusion from transport vesicles. SAM, conventionally thought of as a protein interaction domain, has also been shown to mediate homooligomerization [21]. Although the results are not expected, we conclude that the SMS1 SAM domain is neither necessary nor sufficient for Golgi targeting of SMS. The domains, responsible to subcellular distribution of both enzymes, should be located within the region where SMS1 and SMS2 share the similarity. Furthermore, the SAM domain does not influence SMS activity. As shown in Fig. 5B, Fig. 4. A favorable conformation of active site of human SMS1. The three-dimensional structural model of human SMS1 was built through homology modeling. Two proteins, GlpG protein (PDB entry 2IC8) and PhoN protein (PDB entry 2AKC), with known structures, were used as templates. Multiple sequence alignments and construction of homology models were conducted by using the Modeler module in the Discovery Studio software (version 1.6, Accelrys Inc.). The conserved histidine 285, histidine 328, and histidine 332 along with the respective distances between residues are depicted. C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617 615
C Yeang et al/ Biochimica et Biophysica Acta 1781(2008 )610-617 SMS1 SMS2 國回國画包 MS1ASAM 國國國國 SAM-SMS2 國國國國國 NBD-SM NBD-SM 50 KD 40 kD a Mannosidase‖l Flag TOPRO-3 Merge Cadherin Flag TOPRO-3 Merge Fig. 5. Analysis of potentially important domains for the differential localization of SMSI and SMS2. (A)Schematic of the Flag-tagged constructs used. SAM: Sterile Alpha Motif. TM transmembrane domain. (B)SMS1ASAM-Flag, a mutant with truncation of the first 61 amino acids from SMSl, shows no defect in SMs activity compared with WT SMSl-Flag. Likewise, SAM-SMS2, a fusion protein comprised of the SAM domain from SMSI(amino acids 1-61)and the entirety of sMS2, has unaltered sMs activity compared to wT-SMS2-Flag depicted in red. TOPR ch enzyme is shown at the bottom.(C) SMS1ASAM-Flag remains as a Golgi targeted protein. a Mannosidase ll is depicted in green, while SMSIASAM-Flag is depicted in red. TOPRO-3 is in blue of SAM from SMSI or addition of SAM to SMS2 has no specific properties for both SMSI and SMSZ. This aspect deserves ble impact on SMS activity. Since SMs activity is on. For an unknown reason SMSl-Flag western blot Figs. 2A and 5B). The up band with correct impact on the process of disease development. to of SMSI-Flag disappeared in truncated SMSl-Flag mains could provide a molecular basis for studying isoform- transfected cells( Fig 5B)
deletion of SAM from SMS1 or addition of SAM to SMS2 has no appreciable impact on SMS activity. Since SMS activity is potentially important in certain diseases, and SMS1 and SMS2 activity may have a different impact on the process of disease development, to elucidate these domains could provide a molecular basis for studying isoformspecific properties for both SMS1 and SMS2. This aspect deserves further investigation. For an unknown reason SMS1-Flag western blot showed a doublet (Figs. 2A and 5B). The up band with correct molecular weight of SMS1-Flag disappeared in truncated SMS1-Flag transfected cells (Fig. 5B). Fig. 5. Analysis of potentially important domains for the differential localization of SMS1 and SMS2. (A) Schematic of the Flag-tagged constructs used. SAM: Sterile Alpha Motif. TM: transmembrane domain. (B) SMS1ΔSAM-Flag, a mutant with truncation of the first 61 amino acids from SMS1, shows no defect in SMS activity compared with WT SMS1-Flag. Likewise, SAM-SMS2, a fusion protein comprised of the SAM domain from SMS1 (amino acids 1–61) and the entirety of SMS2, has unaltered SMS activity compared to WT-SMS2-Flag. The protein levels of each enzyme is shown at the bottom. (C) SMS1ΔSAM-Flag remains as a Golgi targeted protein. α Mannosidase II is depicted in green, while SMS1ΔSAM-Flag is depicted in red. TOPRO-3 is in blue. (D) SAM-SMS2-Flag exhibits an identical localization pattern compared to wild type SMS2. Cadherin is depicted in green, while SAM–SMS2-Flag is depicted in red. TOPRO-3 is in blue. 616 C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617
C. Yeang et aL/ Biochimica et Biophysica Acta 1781(2008)610-617 [9 A.H. Schweizer, Clausen, G. van Meer, H.P. Hauri, Localization of O-glycan authors thn was supported by NIH HL-64735 and HL-69817.The This we respect to the endoplasmic reticulum-Golgi intermediate compartment. 」 BioL Chem.269(1994)4035-4041. Dr. Zhiguo Liu from the Shanghai Institute of Organic [101 M. Malgat, A. Maurice. J. Baraud, Sphingomyelin and ceran Chemistry for his help on the homology modeling of the human SMS1 es and plasma membranes from rat liver and brain. [111 P. Moreau, C. Cassagne, Phospholipid trafficking and membrane biogenesis him. Biophys. Acta 1197(1994)257-290. References [12 M ]. Obradors, he subcellular sites of sphingomyelin nthesis in BHK cells, Biochim. Biophys. Acta. 1359(1997)1-12. [11 D.R. Voelker, E.P. Kennedy, Cellular and enzymic synthesis of sphingomyelin, Bio- [13 D. Allan, M. Obradors, Enzyme distributions in infected with Semliki forest virus: evidence for a major fraction of sphing [2]Z.Li, T.K. Hailemariam, H. Zhou, Y. Li, D.C. Duckworth, D.A. Peake, Y. Zhang, M.S. yelin synthase in the trans-Golgi network. Biochim. Biophys. Acta. 1450(1999) tracellular sphingomyelin accumulation and plasma membrane lipid organiza- [14] E Albi, M P Magni, Sphingomyelin synthase in rat liver nuclear membrane and [ 3] T Ding, ZLi, T. Ha Mukherjee, F.R. Maxfield, M P Wu, X C Jiang, SMS [15] E Albi, M.P. Magni, Chromatin-associated in: metabolism in relation [16 E Albi, S Pieroni, M P Magni, C Sartori, Chromatin sphingomyelin changes in cell 4 F.G. Tafesse, K Huitema, M. Hermansson, S. van der Poel J. van den Dikkenberg, A. proliferation and/or apoptosis induced by ciprofibrate. Cell Physiol. 196(2003) S2 are required for sphingomyelin homeostasis and growth in human HeLa [17 K Huitema. ]. van den Dikkenberg, J.F. Brouwers, J.C. Holthuis, Identification of a s. I BioL chem282(2007)17537-17547. 23(2004)33-44 [5 M. Villani, M, Subathra, Y.B. Im, Y. Choi, P. Signorelli, M. Del Poeta, C. Luberto. [18 D Halter, S. Neumann, S M. van Dijk, ]. Wolthoorn, A.M. de Maziere, o V Vieira, P. Sphingomyelin synthases regulate production of diacylglycerol at the golgi. attjus, I Klumperman, G. van Meer, H Sprong, Pre- and post-Golgi translocation lucosylceramide in glycosphingolipid synthesis, I Cell BioL. 179(2007) [6] D Separovic, L Semaan, AL Tarca, M.Y. Awad Maitah, K Hanada, J Bielawski, M. illani, C Luberto, Suppression of sphingomyelin synthase 1 by small interference [19 A.F. Neuwald, An unexpected structural relationship between integral mem- photodamage, Exp Cell Res 314 (2008)1860-1868. [7 D Separovic, K Hanada, M Y. Maitah, B Nagy, L Hang, M-A. Tainsky, J.M. Kraniak]. [20 Qx Zhang, CS. Pilquil, J Dewald, Lo ielawski, Sphingomyelin synthase 1 suppresses ceramide pro ructurally important domains of ate phosphatase-1: implications ost-photodamage, Biochem Biophys Res Commun. 358(2007)196-202. [8] D Jeckel, A Karrenbauer, R. Birk, RR. Schmidt, F. wieland, Sphingomyelin is [211 C D. Thanos, K E Goodwill, J.U. Bowie, Oligomeric structure of the human EphB2 thesized in the cis Golgi, FEBS Lett. 261(1990)155-157. receptor SAM domain, Science 283 (1999)833-836
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