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-32.3. 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 immunoprecipita￾tion, 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