Purification of the Serine Palmitoyltransferase Complex Responsible for Sphingoid Base Synthesis by Using Affinity Peptide Chromatography Techniques*

FLAG peptide, anti-FLAG M2 affinity gel, various acyl-CoAs, and l-serine and its analogs were purchased from Sigma; nickel-nitrilotriacetic acid (Ni-NTA) agarose was from Qiagen GmbH (Hilden, Germany); egg phosphatidylcholine was from Avanti Polar Lipids Inc. (Alabaster AL); and sucrose monolaurate was from Dojindo Laboratories (Kumamoto, Japan).l-[³H(G)]Serine (20 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO), andl-[U-¹⁴C]serine (158 mCi/mmol) was from Amersham Pharmacia Biotech. Chemically synthesized sphingofungin B was a gift from Dr. Shū Kobayashi (Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan) ().

pSV-cLCB1 is a recombinant plasmid for expression of the hamster wild-type LCB1 protein, and the plasmid pSV-HTcLCB1 encodes an NH₂-terminally His₆-tagged LCB1 protein (). For double tagging of the LCB1 protein with the FLAG and His₆ sequences, plasmid pSV-FHcLCB1, encoding the fusion protein, which has the sequence MDYKDDDDKHHHHHH before the first methionine of the wild-type hamster LCB1 protein (Fig. 1 A), was constructed by introducing appropriate codons into pSV-HTcLCB1 by oligonucleotide-directed site-specific mutagenesis. The nucleotide sequence of the construct was verified by using an automated DNA sequencer (ABI PRISM™ 310, Perkin-Elmer Corp.).

The LY-B strain, a CHO cell mutant strain defective in SPT due to the lack of expression of the LCB1 protein, was previously established by us (). LY-B cells were transfected with pSV-FHcLCB1 by using the LipofectAMINE Plus™ reagent (Life Technologies Inc.). After selection with G418 (400 μg/ml), several colonies resistant to the drug were purified, propagated, and assayed for SPT activity. Among transfected LY-B clones showing recovery of SPT activity, one clone designated as LY-B/FHcLCB1 strain was chosen for purification of the SPT enzyme complex.

pMKITNeo, a mammalian expression vector having the SRα promoter, was a gift from Dr. Kazuo Maruyama (Tokyo Medical and Dental University, School of Medicine, Tokyo, Japan). After deletion of the 3′-untranslated region of the hamster LCB2 cDNA (), the LCB2 open-reading frame was inserted in the expression cloning site of pMKITNeo, and the recombinant plasmid was named pMKITNeo-cLCB2.

LY-B/FHcLCB1 cells were cultivated in spinner bottles containing 1 liter of ES medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 2 mml-glutamine, NaHCO₃ (1 g/l), 10 mm Hepes-NaOH (pH 7.4), and 5% (v/v) fetal calf serum at 37 °C. Thereafter, all manipulations were done at 4 °C or on ice. Cells from 5-liter cultures were harvested by centrifugation (600 × g for 5 min), and washed with 250 ml of phosphate-buffered saline. The washed cells were suspended in 30 ml of 10 mm sucrose containing 1 mm EDTA and homogenized with a 40-ml Dounce tissue grinder (pestle type A, Wheaton, Millville, NJ) by 70 strokes. The homogenate was centrifuged (1000 × g for 15 min) to remove cell debris and nuclei. After addition of 2 m sucrose to the supernatant at a final concentration of 0.25 m, the resultant sample was centrifuged (10⁵ × g for 1 h). The precipitate, as intact membranes, was suspended in 10 mmHepes-NaOH buffer (pH 7.5) containing 0.25 m sucrose at ∼10 mg of protein/ml with a syringe equipped with a 24-gauge needle and stored at −70 °C until use.

A stock suspension of egg phosphatidylcholine (5 mg/ml of deionized water) was prepared by sonication at room temperature. Hereafter, all manipulations were done at 4 °C or on ice. The membrane suspension was centrifuged (10⁵ × g for 30 min) and the precipitated membranes (∼20 mg of protein) were suspended in 4 ml of 125 mm sodium phosphate buffer (pH 8.0) containing 188 mm NaCl. After addition of 0.5 ml of 5 mg/ml phosphatidylcholine and 0.5 ml of 20% (w/v) sucrose monolaurate to the membrane suspension, the mixture was incubated for 10 min. After centrifugation (10⁵ × g, 30 min) of the mixture, the supernatant fluid was recovered as the solubilized membrane fraction. The solubilized membrane fraction was incubated with 0.5 ml of anti-FLAG M2 affinity gel, which had been equilibrated with Buffer A (0.1 m sodium phosphate buffer (pH 8.0) containing 50 mm NaCl, 0.1 mg/ml phosphatidylcholine, and 0.1% sucrose monolaurate), for 90 min with gentle shaking. After centrifugation (10⁴ × g for 20 s), the precipitated gel was suspended in 0.5 ml of Buffer A and transferred to a column. The gel in the column was washed with 15 ml of Buffer A and then eluted with 4.5 ml of Buffer A containing 120 μg/ml FLAG peptide. After addition of imidazole to the elution fraction at a final concentration of 10 mm, this sample was incubated with 0.5 ml of Ni-NTA agarose, which had been equilibrated with Buffer B (0.1m sodium phosphate buffer (pH 8.0) containing 0.1m NaCl, 0.1 mg/ml phosphatidylcholine, 0.1% sucrose monolaurate, and 10 mm imidazole), for 1 h with gentle shaking. After centrifugation (1000 × g for 1 min), the precipitated matrix was suspended in 10 ml of Buffer B and precipitated for washing. After a repeat of this washing step twice, the precipitated matrix was suspended with 5 ml of 0.1 msodium phosphate buffer (pH 8.0) containing 0.1 m NaCl, 0.1 mg/ml phosphatidylcholine, 0.1% sucrose monolaurate, and 250 mm imidazole and incubated for 10 min. After precipitation of the matrix by centrifugation (1000 × g and 1 min), the supernatant fraction was collected as the elution fraction of His₆ affinity chromatography. For removal of imidazole, the elution fraction was diluted 5-fold with 10 mm Hepes-NaOH buffer (pH 7.5) containing 0.25 m sucrose and 0.1% sucrose monolaurate and concentrated to 1–2 ml by ultrafiltration with Ultrafree®-4 (Millipore, Bedford, MA). After repeating these dilution and concentration steps twice, the purified SPT fraction was divided and stored at −70 °C until use. The anti-FLAG M2 affinity gel used was regenerated by washing with glycine-HCl buffer (pH 3.5) and subsequent neutralization.

The enzyme source was incubated in 200 μl of a standard SPT reaction buffer (50 mmHepes-NaOH buffer (pH 7.5) containing 5 mm EDTA, 5 mm dithiothreitol, 50 μm pyridoxal phosphate, 25 μm palmitoyl-CoA, and 0.1 mml-[³H(G)]serine (50 mCi/mmol)) at 37 °C for 10 min. After stopping the reaction, lipids were extracted, and the radioactivity of the [³H]KDS that formed was measured as described previously (). The radioactivity extracted from enzyme-negative controls was regarded as a background control.

SDS-PAGE was carried out by a modification of the method of Laemmli (). Samples for SDS-PAGE were incubated in a SDS-sample buffer (0.1 m Tris-HCl buffer (pH 6.8) containing 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% (w/v) glycerol, and 13 μg/ml bromphenol blue) at 37 °C for 15 min. If necessary, acrylamide gel supplemented with 3 m urea was used. Molecular mass standards were purchased from Bio-Rad. Proteins separated on gel were stained with a silver staining kit (Wako, Osaka, Japan). For Western blot analysis, proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride membrane (Bio-Rad). After the blot membrane was blocked with 10% skim milk in phosphate-buffered saline containing 0.1% Tween 20, the LCB1 and LCB2 proteins on the membrane were detected by using an anti-hamster LCB1 antibody and anti-hamster LCB2 antibody (0.5 μg/ml), respectively, as the primary antibody and a horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad; 1:2500 dilution) as the secondary antibody with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). The anti-hamster LCB1 (anti-cLCB1 73/90) and anti-hamster LCB2 (anti-cLCB2 26/45) antibodies we routinely used were raised by immunization of rabbits with the peptides corresponding to the 73–90th amino acid residues of the hamster LCB1 protein and to the 26–45th amino acid residues of the hamster LCB2 protein, respectively, and purified by affinity chromatography with the antigen peptide coupled matrices.

LY-B/FHcLCB1 and CHO-K1 (4 × 10⁶) cells were seeded in 25 ml of Ham's F-12 medium containing 10% (v/v) newborn calf serum and cultured at 37 °C for 24 h. After a wash with Cys/Met-free RPMI 1640 medium (Dainippon Seiyaku Co., Osaka, Japan) supplemented with 2 mml-glutamine, the monolayers were incubated in 10 ml of Cys/Met-free RPMI 1640 medium supplemented with 2 mml-glutamine, 10% (v/v) fetal calf serum, 1% (v/v) Ham'sF-12 medium, and 0.55 mCi of a [³⁵S]Met/Cys protein labeling mix (³⁵S-EasyTag™, 1175 Ci/mmol, NEN Life Science Products, Inc.) at 37 °C for 18 h. Hereafter, all manipulations were done at 4 °C or on ice unless otherwise noted. After the labeled cells had been harvested by scraping, the membranes were prepared from the cells as described previously (). Membranes were solubilized with 1% sucrose monolaurate in 800 μl of Buffer C (0.1 m sodium phosphate buffer (pH 8.0) containing 0.3 m NaCl and 0.1m sucrose). After centrifugation (10⁵ ×g, 30 min), solubilized supernatant fluid was incubated with protein A-Sepharose (a 50-μl bed volume, Amersham Pharmacia Biotech), which had been equilibrated with Buffer C containing 0.1% sucrose monolaurate, for 1 h. After precipitation of the resin (10⁴ × g for 10 s) to remove proteins nonspecifically bound to protein A-Sepharose, 250 μl of the supernatant fluid was mixed with anti-hamster LCB2 antibody-coupled or preimmune IgG-coupled protein A-Sepharose (a 10-μl bed volume) and incubated for 2 h with shaking as described previously. Then, the resin was washed seven times with 1 ml of Buffer C containing 0.1% sucrose monolaurate and incubated in 80 μl of the SDS-sample buffer at 37 °C for 30 min. After precipitation of the resin, the supernatant fluid was subjected to SDS-PAGE. Radioactive images of proteins on gel were analyzed with a BAS2000 Image Analyzer™ (Fuji Film Co., Tokyo, Japan).

Protein concentrations were determined with the Pierce BCA protein assay kit using bovine serum albumin as the standard.

Although we have previously demonstrated that the expression of a His₆-tagged LCB1 protein in CHO cells causes the SPT activity to adsorb a nickel-immobilized matrix (, ), this affinity fractionation step alone was not enough to purify the SPT enzyme near to homogeneity (data not shown). We therefore decided to construct a plasmid encoding another version of the tagged LCB1 protein (designated the FHcLCB1 protein), in which the FLAG- and His₆ peptide sequences were linked in tandem to the NH₂ terminus of the hamster LCB1 protein (Fig.1 A). After transfection of LY-B cells lacking the endogenous LCB1 subunit with the plasmid, a cell line stably expressing the FHcLCB1 protein was isolated and named the LY-B/FHcLCB1 strain. LY-B/FHcLCB1 cells exhibited SPT activity similar to the wild-type level, whereas LY-B cells had no activity (Fig.1 B). Western blot analysis with an anti-hamster LCB1 antibody showed that the expression level of theM _r 58,000 FHcLCB1 protein in LY-B/FHcLCB1 cells was almost equivalent to the level of the endogenousM _r 53,000 LCB1 protein in wild-type CHO-K1 cells (Fig. 1 C). These observations indicate that the FHcLCB1 protein is capable of functioning as a subunit in the SPT enzyme complex in place of the wild-type LCB1 subunit.

Membranes prepared from LY-B/FHcLCB1 cells were incubated with the nonionic detergent sucrose monolaurate, and, after high speed centrifugation, the supernatant fluid as the solubilized membrane fraction was recovered. The solubilization efficiency of SPT activity was ∼100% (Table I). The solubilized membrane fraction was incubated with an anti-FLAG antibody-coupled matrix, and proteins bound to the matrix were eluted with a buffer containing the FLAG peptide. This step was very effective for purification of SPT: ∼1000-fold enrichment of the activity with a ∼30% recovery yield (Table I). The eluent was then incubated with Ni-NTA agarose as a His₆ affinity matrix, and proteins bound to the matrix were eluted with a buffer containing 250 mm imidazole, resulting in a further 2.5-fold enrichment of the activity with an ∼60% recovery yield (Table I). Imidazole in the elution fraction was removed by ultrafiltration, and the resultant fraction was used for characterization of highly purified SPT.

Protein patterns of the purification fractions were analyzed by SDS-PAGE with 10–20% (w/v) gradient acrylamide gel, which is capable of separating proteins ranging from 6.5 to 200 kDa. Most of the proteins of the solubilized membrane fraction did not bind to the FLAG affinity matrix (Fig. 2 A, lanes 2 and3), and the elution fraction of this chromatography displayed four visible bands, a major band at M _r58,000 and minor bands at M _r 47,000, 31,000, and 26,000, by silver stain (Fig. 2 A, lane 4). After further fractionation by His₆ affinity chromatography, only one band at M _r 58,000 was detected in the elution fraction (Fig. 2 A, lanes 5 and 6). The observation that the highly purified SPT fraction displayed only one visible band on silver stain analysis apparently contradicted our previous conclusion that the SPT enzyme complex contains at least two subunit types, the LCB1 and LCB2 proteins. Western blot analysis with anti-hamster LCB1 and LCB2 antibodies revealed that the FHcLCB1 protein overlapped the LCB2 protein in the SDS-PAGE gel (data not shown; see also Figs. 1 C and 3 C). However, SDS-PAGE with 7.5% (w/v) acrylamide gel containing 3 m urea well separated the FHcLCB1 and LCB2 proteins, allowing us to display two bands (M _r 58,000 and 54,000) in the highly purified SPT fraction (Fig. 2 B).

Based on Western blot analysis, the M _r 58,000 protein in the urea-containing SDS-PAGE gel was identified as the FHcLCB1 protein, and the M _r 54,000 protein was identified as the LCB2 protein (Fig.3 A). We checked specificity of the antibodies used as follows. When the membrane fraction of untransfected CHO-K1 cells was blotted, the anti-LCB2 antibody specifically recognized a M _r 54,000 protein in the acrylamide gel containing 3 m urea (Fig. 3 B, lane 1) or a M _r 58,000 protein in the gel containing no urea (Fig. 3 C, lane 1). Transfection of cells with the hamster LCB2 cDNA-expression vector but not an empty vector caused overexpression of the specifically recognized protein (Fig. 3, B and C), indicating that the specific protein was the LCB2 protein. In addition, Western blotting of membrane fractions from wild-type, LY-B, and LY-B/FHcLCB1 cells proved the anti-hamster LCB1 antibody to be specific (Fig. 1 C). Molecular mass values deduced from the cDNAs of the hamster LCB1, LCB2, and FHcLCB1 proteins were 52,519, 62,882, and 54,468 Da, respectively (). It is currently unknown why theM _r values in SDS-PAGE and the deduced molecular mass values of these proteins differ.

The time course of KDS formation by purified SPT was almost linear for at least 10 min (Fig. 4 A), and this activity was also proportional to the amount of the purified enzyme up to 25 ng (Fig. 4 B). The optimum pH for the activity was found to be between pH 7.5 and 8.5 (Fig. 4 C), and purified SPT was highly sensitive to sphingofungin B, a potent inhibitor of SPT (), with an ID₅₀ of 23 nm (Fig. 4 D). These enzymatic properties of purified SPT were well consistent with those of the SPT activity previously characterized in cell homogenates or membranes from wild-type CHO cells (, ).

When the palmitoyl-CoA dependence of KDS formation by purified SPT was examined, the maximum activity was observed at about 25 μm (Fig. 4 E). The double reciprocal plots of KDS formation versus l-serine in the presence of 25 μm palmitoyl-CoA showed that the apparentK _m for l-serine was 0.28 mmand that the apparent V _max was 660 nmol of KDS/mg of protein/min (Fig. 4 F). Based on this apparentV _max value and the assumption that the one-to-one complex of the FHcLCB1 and LCB2 proteins had one catalytic site, the apparent catalytic number of purified SPT was estimated to be ∼80/min. Due to the inhibitory effect of palmitoyl-CoA at higher concentrations (Fig. 4 E), the K _m forl-serine and V _max values could not be determined under excess concentrations of palmitoyl-CoA.

An analysis of the acyl-CoA substrate specificity of purified SPT indicated that palmitoyl-CoA was the best substrate among various acyl-CoAs examined (Table II). Pentadecanoyl- and heptadecanoyl-CoAs were the next most effective, whereas myristoyl- and stearoyl-CoAs and palmitoleoyl-CoA (an unsaturated form of palmitoyl-CoA) were very poor substrates. Arachidoyl-CoA was ineffective.

To assess the amino acid specificity of the SPT reaction, we determined to what extent the production of [³H]KDS from 0.1 mml-[³H]serine was inhibited by excess amounts of nonradioactive competitors. The inhibition of the [³H]KDS production by 5 mm each ofl-alanine, l-threonine,l-glycyl-l-serine, l-serinamide,d,l-serinol, and l-serine methylester was 40% or less, whereas 5 mm nonradioactive l-serine caused ∼90% inhibition (TableIII).

We next attempted to determine the subunit stoichiometry of the SPT enzyme complex. In the silver-stained pattern of the elution fraction of Ni-NTA agarose chromatography, the band of the FHcLCB1 protein appeared much denser than that of the LCB2 protein (Fig. 2 B). Because there was the possibility that a partial population of the FHcLCB1 protein molecules in this fraction did not associate with the LCB2 protein, we further subjected the purified SPT fraction to a matrix conjugated with an anti-LCB2 antibody and analyzed protein patterns of the fractions adsorbed and unadsorbed by the matrix by SDS-PAGE and silver staining. In the adsorbed fraction, the density of theM _r 58,000 FHcLCB1 protein band was similar to that of the M _r 54,000 LCB2 protein band (Fig.5 A, lane 3). Nonspecific adsorption of the FHcLCB1 protein by the anti-LCB2 antibody-conjugated resin was negligible under the experimental conditions used, because no proteins were adsorbed by the control matrix conjugated with a preimmune IgG (Fig. 5 A, lane 4). Consistently, most of the SPT activity applied to the anti-LCB2 antibody-conjugated matrix was adsorbed, whereas ∼70% of the activity applied to the control matrix was not adsorbed (Fig. 5 B). These results suggested that SPT was a complex of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.

It might be inappropriate to conclude the subunit stoichiometry from the silver staining patterns alone, because it remained undetermined whether the stained density per protein molecule was equal between the different subunits. Therefore, we also employed another measure, the radioactivity of metabolically labeled LCB proteins. LY-B/FHcLCB1 cells were incubated with a [³⁵S]Met/Cys protein labeling mix for 18 h, and membranes were prepared from the cells. After solubilization of membranes under conditions in which SPT sustained the activity, the solubilized membrane fraction was subjected to immunoprecipitation with the anti-hamster LCB2 antibody or preimmune control IgG, and the radioactive proteins immunoprecipitated were analyzed by SDS-PAGE and radioactive image analysis. Two proteins corresponding to the FHcLCB1 and LCB2 proteins were specifically immunoprecipitated with the anti-LCB2 antibody (Fig.6 A, lanes 3 and 4). Because the ratio of the radioactivity of the FHcLCB1 protein to that of the LCB2 protein in the specific immunoprecipitate was determined to be 1:1.75 ± 0.65 (n = 3) by image analysis, the molecular ratio of the FHcLCB1 protein to the LCB2 protein in the immunoprecipitate was estimated to be 1:1.1 ± 0.4 by taking account of the numbers of Cys plus Met residues in these proteins. A nearly identical value (1:1.1 ± 0.3, n = 3) was also estimated for the ratio of the endogenous LCB1 protein to the LCB2 protein, when immunoprecipitation analysis was performed with wild-type CHO cells instead of LY-B/FHcLCB1 cells (Fig. 6 B, lanes 1 and2). We previously showed that under similar conditions of immunoprecipitation, the anti-hamster LCB2 protein immunoprecipitated ∼80% of SPT activity along with most of the LCB1 and LCB2 proteins from the solubilized membrane fraction of wild-type CHO cells (), suggesting that the complex of the LCB1 and LCB2 proteins in the immunoprecipitate was relevant to the active SPT enzyme.

Note that the M _r values of the wild-type LCB1 and LCB2 proteins in 3 m urea-containing gel of SDS-PAGE were 53,000 and 54,000, respectively (Fig. 6 A, lane 2; see also Figs. 1 C and 3 B), whereas theM _r values of the LCB1 and LCB2 proteins in gel containing no urea were 53,000 and 58,000, respectively (Fig. 6 B, lane 2; see also Figs. 1 C and 3 C). Therefore, we used the latter gel conditions for separation of the two wild-type LCB proteins by SDS-PAGE. It remains unknown why theM _r of the LCB2 protein changes depending on the existence of 3 m urea in the gel, but one explanation is that the conformation of the LCB2 protein is affected by urea in SDS-PAGE.