Cloning and Initial Characterization of a New Subunit for Mammalian Serine-palmitoyltransferase

Serine-palmitoyltransferase (SPT) catalyzes the rate-limiting step of the de novo synthesis of sphingolipids. SPT is considered to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Here we report the identification of a novel, third, SPT subunit (SPTLC3) that shows 68% homology to the SPTLC2 subunit. Quantitative real-time PCR revealed that SPTLC3 expression is highly variable between different human tissues and cell lines. The highest expression was observed in placenta tissue and human trophoblast cell lines. The overexpression of SPTLC3 in Hek293 cells, which otherwise have very little endogenous SPTLC3, led to a 2- to 3-fold increase in cellular SPT activity. Silencing of SPTLC3 expression in HepG2 cells or human trophoblast cells by transfecting SPTLC3-specific siRNA resulted in a significant reduction of cellular SPT activity. The expression of two SPT isoforms could be a cellular mechanism to adjust SPT activity to tissue-specific requirements of sphingolipid synthesis. Serine-palmitoyltransferase (SPT) catalyzes the rate-limiting step of the de novo synthesis of sphingolipids. SPT is considered to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Here we report the identification of a novel, third, SPT subunit (SPTLC3) that shows 68% homology to the SPTLC2 subunit. Quantitative real-time PCR revealed that SPTLC3 expression is highly variable between different human tissues and cell lines. The highest expression was observed in placenta tissue and human trophoblast cell lines. The overexpression of SPTLC3 in Hek293 cells, which otherwise have very little endogenous SPTLC3, led to a 2- to 3-fold increase in cellular SPT activity. Silencing of SPTLC3 expression in HepG2 cells or human trophoblast cells by transfecting SPTLC3-specific siRNA resulted in a significant reduction of cellular SPT activity. The expression of two SPT isoforms could be a cellular mechanism to adjust SPT activity to tissue-specific requirements of sphingolipid synthesis. Sphingolipids are a ubiquitously distributed class of lipids that can be found in all higher organisms. Sphingoid bases confer important structural properties to membranes and to their partition into microdomains (membrane rafts) and modulate the activities of various enzymes such as protein kinases, protein phosphatases, and phospholipases in cells or cell-free systems (1Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (438) Google Scholar). They are involved in many cellular events, including proliferation, differentiation, senescence, apoptosis, and inflammatory response (2Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar). De novo sphingolipid biosynthesis is initiated by the condensation of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. This pyridoxal 5-phosphate (PLP) 2The abbreviations used are: PLP, pyridoxal 5-phosphate; SPT, serine-palmitoyltransferase; sSPT, soluble, homodimeric isoform of SPT; siRNA, small interference RNA; TX100, Triton X-100; HSN1, hereditary sensory neuropathy type I. 2The abbreviations used are: PLP, pyridoxal 5-phosphate; SPT, serine-palmitoyltransferase; sSPT, soluble, homodimeric isoform of SPT; siRNA, small interference RNA; TX100, Triton X-100; HSN1, hereditary sensory neuropathy type I.-dependent reaction is catalyzed by the serine-palmitoyltransferase (SPT, EC 2.3.1.50). SPT is believed to be a heterodimer and intracellularly bound to the outer membrane of the endoplasmic reticulum (3Hanada K. Biochim. Biophys. Acta. 2003; 1632: 16-30Crossref PubMed Scopus (438) Google Scholar, 4Han G. Gable K. Yan L. Natarajan M. Krishnamurthy J. Gupta S.D. Borovitskaya A. Harmon J.M. Dunn T.M. J. Biol. Chem. 2004; 279: 53707-53716Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The two SPT subunits SPTLC1 (55 kDa) and SPTLC2 (65 kDa) show a mutual similarity of ∼20% and are highly conserved among species. Although both subunits seem to be required for enzyme activity, SPTLC2 is considered to be the catalytic subunit due to the presence of a PLP binding site (3Hanada K. Biochim. Biophys. Acta. 2003; 1632: 16-30Crossref PubMed Scopus (438) Google Scholar). SPT activity was detected in various tissues, including brain, lung, liver, kidney, and muscle (5Merrill Jr., A.H. Nixon D.W. Williams R.D. J. Lipid Res. 1985; 26: 617-622Abstract Full Text PDF PubMed Google Scholar) and is essential for embryonic development, because homozygous SPTLC1 and SPTLC2 knock-out mice die during embryogenesis (6Hojjati M.R. Li Z. Jiang X.C. Biochim. Biophys. Acta. 2005; 1737: 44-51Crossref PubMed Scopus (132) Google Scholar). In contrast to the membrane-bound mammalian isoform a soluble, homodimeric SPT isoform (sSPT) was found in the sphingolipid producing prokaryote Sphingomonas paucimobilis (7Ikushiro H. Hayashi H. Kagamiyama H. J. Biol. Chem. 2001; 276: 18249-18256Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8Ikushiro H. Hayashi H. Kagamiyama H. Biochim. Biophys. Acta. 2003; 1647: 116-120Crossref PubMed Scopus (26) Google Scholar). A third 10-kDa subunit was identified in yeast, but no mammalian homologue has yet been described (9Gable K. Slife H. Bacikova D. Monaghan E. Dunn T.M. J. Biol. Chem. 2000; 275: 7597-7603Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Here we report the identification and initial characterization of a previously unknown third SPT subunit (SPTLC3) in mammalian cells. General—All Chemicals, unless otherwise stated, were purchased from Sigma. The direct Topo Cloning Vector pcDNA3.2 was from Invitrogen. Protein A-agarose was from Roche Applied Science. Anti-V5 tag monoclonal antibodies were from Serotec (Oxford, UK). The data base search was done with NCBI Blast (www.ncbi.nlm.nih.gov/blast), and subsequent analysis was done in the program BioEdit (version 5.0.9, Dept. of Microbiology, North Carolina State University). Cloning—All constructs were amplified out of a human cDNA library (Matchmaker cDNA Library Muscle/Brain, Clontech, Mountain View, CA) by PCR using appropriate primers and the Long-Expand Mix (Roche Applied Science). The following primers were used for cloning: SPTLC1fw, 5′-caccatggcgaccgccacggagcagtgggttc-3′; SPTLC1rv, 5′-gagcaggacggcctgggctacctcc-3′; SPTLC2fw, 5′-caccatgcggccggagcccggaggctgctgctgc-3′; SPTLC2rv, 5′-gtcttctgtttcttcatacgtcgtctcg-3′; SPTLC3fw, 5′-caccatggctaaccctggaggtggtgc-3′; and SPTLC3rv, 5′-atcttcgagttcaaagctcgtctcatcatagagc-3′. The PCR product was controlled for purity by agarose gel electrophoresis and cloned directly into a pcDNA3.1 Expression Vector according to the manufacturer's instructions (Directional TOPO cloning kit, Invitrogen). The cloning was done in this way so that all constructs were expressed with a C-terminal His-V5 tag. All constructs were subsequently verified by sequencing. SPTLC1–3 Expression in HEK293 Cell Lines—Hek293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Transfections were done using Metafectene (Biontex, Munich, Germany). Selection was performed in the presence of G418 according to the manufacturer's instructions. siRNA Transfection—The following sequence was used for silencing SPTLC3 expression: UAAACGUCCAGUUAUAGUCGUCUGA. The sequence showed no homology to the sequences of SPTLC1 and SPTLC2. The siRNA was transfected using Lipofectamine (Invitrogen) by the standard protocol. SPT activity was determined 48 h after transfection. Immunization and Affinity Purification of Antibodies—Antibodies were custom made by Eurogenetec (Liége, Belgium) according to their standard protocol. The following peptide sequences were used for immunization: anti-SPTLC1, FSTKTYKLQERSDLTC and CQLEESTGSREQDVR; anti-SPTLC2, GGLYKRPFNEAFEC and CDRPFDETTYEETED; and anti-SPTLC3, CNGKLHNHKKQSNGSQ and CKSARPELYDETSFEL. All antibodies were affinity purified by the company according to the Eurogenetec protocol. The specificity of the antibodies was verified by testing the pre-immune serum and, additionally, by competing the antigen interaction with an excess of free immunization peptide. All used antibodies showed a specific binding to the target protein. Preparation of Human Placenta Extract—Human placenta tissue, obtained freshly after cesarean section, was cut into small pieces and washed several times in ice-cold phosphate-buffered saline. The tissue pieces were homogenized in a blender and filtered through a gaze. The filtrate was centrifuged (2000 × g, 5 min, 4 °C), washed twice in phosphate-buffered saline, and frozen in liquid N2. Quantitative Reverse Transcription-PCR—Tissue-specific SPT mRNA expression was quantified using a normalized commercial cDNA panel for 24 human tissues (OriGene, Rockville, MD). The mRNA from cells and tissue was prepared using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) and transcribed to cDNA using oligo(dT) primers and Superscript III (Invitrogen) according to the manufacturer's instructions. Specific primers for the different SPT subunits were designed using the Oligo6.0 software (Molecular Biology Insights, Cascade, CO). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used as the reference gene for quantification. LightCycler PCR was performed with a DNA SYBR Green kit following the manufacturer's instructions (Roche Diagnostics). The following primers (0.4 μm each) were used: SPTLC1fw, 5′-aagaagccattatatactcatat-3′; SPTLC1rv, 5′-ggcactgataagatcaata-3′; SPTLC2fw, 5′-gagtgtgtacaacagttagctg-3′; SPTLC2rv, 5′-tggctcacaaaggccac-3′; SPTLC3fw, 5′-tgcagccaagtatgatgagtcta-3′; and SPTLC3rv, 5′-gcagatgcacgatggaac-3′. Amplification was carried out for SPTLC1: 40 cycles, each consisting of 10 s at 95 °C, 10 s at 58 °C, and 20 s at 72 °C; for SPTLC2: 40 cycles, each consisting of 10 s at 95 °C, 10 s at 58 °C, and 20 s at 72 °C; and for SPTLC3: 50 cycles, each consisting of 10 s at 95 °C, 10 s at 61 °C, and 20 s at 72 °C. The linearity of the assays was determined by serial dilutions of the templates for each primer set separately. Activity Assay—A reaction mixture containing final concentrations of 25 mm Hepes (pH 8.0), 2.5 mm EDTA, 0.5 mm l-serine, 0.05 mm palmitoyl-CoA, 50 μm pyridoxal 5′-phosphate, 0.2 μCi of l-U-14Cserine (Amersham Biosciences). The reaction was performed on ∼400 μg of total extract at 37 °C for 60 min. For some assays the buffer was adjusted to 0.2% Triton X-100 (TX100) as stated in the text. The control samples included myriocin (40 μm) to specifically inhibit SPT activity. Total assay volume was 200 μl. The reaction was stopped by adding 400 μl of 0.5 n NH4OH, followed by the addition of 2.25 ml of chloroform:methanol (1:2). Long-chain bases were extracted by adding 0.75 ml of NH4OH and 0.75 ml of chloroform, vortexing, and centrifuging briefly. The upper aqueous layer was aspirated, and the lower layer was washed two times with 2 ml of 0.1 m KCl. The lower organic phase was removed and counted for radioactivity. By using a systematic screen of the genomic data base for sequences that are homologous to SPTLC1 and SPTLC2 we identified a short open reading frame of 134 amino acids (NICB accession number BAC11509). This short sequence showed 65% identity (86% similarity) with the C-terminal part of SPTLC2. Three further overlapping expressed sequence tags were identified upstream (accession numbers: CF994231, AL109983, and AK075271), which revealed a continuous open reading frame of 1660 bp. This open reading frame encodes for a polypeptide with a theoretical molecular mass of 63 kDa and an overall identity to the SPTLC2 subunit of 68% (84% similarity). The identity to the SPTLC1 subunit was 21% (45% similarity). A data base scan for conserved structural elements (NCBI) assigned the relationship to the family of aminotransferases type II in accordance with the identification of a conserved PLP binding domain at position 371. Based on its homology to SPTLC2 the new protein was named SPTLC3. No overlapping expressed sequence tags were found further upstream. The expression of the in silico identified cDNA was verified by PCR using specific primer sets. Based on a human cDNA library we amplified a DNA fragment that matched to the expected size of 1.7 kbp. Subsequent DNA sequencing proofed the identity of this product with the sequence in the data base. Similar sequences were also found for other species like rat, mouse, and dog (accession numbers: AY417571.1, XM542889.1, AY417572.1, and XM230620.3). The alignment between SPTLC2 and SPTLC3 (Fig. 1a) revealed a highly homologous region within the core of the two proteins and less conserved N- and C-terminal tails. A genomic analysis showed that the SPTLC3 gene is located on chromosome 20 p12.1–12.3 and composed of 12 exons (Fig. 1b). The mRNA expression levels of SPTLC1, -2, and -3 were analyzed by quantitative reverse transcription-PCR in a commercially available 24-human tissue cDNA panel. For each subunit a specific primer set was and PCR were For all primer a PCR product of the expected size was on the agarose primer were The analysis showed a for each primer which specific of the SPTLC2 was detected in all 24 tissues, SPTLC1 could be detected in small SPTLC3 was detected in tissues cells and (Fig. the expression of the SPT subunits between the tissues we normalized the expression of SPTLC2 and SPTLC3 to the expression levels of SPTLC1 mRNA (Fig. expression levels of SPTLC3 were observed in kidney, liver, and tissue but in placenta the was higher the For tissues the SPTLC2: SPTLC1 between 5 and the showed a higher with between 2 in and to in higher were observed in tissues that SPTLC3 cells and Expression of SPTLC1–3 in Human Cell mRNA expression of SPTLC1, and SPTLC3 was in Hek293 and HepG2 cells. the tissue panel showed a SPTLC3 expression in placenta tissue we also determined the expression in two human cell and and in human placenta tissue, which we obtained freshly after a cesarean (Fig. the SPTLC1 and SPTLC2 expression levels were in the cell the SPTLC3 expression showed Hek293 cells expressed SPTLC3 in HepG2 cells showed a higher SPTLC3 The expression of SPTLC3 in the trophoblast cell and was to higher in HepG2 cells. In with the tissue panel data we found the highest expression in the placenta verified at the protein by peptide antibodies the N- and C-terminal of the subunits SPTLC1–3 and The peptide sequences were from that are conserved between the subunits to of the different All antibodies were from the using a peptide affinity with the immunization All antibodies specifically detected proteins in the expected size on a (Fig. SPTLC1 was detected at kDa and SPTLC2 at SPTLC3 to a size of 63 The specificity of the antibodies was verified by the with serum and, by competing the antigen binding with an excess of free immunization peptide. In both no was observed Although all antibodies specifically the proteins the of the antibodies SPTLC1 and SPTLC2 were higher SPTLC3. This resulted in a for SPTLC3 on the with SPTLC1 or the of SPTLC3 mRNA in cells was or In with the mRNA expression data the cell showed a variable expression of the SPTLC3 subunit on the protein (Fig. No for SPTLC3 was in Hek293 cells, which also levels of SPTLC3 HepG2 cells show a and a mRNA The cells and showed a for SPTLC3, and in with the reverse transcription-PCR data we observed the in placenta In contrast to SPTLC3, SPTLC1 and SPTLC2 were expressed in in the different cell lines. SPTLC3 SPT Activity 2- to SPTLC3 SPT activity, we cloned the for SPTLC1, and SPTLC3 into a mammalian expression with a C-terminal tag of the subunits was transfected and in Hek293 cells. in the expression levels due to in the we cell for all the By using an all subunits were specifically detected at the expressed proteins according to their expected size the mass addition of 5 kDa due to the and revealed no of (Fig. SPT activity (Fig. was determined with the of an assay using as the 2000; PubMed Scopus Google Scholar). No increase in SPT activity was in the cells. The overexpression of SPTLC2 and SPTLC3 resulted in a 2- to 3-fold higher SPT activity with the the increase was a less in the cells with the cells. This shows that the identified SPTLC3 like a in the SPT Silencing of SPTLC3 by siRNA SPT the overexpression of SPTLC3 SPT activity, we were silencing the expression of this subunit has the transfected and cells with SPTLC3-specific The SPTLC3 siRNA sequence no similarity to the sequences of SPTLC1 and SPTLC2 to of The of the siRNA resulted in a reduction of SPTLC3 mRNA expression with the transfected (Fig. The expression of SPTLC3 mRNA also led to protein expression as on the Silencing of SPTLC3 expression resulted in a significant in SPT activity (Fig. The activity reduction with the of expressed SPTLC3 No silencing was in Hek293 cells, which no SPTLC3, a reduction could be observed in HepG2 cells. A reduction was in and cells, which also the highest levels of SPTLC3. further the that SPTLC3 is a to cellular SPT activity. Triton X-100 with the SPTLC3 but SPTLC2 observed that the addition of 0.2% to the activity assay SPT activity in Hek293 cells (Fig. This increase in activity could be by an of the membrane-bound SPT which subsequently in a higher specific SPT activity. the other SPT activity in placenta extract was in the presence of the activity of the SPT isoform from paucimobilis (sSPT) is in the presence of the of on SPT activity, we the activity in the cells in the presence or of 0.2% the presence of no on the cells, the of SPTLC3 overexpression was (Fig. No in activity was observed in cells. This that the reaction from the can also to a in Hek293 cells but to a in placenta to the of SPTLC3 expression in Hek293 cells (Fig. SPT activity on the reaction (Fig. which is by In placenta tissue SPTLC3 but little SPTLC2. The addition of to an of the SPTLC3 and in to an of the we in this tissue a reduction of in the presence of in the SPTLC1 gene and were to be for the of an sensory neuropathy sensory neuropathy type M. 2001; PubMed Scopus Google Scholar, K. C. G. L. de Jr., 2001; PubMed Scopus Google Scholar). is by a sensory which to and from other such as type 2 G. G. J. 2002; PubMed Scopus Google Scholar). Although SPT activity is essential for embryogenesis and cell (6Hojjati M.R. Li Z. Jiang X.C. Biochim. Biophys. Acta. 2005; 1737: 44-51Crossref PubMed Scopus (132) Google the of are to the and other tissues like lung, or This variable of the different tissues for an SPT activity. of this could be the of different SPT isoforms with tissue-specific structural and This led to the identification of a previously unknown third SPT SPTLC3. SPTLC3 shows 68% homology to the SPTLC2 subunits, including a conserved PLP binding show an consisting of 12 exons but are located on different and SPTLC3 SPTLC3 can be considered as an isoform of SPTLC2. The structural similarity that SPTLC3 has a as the SPTLC2 subunit. The overexpression of SPTLC3 in Hek293 cells, which SPTLC3 in led to a 2- to 3-fold increase in cellular SPT activity. A increase in SPT activity was after the SPTLC2 subunit but by This is by that mice SPT activity A. D. A. Gable K. Harmon J.M. Dunn T.M. Jr., 2005; PubMed Scopus Google Scholar). In with silencing of SPTLC3 expression by transfecting a SPTLC3-specific siRNA cellular SPT activity. The activity reduction showed a to the of expressed SPTLC3 mRNA in the various cell lines. Although SPTLC3 and SPTLC2 show we also some different properties between the two the SPT reaction but the that with binding or the site in SPTLC3, because the of was also observed for the SPT isoform of The that the expressed SPTLC2 and SPTLC3 subunits increase SPT activity that both subunits can This the on the of the different SPT was in that the SPTLC1 subunit two and is bound to the outer membrane of the endoplasmic reticulum G. Gable K. Yan L. Natarajan M. Krishnamurthy J. Gupta S.D. Borovitskaya A. Harmon J.M. Dunn T.M. J. Biol. Chem. 2004; 279: 53707-53716Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, M. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The overexpression of SPTLC1 increase SPT activity (Fig. that SPTLC1 is directly involved in the catalytic SPTLC1 as an that SPTLC2 and SPTLC3 to the endoplasmic reticulum the to be The tissue expression showed by contrast to the of the of expression is highly variable between various are the very expression levels in placenta and the trophoblast cell lines. This for a tissue-specific of SPTLC3 of the for a highly sphingolipid a specific for SPT, shows activity, and several other a between SPT activity and response 2004; PubMed Scopus Google Scholar, A. J. M. Biophys. 2003; PubMed Scopus Google Scholar). In this is that SPTLC3 is or little expressed in tissues that are to or response like cells, and the of SPTLC3 expression to be by expression of SPTLC2 in The highly variable SPTLC3 expression in various tissues further the and of the SPT SPT is to be a composed of SPTLC1 and SPTLC2. to the similarity between SPTLC2 and SPTLC3 is that SPTLC3 also with the of the enzyme is an in the of the highly variable of SPTLC3 to SPTLC1 mRNA SPTLC3 shows an expression of to higher on the mRNA SPTLC1, is that all SPTLC3 subunits can to SPTLC1 in a the mRNA of SPTLC2 shows higher expression levels the SPTLC1 for this be that the SPTLC2 and SPTLC3 subunits are within the This is by the that an SPT cell K. M. A. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google which shows no expression of SPTLC1, also has a very expression of the mRNA levels for SPTLC2 are of cells with SPTLC1 cDNA both SPT activity and SPTLC2 expression levels M. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google that the of SPTLC2 on the presence of the other the that overexpression of SPTLC2 or SPTLC3 in a 2- to 3-fold increase in SPT activity that SPTLC1 expression but SPTLC2 and SPTLC3 are the for SPT activity. could be that the SPT is a but a higher that of several This was by and (6Hojjati M.R. Li Z. Jiang X.C. Biochim. Biophys. Acta. 2005; 1737: 44-51Crossref PubMed Scopus (132) Google Scholar). further are to the of the SPT enzyme and the of SPTLC1 in this also be to the in the SPTLC1 gene have the on the and The that SPTLC2 and SPTLC3 have but different could an tissues are for the to in the SPTLC1 gene were identified as the of in several are further of no was found in the SPTLC1 gene or the SPTLC2 gene M. K. K. De M. E. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). be to the SPTLC3 gene shows in In show that SPT is in a way The of a third subunit new as to the and cellular of this are to a on the and cellular of this also H. Kagamiyama for with the expression for the SPT from