A Mammalian Homolog of the Yeast LCB1 Encodes a Component of Serine Palmitoyltransferase, the Enzyme Catalyzing the First Step in Sphingolipid Synthesis
1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês
10.1074/jbc.272.51.32108
ISSN1083-351X
AutoresKentaro Hanada, Tomoko Hara, Masahiro Nishijima, Osamu Kuge, Robert C. Dickson, M. Marek Nagiec,
Tópico(s)Cellular transport and secretion
ResumoSerine palmitoyltransferase (SPT; EC2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 and LCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT. Serine palmitoyltransferase (SPT; EC2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 and LCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT. Sphingolipids are ubiquitous constituents of membrane lipids in mammalian cells and are also distributed widely in other animals, plants, and microbes (1Karlsson K.-A. Lipids. 1970; 5: 878-891Crossref PubMed Scopus (198) Google Scholar). Previous studies with mutant cells defective in sphingolipid biosynthesis have revealed that sphingolipids are essential for the growth of Saccharomyces cerevisiae (2Wells G.B. Lester R.L. J. Biol. Chem. 1983; 258: 10200-10203Abstract Full Text PDF PubMed Google Scholar, 3Pinto W.J. Wells G.W. Lester R.L. J. Bacteriol. 1992; 174: 2575-2581Crossref PubMed Google Scholar) and Chinese hamster ovary (CHO) 1The abbreviations used are: CHO, Chinese hamster ovary; SPT, serine palmitoyltransferase; bp, base pair(s); kbp, kilobase pair(s); RACE, rapid amplification of cDNA ends; His6, six consecutive histidine residues; kb, kilobase(s); ORF, open reading frame; GM3, N-acetylneuraminyl lactosylceramide; RI, relative intensity. cells (4Hanada K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1990; 265: 22137-22142Abstract Full Text PDF PubMed Google Scholar, 5Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar), implying that sphingolipids play crucial roles in eukaryotes. It has also been demonstrated that sphingoid bases and ceramide modulate activities of various enzymes such as protein kinases, protein phosphatases, and phospholipases in cells or in cell-free systems and that these sphingolipids appear to participate in various cellular events including proliferation, differentiation, senescence, apoptosis, and inflammatory responses (for review, see Refs. 6Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar and 7Merrill Jr., A.H. Sweeley C.C. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier Science, Amsterdam1996: 309-339Google Scholar). Moreover, sphingomyelin and glycosphingolipids have been suggested to be involved in the formation of detergent-resistant membrane subdomains (8Brown D. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2618) Google Scholar, 9Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar, 10Hanada K. Izawa K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1993; 268: 13820-13823Abstract Full Text PDF PubMed Google Scholar, 11Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), where localized signaling events may occur (12Anderson R.G. Curr. Opin. Cell Biol. 1993; 5: 647-652Crossref PubMed Scopus (171) Google Scholar, 13Lisanti M.P. Scherer P.E. Tang Z. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (590) Google Scholar, 14Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar). Sphingolipid biosynthesis is initiated by condensation ofl-serine with palmitoyl coenzyme A, a reaction catalyzed by serine palmitoyltransferase (SPT; EC 2.3.1.50) to generate 3-ketodihydrosphingosine (for a review of sphingolipid biosynthesis, see Refs. 7Merrill Jr., A.H. Sweeley C.C. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier Science, Amsterdam1996: 309-339Google Scholar and 15Merrill Jr., A.H. Jones D.D. Biochim. Biophys. Acta. 1990; 1044: 1-12Crossref PubMed Scopus (396) Google Scholar). 3-Ketodihydrosphingosine is reductively converted to dihydrosphingosine, which is N-acylated and then dehydrogenated to form ceramide. Ceramide is converted to various complex sphingolipids, for instance, sphingomyelin and glycosphingolipids in mammalian cells. The structure of the polar head groups of complex sphingolipids is highly diverse, and more than 300 different types of complex sphingolipids have been reported (16Hakomori S. Kanfer J.D. Hakomori S. Sphingolipid Biochemistry. 3. Plenum Press, New York1983: 1-165Google Scholar, 17Hori T. Sugita M. Prog. Lipid Res. 1993; 32: 25-45Crossref PubMed Scopus (45) Google Scholar, 18Lester R.L. Dickson R.C. Adv. Lipid Res. 1993; 26: 253-274PubMed Google Scholar). By contrast, the structure of the ceramide moiety is relatively well conserved from mammals to single-cell eukaryotes, although there are heterogeneities in the acyl chain length and the number of the hydroxyl groups (1Karlsson K.-A. Lipids. 1970; 5: 878-891Crossref PubMed Scopus (198) Google Scholar, 17Hori T. Sugita M. Prog. Lipid Res. 1993; 32: 25-45Crossref PubMed Scopus (45) Google Scholar, 18Lester R.L. Dickson R.C. Adv. Lipid Res. 1993; 26: 253-274PubMed Google Scholar). Such structural conservation most likely reflects an evolutional conservation of the biosynthetic pathway for ceramide. SPT is suggested to be a rate-determining enzyme in the sphingolipid synthetic pathway and, thus, to be a key enzyme for regulating cellular sphingolipid content (15Merrill Jr., A.H. Jones D.D. Biochim. Biophys. Acta. 1990; 1044: 1-12Crossref PubMed Scopus (396) Google Scholar). However, the actual mechanisms for regulating ceramide and complex sphingolipid synthesis remain unknown largely because few biosynthetic enzymes have been purified, and a limited number of genes encoding the enzymes have been isolated. Two S. cerevisiae genes, LCB1 and LCB2, have been isolated by complementation of mutant strains defective in SPT activity (19Buede R. Rinker-Schaffer C. Pinto W.J. Lester R.L. Dickson R.C. J. Bacteriol. 1991; 173: 4325-4332Crossref PubMed Scopus (157) Google Scholar, 20Nagiec M.M. Baltisberger J.A. Wells G.B. Lester R.L. Dickson R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7899-7902Crossref PubMed Scopus (186) Google Scholar). That the LCB1 and LCB2genes are indispensable for SPT activity suggests that SPT is composed of subunits including at least the Lcb1 and Lcb2 proteins (19Buede R. Rinker-Schaffer C. Pinto W.J. Lester R.L. Dickson R.C. J. Bacteriol. 1991; 173: 4325-4332Crossref PubMed Scopus (157) Google Scholar, 20Nagiec M.M. Baltisberger J.A. Wells G.B. Lester R.L. Dickson R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7899-7902Crossref PubMed Scopus (186) Google Scholar). Independently, Zhao et al. (21Zhao C. Beeler T. Dunn T. J. Biol. Chem. 1994; 269: 21480-21488Abstract Full Text PDF PubMed Google Scholar) isolated the SCS1gene, which is identical to the LCB2, as an allele whose mutations suppressed the hypersensitivity of a yeast mutant to high concentrations of calcium. Recently, a full-length mouse (m LCB2) and partial human (h LCB2) cDNA homologs of LCB2 have been isolated (22Nagiec M.M. Lester R.L. Dickson R.C. Gene (Amst.). 1996; 177: 237-241Crossref PubMed Scopus (71) Google Scholar). Here we present evidence for the isolation of mammalian homologs of LCB1from mouse and CHO cells. In addition, we present biochemical evidence that the CHO LCB1 protein is a component of the SPT enzyme. A mouse LCB1 homolog was isolated by screening a testis cDNA library for hybridization to a 32P-labeled 953-bp human cDNA fragment from the expressed sequence tag clone 6H6 (GenBank accession no. T24597) predicted to encode the LCB1 protein. The human sequence was identified by a computer search of the dbest data base of expressed sequence tags at the National Center for Biotechnology Information using the tblastn algorithm (23Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71458) Google Scholar) and the S. cerevisiae Lcb1 protein sequence (19Buede R. Rinker-Schaffer C. Pinto W.J. Lester R.L. Dickson R.C. J. Bacteriol. 1991; 173: 4325-4332Crossref PubMed Scopus (157) Google Scholar) as a query. The largest mouse cDNA clone, 1.2 kbp, lacked a 5′-coding region. The missing 442 bp were obtained by performing the polymerase chain reaction in the presence of a 5′-RACE-ready cDNA, prepared from mouse kidney (CLONTECH), and the LCB1-specific primers 5′-CCCCTCTCTTAGAGTACGCAG-3′ and 5′-GCCAGGCGCTCTTCTAAATC-3′ plus an anchor primer (CLONTECH). The sequence of the mouse LCB1cDNA was determined by using a cycle sequence protocol (Life Technologies, Inc.). A cDNA library, made from mRNA of CHO-K1 cells, was prepared by using a SuperScriptTM plasmid system (Life Technologies, Inc.) according to the manufacturer's protocol. Primers used for amplification of c LCB1 were: primer A, 5′-TTAGTCGACATATCTGGATTCTCTTCCATGAT-3′ (reverse); primer B, 5′-TTAGAATTCAGAACTGATTGAAGAGTGGCA-3′ (forward); primer C, 5′-TTAGAATTCATTGAAGAGTGGCAGCCAGAGCC-3′ (forward). The CHO cDNA library was subjected to polymerase chain reaction by using primers A and B (themocycling conditions were 94 °C for 45 s, 57 °C for 45 s, and 72 °C for 90 s at 40 cycles). The initial amplified DNA was diluted 20,000-fold, and then the diluent was subjected to a secondary polymerase chain reaction with primers A and C (94 °C for 45 s, 57 °C for 45 s, and 72 °C for 90 s at 30 cycles). After digestion with EcoRI and SalI, the amplified 0.9-kbp DNA was cloned into pBlueScript SK, and its DNA sequence was determined. Based on the sequence of the cloned 0.9-kbp DNA, a 25-mer oligonucleotide (5′-CCATCCTGCTCTCAACTACAACATC-3′), which should perfectly match a portion of the c LCB1 sequence, was synthesized, biotinylated, and used with the GeneTraperTM cDNA positive selection system (Life Technologies, Inc.) to isolate c LCB1. cDNAs hybridizable to the 25-mer oligonucleotide were retrieved from the CHO-K1 cDNA library, using the procedures described in the Life Technologies, Inc. manual, and transformed into Escherichia coli. Bacterial colonies harboring the c LCB1 coding sequence were detected by colony hybridization using the cloned 0.9-kbp fragment of c LCB1, random prime-labeled with [α-32P]CTP, as a probe. In this way, three bacterial colonies with a 2.7-kbp cDNA corresponding to c LCB1 were isolated. The nucleotide sequence of the cloned cDNAs was determined by using an automated DNA sequencer (ABI PRISMTM 310 genetic analyzer, Perkin-Elmer Applied Biosystems). c LCB2 was also isolated by essentially the same procedures as described above with the following exceptions: primer A, 5′-TTAGAATTCTATCAGGATTTTGAAAACTTCTATAC-3′ (forward); primer B, 5′-TTACAGCTGCAGCAGATCCCCAACTTC-3′ (reverse); primer C, 5′-TTAGTCGACAGTCTTCATTGCCATAGATGATGAA-3′ (reverse). A 1.1-kbp DNA fragment of c LCB2 was amplified by polymerase chain reaction, cloned, and its DNA sequence was determined. Then, a 25-mer oligonucleotide sequence (5′-AGCCAGGGTGGATATCATGGAGAGA-3′) was synthesized for the cDNA positive selection system. The previously cloned 1.1-kbp fragment of c LCB2 was used for a32P-labeled probe for the colony hybridization. Eventually, one 2-kbp clone of c LCB2 was isolated. Mutant SPB-1 (4Hanada K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1990; 265: 22137-22142Abstract Full Text PDF PubMed Google Scholar) and its parent CHO-K1 (ATCC CCL 61) cell lines were routinely maintained in Ham's F-12 medium supplemented with 10% newborn calf serum, penicillin G (100 units/ml,) and streptomycin sulfate (100 μg/ml) in a 5% CO2 atmosphere in 100% humidity at 33 °C. pSV-OKneo was constructed by inserting a G418-resistant determinant from pMC1neo poly(A) (Stratagene) into a blunt-ended ClaI site of pSV-SPORT1 (Life Technologies, Inc.), a mammalian expression vector. To construct a c LCB1-expressing plasmid, the SalI-NotI 2.7-kbp fragment of c LCB1, which was originally cloned into pSPORT1, was transferred between the SalI and NotI sites of pSV-OKneo, and the resultant plasmid was designated pSV-cLCB1. Similarly, the SalI-NotI 2-kbp fragment of c LCB2 was transferred from pSPORT1 to pSV-OKneo, and the resultant c LCB2-expressing construct was designated pSV-cLCB2. SPB-1 cells were transfected with these expression plasmids by lipofection with LipofectinTM reagent (Life Technologies, Inc.) and, after selection for G418 resistance (400 μg/ml), transfectant colonies were purified with cloning cups. As a control, SPB-1 cells were transfected with the vector, pSV-OKneo, and one G418-resistant SPB-1 clone was isolated. A Northern blot containing about 2 μg/lane poly(A)+ RNA from mouse tissues (CLONTECH) was hybridized separately with a 32P-labeled 1,308-bp EcoRI-XhoI fragment of the mouse LCB1, a 1,458-bp EagI-XhoI fragment of the mouse LCB2, and a 2-kbp fragment of human β-actin cDNA. Stringent hybridization conditions were used as recommended by the manufacturer (CLONTECH). A Hewlett-Packard Jetscan II scanner was used to scan the autoradiograms, and the intensity of hybridizing bands was measured using SigmaGel software (Jandel Scientific). Total RNA was prepared from CHO cells with an RNA isolation kit (ISOGEN, Nippon gene, Toyama, Japan). After electrophoresis in an agarose gel, RNA was blotted onto a Nylon membrane (Hybond N, Amersham). The membrane was hybridized with the 32P-labeled 0.9-kbp fragment of c LCB1 under stringent conditions, and hybridizing c LCB1 mRNA was detected by autoradiography. CHO cells were cultivated in F-12 medium containing 10% serum at 40 °C for 2 days to subconfluence, and then SPT activity of lysates prepared from the cells was determined as described previously (24Merrill Jr., A.H. Biochim. Biophys. Acta. 1983; 754: 284-291Crossref PubMed Scopus (118) Google Scholar). In some experiments, the cell lysates were incubated with various concentrations of sphingofungin B (a gift from Dr. Shu Kobayashi, Department of Applied Chemistry, Science University of Tokyo, Tokyo) at 4 °C for 10 min before the SPT assay. Replicas of CHO cell colonies were prepared on polyester discs and assayed in situ for SPT activity as described previously (4Hanada K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1990; 265: 22137-22142Abstract Full Text PDF PubMed Google Scholar). CHO cells were seeded in 5 ml of F-12 medium containing 10% serum in 60-mm dishes and cultured at 40 °C for 2 days to subconfluence. After washing twice with 2 ml of F-12 medium, the cell monolayers were incubated in 1.3 ml of F-12 medium containing 1% Nutridoma-SPTM (Boehringer Mannheim) and 24 kilobecquerels of l-[U-14C]serine (Amersham) at 40 °C for 2 h. The monolayers were washed twice with 2 ml of cold phosphate-buffered saline, harvested by scraping, and precipitated by centrifugation at 4 °C. The cell pellets were suspended in 0.9 ml of cold phosphate-buffered saline, and 0.1 ml and 0.8 ml of the suspension were used for protein determination and lipid extraction, respectively. Lipids extracted from the cells by the method of Bligh and Dyer (25Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar) were separated on TLC plates (Silica Gel 60, Merck) with a solvent of methyacetate, propanol-1,chloroform, methanol, and 0.25% KCl (25/25/25/10/9 v/v). Radioactive lipids on the TLC plates were visualized, and their relative radioactivity was determined by using a BAS2000 Image AnalyzerTM (Fuji). CHO cells (1.5–3.5 × 106 cells) were seeded in a 150-mm culture dish containing 15 ml of 10% serum and F-12 medium and cultured at 33 °C for 1 day. Then, after washing twice with 10 ml of phosphate-buffered saline, the cell monolayers were cultivated in 25 ml of Nutridoma-BO containing gentamicin (10 μg/ml), a sphingolipid-deficient medium, at 40 °C for 2 additional days. After washing twice with 10 ml of cold phosphate-buffered saline, the cell monolayers were harvested by scraping, phospholipids were extracted from the harvested cells (25Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar), and the extracted phospholipids were separated by TLC with a solvent of chloroform/methanol/acetic acid (65/25/10 v/v). The phosphorus content of the phospholipids was determined by the method of Rouser et al. (26Rouser G. Siakotos A.N. Fleischer S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1320) Google Scholar). c LCB1cloned into pSPORT1 was digested with BsrGI and self-ligated to delete the NcoI site in the 3′-untranslated region. The resultant plasmid, digested with NcoI and SalI, was ligated with annealed oligonucleotides (5′-TCGACCATGGCGCATCACCATCACCATCA-3′ and 5′-CATGTGATGGTGATGGTGATGCGCCATGG-3′) encoding a His6-tag sequence. The SalI-NotI fragment of the tagged c LCB1 was transferred from pSPORT1 to pSV-OKneo, and the resultant construct was designated pSV-HTcLCB1. The fusion protein encoded by pSV-HTcLCB1 has the sequence Met-Ala-His-His-His-His-His-His before the first methionine of the wild type cLCB1 protein. SPB-1 cells were transfected with pSV-HTcLCB1, and G418-resistant transformants were selected as described above. By an in situ SPT assay of the drug-resistant colonies, transformants having SPT were identified, and one purified clone was designated SPB-1/HTcLCB1. All manipulations were done at 4 °C or on ice unless noted otherwise. After CHO cells were cultured at 40 °C for 2 days, membranes were prepared from the cells as described previously (11Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The membranes (1 mg of protein) were incubated in 0.5 ml of a solubilization buffer consisting of 0.1 m sodium phosphate buffer (pH 8.0), 50 mm NaCl, 10 mm imidazole, and 1% sucrose monolaurate (Mitsubishi Kasei Shokuhin Inc., Tokyo) for 10 min. After centrifugation (105 × g, 30 min) of the sample, 0.4 ml of the supernatant fluid was incubated with 50 μl of Ni2+-nitrilotriacetic acid agarose resin (Qiagen, Hilden) in a microcentrifuge tube for 1 h with gentle shaking. The resin was pelleted by centrifuging for 1 min at 2,000 ×g, and the supernatant fluid was stored as an unabsorbed fraction. After washing twice with 1 ml of the solubilization buffer, the resin was incubated with 0.3 ml of an elution buffer consisting of 0.1 m sodium phosphate buffer (pH 8.0), 0.1 mimidazole, and 1% sucrose monolaurate for 10 min with gentle shaking. After precipitating the resin, the supernatant fluid was recovered as an absorbed fraction. The recovered fractions were assayed for SPT activity. Protein concentrations were determined by the method of Lowry et al. (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as the standard. A computer search for sequences with similarity to the yeast Lcb1 protein identified a human expressed sequence tag (GenBank accession no.T24597). Most of the base sequence in this expressed sequence tag clone was determined and then used to identify a 1.2-kbp cDNA in a mouse testis cDNA library by nucleic acid hybridization. Sequence analysis of the mouse 1.2-kbp cDNA indicated that it potentially encoded a homolog of the Lcb1 protein and suggested that it lacked a 5′-end because no ATG start codon was present. The missing 5′-end of the mouse cDNA was cloned using the RACE technique yielding a 1.7-kbp mouse cDNA (designated m LCB1, GenBank accession no. AF003823). Because the size of m LCB1 mRNA was estimated to be about 2.7 kb by Northern blot analysis (see below), the 1.7-kbp clone could not represent a full-length cDNA. However, the 1.7-kbp mouse cDNA appears to encode a complete open reading frame (ORF) but lacks the 3′-untranslated region (see below). We next screened a CHO cell cDNA library, consisting of about 107 independent cDNA clones, and obtained three 2.7-kbp clones. Because these three cDNA clones were found to have identical 5′- and 3′-terminal sequences by nucleotide sequence analysis, we chose one cDNA clone, designated c LCB1(GenBank accession no. AF004831), for further analysis. c LCB1 contains a 1,419-bp ORF predicted to encode a protein of 473 amino acid residues with a molecular mass of 52,519 Da (Fig.1). The ACCATGG sequence around the first ATG codon of the ORF corresponds to a Kozak sequence (28Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar) for efficient translational initiation, and there is a poly(A) attachment signal (AATAAA) 33 bases upstream of the poly(A) tail. The size of mRNA for c LCB1 was estimated by Northern blot analysis to be about 2.7 kb (data not shown). Thus, the 2.7-kbp c LCB1 is suggested to encode a complete ORF. m LCB1 also encodes a homologous 1,419-bp ORF, and there is 95% amino acid identity between the predicted cLCB1 and mLCB1 proteins (Fig. 1). The putative products of c LCB1 and m LCB1 have about 35% amino acid identity to the yeast Lcb1 protein (Fig. 1), suggesting that these mammalian cDNAs are homologs of the yeast LCB1 gene. The ALOM algorithm (29Nakai K. Kanehisa M. Genomics. 1992; 14: 897-911Crossref PubMed Scopus (1368) Google Scholar) predicts that the cLCB1 and mLCB1 proteins have one transmembrane domain near their amino terminus (Fig. 1), and another algorithm for predicting protein localization sites (29Nakai K. Kanehisa M. Genomics. 1992; 14: 897-911Crossref PubMed Scopus (1368) Google Scholar) predicts that these mammalian LCB1 proteins are located in the endoplasmic reticulum membrane. These predictions are in agreement with SPT being a membrane-bound enzyme enriched in the endoplasmic reticulum (30Mandon E.C. Ehses I. Rother J. van Echten G. Sandhoff K. J. Biol. Chem. 1992; 267: 11144-11148Abstract Full Text PDF PubMed Google Scholar). In yeast cells, both the LCB1 and LCB2 genes are necessary for expression of SPT activity (19Buede R. Rinker-Schaffer C. Pinto W.J. Lester R.L. Dickson R.C. J. Bacteriol. 1991; 173: 4325-4332Crossref PubMed Scopus (157) Google Scholar, 20Nagiec M.M. Baltisberger J.A. Wells G.B. Lester R.L. Dickson R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7899-7902Crossref PubMed Scopus (186) Google Scholar). A mouse LCB2homolog (m LCB2) has been described recently (22Nagiec M.M. Lester R.L. Dickson R.C. Gene (Amst.). 1996; 177: 237-241Crossref PubMed Scopus (71) Google Scholar). We isolated an LCB2 homolog from a CHO cell cDNA library by procedures similar to those used to isolate c LCB1. The 2,044-bp cDNA (designated c LCB2; GenBank accession no.AF004830) contains a 1,680-bp ORF that potentially encodes a polypeptide consisting of 560 amino acid residues with a molecular mass of 62,882 Da. The cLCB2 protein is 98% identical to the mLCB2 protein and 43% identical to the yeast Lcb2 protein (Fig.2), suggesting that the c LCB2is a CHO LCB2 homolog. The cLCB2 and mLCB2 proteins are predicted to have one transmembrane domain (Fig. 2) and to be located in the endoplasmic reticulum membrane. The distribution of the LCB1 mRNA in mouse tissue was analyzed and compared with the LCB2 mRNA (22Nagiec M.M. Lester R.L. Dickson R.C. Gene (Amst.). 1996; 177: 237-241Crossref PubMed Scopus (71) Google Scholar). Northern blot analysis of mRNA isolated from various mouse tissues revealed a 2.7-kb LCB1-specific mRNA in all tested tissues (Fig. 3). The highest level of mRNA, measured relative to the level of the 2-kb β-actin mRNA, was found in kidney followed by brain and liver. Two LCB2-specific messengers were detected. The 2-kb mRNA, whose size corresponds to the cloned 1.89-bp cDNA (22Nagiec M.M. Lester R.L. Dickson R.C. Gene (Amst.). 1996; 177: 237-241Crossref PubMed Scopus (71) Google Scholar), was found in all tissues, whereas the 6.5-kb mRNA was present in high level in some tissues but was undetectable in others (Fig. 3). The highest level of the LCB2 mRNA was also found in kidney and brain. We previously isolated an SPT-defective CHO mutant strain, SPB-1 (4Hanada K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1990; 265: 22137-22142Abstract Full Text PDF PubMed Google Scholar). The SPT activity in SPB-1 cells is thermolabile, and thus sphingolipid synthesis stops almost completely at nonpermissive temperatures (4Hanada K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1990; 265: 22137-22142Abstract Full Text PDF PubMed Google Scholar, 5Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar). To determine if expression of c LCB1 or c LCB2 complemented the SPT defect in SPB-1 cells, we constructed plasmids pSV-cLCB1 and pSV-cLCB2, in which c LCB1 and c LCB2, respectively, were cloned into pSV-OKneo, a mammalian expression vector with a G418-resistant determinant. SPB-1 cells were transfected with these plasmids, and G418-resistant clones were isolated. Colonies of the drug-resistant cells were replicated on polyester discs, and in situ SPT assays using replicated colonies were carried out to determine the population of colonies having SPT activity. When transfected with plasmid pSV-cLCB1, about 50% of the population of G418-resistant SPB-1 colonies had SPT activity. In contrast, no colonies of G418-resistant SPB-1 cells transfected with pSV-cLCB2 showed any recovery of SPT activity. These results demonstrate specific complementation of the SPT defect of SPB-1 cells by the c LCB1 and eliminate the possibility that the recovery of SPT activity in SPB-1 cells transfected with pSV-cLCB1 cells is caused by spontaneous reversion events. For further analysis of SPB-1 cells transfected with pSV-cLCB1, one stable transformant designated SPB-1/cLCB1 was chosen. As a control, a G418-resistant isolate of SPB-1 cells transfected with the pSV-OKneo vector was also isolated. After cells were cultivated at the nonpermissive temperature (40 °C) for 2 days to inactivate the endogenous SPT activity of SPB-1 cells, lysates were prepared from the cells for SPT assay. SPT activity in SPB-1 cells was less than 5% of that in the parental CHO-K1 cells (TableI) as described previously (4Hanada K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1990; 265: 22137-22142Abstract Full Text PDF PubMed Google Scholar). In contrast, SPT activity in SPB-1/cLCB1 cells was more than 90% of that in CHO-K1 cells, whereas the vector-transfected control cells exhibited no recovery of SPT activity (Table I). Sensitivity of SPT activity to sphingofungin B, a potent inhibitor of SPT (31Zweerink M.M. Edison A.M. Wells G.B. Pinto W. Lester R.L. J. Biol. Chem. 1992; 267: 25032-25038Abstract Full Text PDF PubMed Google Scholar), was identical between CHO-K1 and SPB-1/cLCB1 cells (the dose producing 50% inhibition of SPT activity was about 10 nm in both cell types; data not shown), indicating that SPB-1/cLCB1 cells produce SPT activity that behaves like the wild type.Table IcLCB1 restores SPT activity to SPB-1 mutant CHO cellsStrainKDS producedpmol/mg protein/10 minCHO-K1548 ± 5SPB-1<20SPB-1/cLCB1501 ± 21SPB-1/pSV-OK 90 90<10 Open table in a new tab The recovery of SPT activity in SPB-1 cells after transfection with the c LCB1-expressing plasmid but not with the c LCB2-expressing plasmid suggests specific complementation of the SPT defect of SPB-1 cells by the c LCB1. Possibly, the endogenous LCB1 protein in SPB-1 cells is thermolabile, and in SPB-1/cLCB1 cells the LCB1 protein derived from pSV-cLCB1 complexes with the endogenous LCB2 protein to form a functional SPT complex, thereby restoring SPT activity to the wild type level. The nature of the genetic defect in SPB-1 cells leading to loss of SPT activity is not known. It does not appear to affect transcription because there was no difference in the size (2.7 kb) of the LCB1 messenger or in its expression level between SPB-1 and CHO-K1 cells even after culture at the non-permissive temperature, as judged by Northern blot analysis.2 SPT catalyzes the initial step dedicated to sphingolipid biosynthesis and is suggested to be rate-determining for de novo sphingolipid synthesis (for review, see Ref. 15Merrill Jr., A.H. Jones D.D. Biochim. Biophys. Acta. 1990; 1044: 1-12Crossref PubMed Scopus (396) Google Scholar). However, little is known about the regulation of SPT activity. Our finding that the tissue distribution of both LCB1 and LCB2messengers follows a similar pattern in mouse (Fig. 3), as expected for mRNAs encoding subunits of the same enzyme, suggests that transcription of the two genes is coordinately controlled in vivo. The high relative level of LCB messengers (Fig. 3) in mouse kidney, brain, and liver (LCB1 mRNA only) as well as the low level in testis correlates well with the relative level of SPT specific activity found in these tissues in rat (33Merrill Jr., A.H. Nixon D.W. Williams R.D. J. Lipid Res. 1985; 26: 617-622Abstract Full Text PDF PubMed Google Scholar). The highest SPT specific activity among rat tissues was found in lung (33Merrill Jr., A.H. Nixon D.W. Williams R.D. J. Lipid Res. 1985; 26: 617-622Abstract Full Text PDF PubMed Google Scholar). Both LCB messengers are also present at a high level in lung relative to total poly(A)+ RNA (relative intensity (RI) value, Fig. 3), but the level becomes low when calculated relative to the 2-kb actin mRNA (actin-normalized RI value, Fig. 3), whose concentration is high in this tissue. The nature of the relationship between the level of LCB messengers and the specific activity of SPT in various cells and tissues requires further study. There is high amino acid conservation between the mammalian LCB1 proteins and between residues 150–449 of the yeast Lcb1 protein (Fig.1). Such strong conservation, despite a long phylogenetic distance between S. cerevisiae and mammals, leads us to speculate that this region of the LCB1 protein may be necessary for SPT catalytic activity, for regulation of SPT activity, or for self-interaction or interaction with other subunits. That the cLCB2 and mLCB2 proteins are predicted to have about 40% amino acid identity to the yeast Lcb2 protein (Fig. 2) suggests their functional resemblance. The mammalian LCB2 proteins contain a motif, G373TFTKSFG380 (Fig. 2) which is related to a pyridoxal phosphate binding motif found in the E. coli 2-amino-3-ketobutyrate coenzyme A ligase (34Mukherjee J.J. Dekker E.E. Biochim. Biophys. Acta. 1990; 1037: 24-29Crossref PubMed Scopus (21) Google Scholar). The underlined Lys377 of the mammalian LCB2 proteins is predicted to correspond to a lysine residue that forms a Schiff base with pyridoxal phosphate in the 2-amino-3-ketobutyrate coenzyme A ligase (34Mukherjee J.J. Dekker E.E. Biochim. Biophys. Acta. 1990; 1037: 24-29Crossref PubMed Scopus (21) Google Scholar). This motif is conserved perfectly among the predicted yeast, mouse, and CHO LCB2 proteins (Fig. 2), implying that these proteins are a catalytic component of SPT. However, it remains to be shown biochemically that these mammalian LCB2 proteins are a component of the SPT enzyme. The availability of both the mammalian LCB1 and LCB2 cDNA should enable us not only to address this issue but also to elucidate the mechanism(s) for regulating SPT activity in mammalian cells. Purification of SPT may be facilitated by using the His6-tagged cLCB1 protein, and examination of purified SPT should allow identification of all components of SPT. We thank Dr. Juan L. Iovanna (INSERM) for plasmid 6H6 and Dr. Debra J. Wolgemuth (Columbia University) for the mouse cDNA library.
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