Vol. 44, No. 6, May 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 1193-1202
MOLECULAR CLONING AND EXPRESSION OF MOUSE CERAMIDE GLUCOSYLTRANSFERASE Shinichi Ichikawa*, Katsuya Ozawa, and Yoshio Hirabayashi Laboratory for CelluLar Glycobiology, Frontier Research Program, The Institute of Chemical and Physical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan Received November 12, 1997 Received after revision, January 7, 1998
SUMMARY: Ceramide glucosyltransferase (EC 2.4.1.80) catalyzcs the first glycosylation step of glycosphingolipid (GSL) synthesis, the transfer of glucose from UDP-Glucose to hydrophobic ccramide and generate gtucosylceramide (GlcCer). We have chined mouse ceramide glucosyltransferase cDNA from a brain cDNA library by PCR based homology cloning. The nucleotide sequence determination revealed that mouse ceramidc glucosyltransfcrase eDNA encodes 394 amino acids with a calculated molecular mass of 45 kDa. The amino acid sequence of mouse ceramide glucosyltransferase showed 98% identity with the human sequcncc. Homology searches against currently available databases identified three homologous proteins in Caenorhabditis elegans and one homologous protein in Cyanobacteria. Highly conserved sequences of ceramide glucosyltransferases and the homologs among a wide variety of organisms suggest biological significance of the lipid glucosylation system.
Key words: ceramide, glucosylceramide, glucosyltransferase, cDNA, mousc INTRODUCTION
Glycosphingolipids (GSLs) are a class of membrane lipid composed of hydrophobic ceramide and a hydrophilic sugar chain. The lipids arc found virtually on all eukaryotic cell surfaces and believed to play important roles in a variety of cellular processes including ceil recognition, cell growth, development, and differentiation (1, 2). Among over 400 GSLs, the simplest GSL glucosylceramide (GlcCer) is of particular importance since the most of GSLs are derived from this lipid. These "glucosphingolipids" were found ubiquitously in animal organs and tissues. GlcCer are synthesized from ceramide and UDP-GLc by the action of ccramidc glucosyltransfcrase (UDP-Glc: ceramide 131-1' glucosyltransferase, EC 2.4.1.80) (3). To understand the structure of ceramide glucosyltransferase protein, isolation of the cDNA is necessary. Recently, we have cloned eDNA for human ceramide glucosyltransferase by complementatkm cloning using a mouse melanoma mutant cell line deficient in the enzyme activity (4-6). The sequcncc analysis revealed that ceramide glucosyltransferase was a novel protein and had no sequence similarity to other known glycosyltransferases. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence data base with following accession no. D89866. *Corresponding author. Phone: (+81) 48 462 1111(ext.6235), FAX: (+81) 48 462 4690, E-mail:
[email protected] or
[email protected] 1039-9712/98/061193-10505.00/0 1193
Copyright 9 1998 by Academic" Press Australia. All rights of reproduction in any form reserved.
Vol. 44, No. 6, 1998
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL
A laboratory mouse is one of the most important animal as a standard experimental system, and the study of mouse genetics becomes increasingly important by the advent of the gene-targeting technology (7). However, mouse ceramide glucosyltransferase has not been studied satisfactorily. In this communication, we report on cDNA cloning and expression of mouse ceramide glucosyltransferase. MATERIALS AND METHODS
Materials: Mouse brain eDNA library in pCMV.SPORT2 vector (107 independent clones, C57BL/6J mouse) was purchased from Life Technologies Inc. (MD, USA). Expand High Fidelity PCR System and mixture of proteinase inhibitors, Complete, were obtained flom Boehringer Mannheim (Tokyo, Japan). 6-{[(N-7-Nitrobenz-2-oxo-l,3-diazol-4yl)amino]caproyl}sphingosine (C6-NBD-Cer) was purchased from Molecular Probes (OR, USA). The I.M.A.G.E. Consortium cDNA clone, clone ID 577841(8) was obtained from Gcnome Systems Inc. (MI, USA). PCR cloning: For the isolation of mouse ceramide glucosyltransferase eDNA, we used the PCR cloning (9). The template DNA was prepared from the mouse brain eDNA library by the alkaline lysis method (10). Sequences of primers used in these experiments are shown as follows. Mouse primers, e, f, g, and i were designed using the sequences of the mouse exons (detail,; will be published elsewhere). a: 5" dTCACACAGGAAACAGCTATGAC3' (M13 reverse primer) b: 5" dTITCAGTGGTITCAGAAGAGAGAY [complementary to the human sequence 158180] (6) c: 5' dCTGTWFGTCAGTTGCCTTCTTG3' (complementary to the human sequence 113-134) d: 5" dGATGGCGCTGCTFACCTG3' (identical to the mouse sequence 1(-1)-18] e: 5" dTTCACAGATGCAAGTGCCATGC3" (complementary to the mouse sequence 13191340) f: 5" dTATTGTAGCAGGAAGCATGXTAAT3" (complementary to the mouse sequence 841864) g: 5" dXTGCTGAAGATI'ACTTTATGGCC3' (identical to the mouse sequence 698-720) h: 5" dCATACCATGGCGCTGCTGGACCTGGCC3' (identical to the mouse sequence 1-21, Ncol site at the 5" end) i: 5" dACCOGCTGAGCTCCATTCCACACFGTGCOCCAT3" (complementary to the mouse sequence 1154-1224, Bpu1102I site at the 5" end) Expand High FideIity PCR System (Boehringer Mannheim, Tokyo, Japan) was used according to manufacture's instruction. PCR was performed in 1(10 ~1 of a reaction mixture containing template DNA (1 ~g of DNA prepared from the amplified library or i ~tl of the first step reaction mixture) 1X Expand HF buffer, 1.5 mM MgCI2, 200 ~tM each of dNTPs (dATP, dTTP, dCTP, and dGTP), a pair of primers (40 pmoles each), and the enzyme mix (0.75 U). For the amplification of the G+C-rich 5'-untranslated region, dimethylsulfoxide was added to 5% of the reaction mixture. PCR conditon is 30 cycles of 94~ 1 rain, 60~ 1 rain, and 72~ 2 rain. A primer set used for the amplification of each fragment is shown as follows. 5" -untranslated region: the first step, primers a and b, the second step, primers a and c. N-terminal half: the first step, primers d and e, the second step, primers d and f. C-terminal half: the first step, primers d and e, the second step, primers g and e. A full-length coding region: the first step, primers d and e, the second step, primers h and i. D N A manipulation: DNA manipulation was carried out according to Sambrook et al. (10).
Nucleotide sequence determination: Nucleotide sequence was determined in both directions using the cycle sequencing kit (Amersham Life Technologies, IL, USA) based on the dideoxy chain termination method (11). LI-COR 4000L sequencer (NB, USA) was used for the analyses. 1194
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E x p r e s s i o n o f ceramide g l u c o s y l t r a n s f e r a s e in E.coli: Mousc ceramidc glucosyltransferase was expressed in E.coli using the pET system (Novagcn, WI, USA) (12). NcoI and Bpu 11021 sites were introduced into the cnd of 5' and 3' coding regions, respectively, by PCR using d and i primers. The generated eDNA fragment was cloned into Smal site of Blucscript KS vector (Stratagene, CA, USA) and amplified in E. coll. The insert was cxciscd with Ncol and Bpu1102I. The fragment was then cloned into NcoI and Bpu 1 t02I sites of pET3d vector. The resulting plasmid pET-CG-lm was transformed into E. coil strain BL21 (DE3) LysS. For the expression of the cloned ceramidc glucosyltransferasc in E. coil, the cells harboring the plasmid were grown at 37~ in 500 ml NZCYM medium containing 400 ~t~ rnl ampicillin (10). Whcn cell density reached OD600=I1.3, isopropyl [~-thiogalactopyranosktc (IPTG) was added to a final concentration of 1 mM and incubated for an additional 5 h at 30~ After thc incubation, the cells were harvested and lysed by sonication in 5 ml of 20 mM Tris-HCl, pH 7.5 containing 0.25 M sucrose, 100 ~g/ml lysozymc, and 1X Completc (Boehringer Mannhcim). The lysatc was centrifuged at 100,0I)0 :~: g for 1 h. Thc pcllet was rcsuspcndcd in 5 ml of the same buflcr and ccntrifugcd at 100,000 x g for 1 h again. The pellet was resuspended in 600 ~I of the buffer and used as an enzyme source. E n z y m e assay: Ceramidc glucosyltransfcrase activity was assayed according to Lipsky et al. (13) with slight modifications. A synthetic fluorescent substrate, C6-NBD-Ccr was ~lsed for the assay as liposomes. C6-NBD-Cer (50 ~tg) and lecithin (500 p.g) were mixed together in 100 ~i ethanol and the solvent was evaporated. To this, 1 ml of water was added and was sc)nicated to form liposomes. Standard reaction mixture (1(10 ~1) wax 20 rnM Tris-HCl (pH 7.5)/500 gM UDP-GIc/20 gl liposomes/800 ~g protein of the membrane fraction as enzyme source. The mixture was incubated for 12 h at 30~ For the assay of the substrate specificity, 500 ~M UDPGal, UDP-glucuronate, or UDP-Xyl were used instead of UDP-Glc. Aftcr the incubation of thc reaction mixture, lipids were extractcd with [CHCI3/CH3OH, 2:1, (v/v)] and applicd to silica gel 60 plates (E. Merck). NBD=lipids were scparated in [CHCI3/CH3OH/H20, 65:25:4, (v/v)]. The lipids were visualized by UV illumination. Protein assay: Proteins were assayed using Micro BCA Protein Assay Rcagcnt Kit (PIERCE, IL, USA) (14). Northern blot analysis: Northcm blot analyses werc performed as described previously (10). A pre-madc mcmbranc was used for the analysis of mRNA in various tissues (Mouse Multiple Tissue Northern Blot, Clontech, CA, USA). The DIG-labelcd m~)usc antisensc RNA probe corresponding to nucleotide positions bctween 186 and 1340 was generated by T3 RNA polymerase (Stratagcne, CA, USA) with the DIG RNA labeling kit (Boehringcr Mannhcinq, Tokyo, Japan). Hybridization was carried out at 65~ for 30 h in 5x SSC containing, 50% formaldehyde, 4% SDS, 10% blocking solution (Boehringer Mannhcim, Tokyo, Japan), 100 ~g/ ml of salmon sperm DNA and 1 ~g/ml DIG-labeled probe. After hybridization, the membrane was washed with 2x SSC/0.5% SDS and 0.1x SSC/0.5% SDS each for 30 rain at 65~ The membrane was exposed to X-ray film for 30 rain and developed. RESULTS AND DISCUSSION
To clone a cDNA encoding mouse ceramide glucosyltransferase, we uscd the PCR cloning method. Plasmids prepared from a mouse brain eDNA library was used as a template. First, we have isolated 5" region[(-185)-134] of mouse ceramidc glucosyltransferase by nested PCR using a vector primer and two primers complementary to human ceramide glucosyltransferase sequence. The generated fragments were cloned into Bluescript KS vector and the nucleotidc sequences were determined. The information obtained from this sequence was used to design a forward primer
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BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
corresponding to the first 18 nucleotide scquencc of the coding rcgion. Other primcrs idcntical or complementary to the mouse cDNA were synthesized using the sequence information obtained from the genomic sequence (details will be published clsewhere). A full-lcngth coding rcgion (11224), N-terminal half [(-1)-864], and C-terminal half of the cDNA (698- i340) were amplified using nested PCR and cloned into Bluescript KS vector. Fragments isolated by PCR. covered 1.5 kb of the mouse ceramide glucosyltransfcrase cDNA containing ml entire coding rcgiun. The nuclcotide sequence was determined in both directions at least twice using fragments obtained from independent PCR amplifications. The nucleotide sequence betwecn positions 181 and 1318 of the cDNA was idcntical to the exon sequences of the mouse ceramidc glucosyltransfcrasc genc (details will be published elsewhere). Similar to the human sequence, G+C-rich sequence was found in the 5' -untranslated region (6, 15). The nucleotide sequence and the deduccd amino acid sequencc of mouse ceramide glucosyltransferase arc shown in Fig. 1. When thc firs1: ATG is used as the initiation codon, mouse ceramide glucosyltransferase codes for 394 amino acids and has a calculated molecular mass of 45 kDa. There was a hydrophobic region close to the N-terminal, which is presumably a signal-anchor sequence (Fig. 1). The featurc wl,s also found in the human enzyme (6). Mouse ceramide glucosyltransferase was highly homologous with the human enzyme and it exhibited 98 % identity on the basis of amino acid sequencc (Fig. 2). At the time we cloned human ceramide glucosyltransferase, there were no homologus sequences in data bases (6). However, homology searches of mouse ceramidc glucosyltransfcrase against currently available nucleotide and protein databases in National Center for Biological Information (NCBI) revealed three homologous genes in C. elegans (41%, 39%, and 21% identitics on the basis of amino acid sequence) and one in Cyanobactcria Synechocystis sp. strain PCC6803 (24% identity with 60% homology on the basis of amino acid sequence) (Fig. 2). The C. elegans genc which had the highest homology with the mouse enzyme was identified as the ceramide glucosyltransferase gene (dctails will be published elsewhere). Since the existence of GSLs has not bccn rcportcd in Cyanobacteria, the homolog in this bacteria might be responsible for the synthesis of glucosyl diacylglycerol found in this organism (16). The cDNA encoding galactosyl diacylglycerol synthase has been cloned from plant recently, however the sequence showcd no homology with ceramide glucosyltransferase (17). In addition, there was a mouse expression sequence tag (EST) clone (the I.M.A.G.E. Consortium cDNA clone, clone ID 577841) (17) which has high homology with mouse ceramide glucosyltransferase cDNA in the database. This clone was obtained from Genome Systems Inc. and the nucleotide sequence was determined. Sequences of mouse ceramide glucosyltransferase cDNA and the EST clone were identical between nucle{~tide positions 104 and 826. The result indicated that the EST clone was a partial ceramide glucosyltransferase cDNA. As shown in Fig. 2, there are highly conserved segments among ceramide glucosyltransferases and its homologs from different species of organisms. Mouse ceramide glucosyltransferase was expressed in E. coli and examined for the enzyme activity. The cDNA was cloned into an E. coli expression vector pET 3d. The resulting plasmid
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BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL
-I 85 (5') GTGGCCGGGGGCGCGCAGGCCCTGCCCGCCCCI-rCCGTCCT -145 -144 CACGCCCGCCGCCCCGCCGGCCCIq'CCTCTCCCACCTTCCACTCGCGGCCCGCC-CCCCGCACCCGC-CGGCCC -73 -72 CCGCGTCCTCCTCCCGCGGCAGCGCTGTCCGCGGCGGCCGGAGCGGC-CCGGGCCGGGCCAGCGGGCCGGGGG -I I ATGGCGCTGCTGGACCTGC-CCCAGGAGGGAATGGCCTTGI-rCGGCI-FCGTC-CTCI-TCGTGGTGCTGTGGCTG 1 M A L L 0 L A Q E G M A L F G F V L F V V L W L
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73 ATGCAI-FTCATGTCCATCATCTACACCCGGI-FACACCTCAACAAGAAGGCAACAGACAAACAGCCGTATAGC 144 25 M H F M S I I Y T R L H L N K K A T D K O P Y S 48 145 AAGCTCCCTGG-fGTCTCTCTTCTGAAGCCACTGAAGGGGGTGGATCCTAACCTAATCAACAACTTGGAGACA 216 49 K L P G V S L L K P L K G V D P N L I N N L E T 72
217 TTCI-tTGAACTGGATTATCCCAAATATGAAGTACTCCT-FrGTGTACAAGATCATGATGATCCAC-CCAI-rGAT 288 73
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361 ATTAACCCTAAAATTAATAATTTGATGCCAGCATATGMGFrC-CAAAATATGATCTCATATGGATTTGTGAT 432 121 I N P K I N N L B P A Y E V A K Y D L t W I C D 144 433 AGTGGMTAAGAGTCATCCCAGACACAlqAACTGACATGGTGMTCAGATGACAGAGA,~,GTGGGGTTGGTC 504 145 S G I R V I P D T L T D M V N 0 M T E K V G L V 168 505 CACGGGCTGCCGTATGTAGCCGACAGACAAGGCTTTGCTGCCACCIq-AGAGCAGGTATATTTTGGAACFFCA 576 169 H G L P Y V A 0 R O G F A A T L E Q V Y F G T S 192 577 CACCCAAGATCCTATATCTCTGCCMTGTAACTGGClq'CAAATGTGTGACGGGGATGTCTTGTTTGATGAGG 648 193 H P R S Y ~ S A N V T G F K C V T G M S C L M R 216 649 AAGGATGTGCTAGATCAGGCAGGAGGGCTCATAGCCTFFGCTCAGTACATTGCTGAAGATTAC'FFFATGGCC 720 217 K D V L D 0 A G G L I A F A Q Y I A E D Y F M A 240 721 AAAGCAATAGCCGACCGAGGlqGGAGGTTTTCAATGTCTACTCAAGFFGCCATGCAAAACTCTGGlqCGTAC 792 241 K A I A D R G W R F S M S T O V A M O N S G S Y 264 793 TCAAlqqCTCAG1TTCAATCCAGAATGATCAGGTGGACCMAtqGAGAA1-FAACATGCTFCCTGCTACAATA 864 265 S I S Q F O S R M I R W T K L R I N M L P A T I 288 865 AI-FFGTGAGCCAATTTCAGAATGCI-FTGTTGCCAGFFFAAI-FA'FFGGGTGGGCAGCCCACCATGTAIqCAGA 936 289 I C E P I S E C F V A S L I I G W A A H H V F R 312 937 TGGGATATCATGGTCi-FClqCATGTGCCACTGCCTGGCATGGlqq'ATA1TFGACTACATTCAACTCAGGGGT1008 313 W 0 I M V F F M C H C L A W F I F D Y 1 O L R G 336 1009 GTCCAGGGTGGCACACTGTGII I I ICAAAAClqGATTATGCTGTGGCCTGGlq-CATCCGTGAATCCATGACA 1080 337 V O G G T L C F S K L D Y A V A W F t R E S t~ T 360 1081 ATCTACATTFTs 361 t Y I F L
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1t 53 GGGGGGACAGCAGAGGAGATCCTGGATGTGTAAGAAGACCTCTGTGACTGATGGCGCACAGTGTGGAATGGA1224 385 G G T A E E I L D V * 395 1225 AGTGTTATAAAI-iATGTTTATAGAGACACTTTCCAGGGTCTCCCFFCAGTAG1TiATCACATGTATGTTTTG1296 1297 GTATCTGCTCTTTAATTTATTTGCATGGCACI-rGCATCTGTGM (3') 1340
Fig. 1. Nucleotide sequence and deduced amino acid sequence of mouse ceramide glucosyltransferase cDNA. Putative signal-anchor sequence is underlined.
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