Purification, cDNA Cloning, and Expression of UDP-Gal: Glucosylceramide beta -1,4-Galactosyltransferase from Rat Brain*

Tomoko NomuraDagger §, Minoru TakizawaDagger , Junken Aoki, Hiroyuki Arai, Keizo Inoue, Etsuji WakisakaDagger , Naonobu YoshizukaDagger , Genji ImokawaDagger , Naoshi Dohmaeparallel , Koji Takioparallel , Michihiro HattoriDagger , and Noboru MatsuoDagger

From the Dagger  Biological Science Laboratories, Kao Corporation, 2606, Akabane, Ichikaimachi, Haga, Tochigi 321-3497, Japan, the parallel  Division of Biomolecular Characterization, Institute of Physical and Chemical Research (RIKEN), 2-1, Hirosawa, Wako, Saitama 351-0198, Japan, and the  Faculty of Pharmaceutical Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29.7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N-glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn2+ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse beta -1,4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to beta -1,4-GlcNAc in glycoproteins.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Glycosphingolipids (GSLs),1 a family of complex lipids composed of ceramide and mono- or oligosaccharide moieties, have been demonstrated to play an important role in various cellular functions including recognition, cell adhesion, proliferation, and differentiation (1). GSLs are synthesized by the sequential addition of sugar residues to ceramide by glycosyltransferases, which are specific to each glycosidic linkage.

Glucosylceramide is a product of the first glycosylation step in GSL biosynthesis, which is catalyzed by glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase (EC 2.4.1.80)). The purification (2) and cDNA cloning (3) of this enzyme has recently been reported. Lactosylceramide (LacCer), synthesized after glucosylceramide, is a precursor for the synthesis of four different major classes of GSLs: the ganglio series, the lacto and neolacto series, the globo series, and sulfated GSLs. The enzyme synthesizing LacCer is therefore a key enzyme, in addition to glucosylceramide synthase, for the synthesis of these GSL families. Lactosylceramide synthase (UDP-galactose:glucosylceramide beta -1,4-galactosyltransferase) transfers galactose from UDP-Gal to glucosylceramide (GlcCer), generating a beta -1,4-glycosidic linkage. Several groups have detected the activity of this enzyme in embryonic chicken brain (4), rat liver (5), rat bone marrow cells (6), and human proximal tubular cells (7). beta -1,4-Galactosyltransferase (EC 2.4.1.22), which generates an identical glycosidic linkage when GlcNAc is associated with glycoproteins, has also been reported to be able to catalyze LacCer synthesis (8). On the contrary, Nakazawa et al. (9) showed that recombinant glycopeptide beta -1,4-galactosyltransferase (beta -1,4-GalT) had little activity toward GlcCer, indicating that LacCer synthase and glycopeptide beta -1,4-GalT are different enzymes. In order to draw a clear conclusion on this point, it is essential to purify LacCer synthase and to clone LacCer synthase cDNA. Chatterjee et al. (10) reported a purification of LacCer synthase from human kidney. However, its relation to other galactosyltransferases and its cDNA structure have not been revealed yet.

Here, we report the purification of LacCer synthase from rat brain and its cDNA cloning. We found that purified LacCer synthase preferred GlcCer as a substrate and exhibited low activity toward a glycoprotein. The deduced amino acid sequence showed that LacCer synthase was different from glycopeptide beta -1,4-GalT, although there was some homology between the C-terminal domains of both enzymes.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

UDP-galactose, CDP-choline, ceramide, glucosylceramide, lactosylceramide, globoside, GM2, GA2, ovalbumin, Triton X-100, UDP-hexanolamine-agarose, sodium cacodylate, and dioleoyl phosphatidylcholine were purchased from Sigma. WGA-agarose was obtained from Wako Pure Chemical (Osaka, Japan), and hydroxylapatite was from Seikagaku-kogyo Co., Ltd. (Tokyo, Japan). DTT and Pefabloc SC were purchased from Merck. UDP-[14C]galactose (10.5 GBq/mmol), [alpha -32P]dCTP, and the Rediprime DNA labeling system were obtained from Amersham Pharmacia Biotech. The PD-10 and Mini Q columns were purchased from Amersham Pharmacia Biotech. Rat brains (dissected from 2-3-week-old male and female Wistar rats) were obtained from Oriental Bio Service Kanto (Tsukuba, Japan). Recombinant N-glycanase (EC 3.5.1.52) was from Genzyme (Cambridge, MA). Rat brain Marathon-ReadyTM cDNA and rat multiple tissue Northern blot were from CLONTECH Laboratories, Inc. (Palo Alto, CA). SuperScriptTM Rat Brain cDNA Library was from Life Technologies, Inc. The other chemicals used were of analytical grade.

Assay for LacCer Synthase Activity

LacCer synthase activity was determined as described previously (5) with a minor modification. The standard 0.1-ml incubation mixture contained 20 µmol of sodium cacodylate (pH 7.2), 1.0 µmol of MnCl2, 0.75 µmol of CDP-choline, 0.4 mg of Triton X-100, 1.9 µmol of dioleoyl phosphatidylcholine, 27 nmol of GlcCer, and 2.1 nmol (23 kBq) of UDP-[U-14C]galactose. Incubation was carried out at 37 °C for 30 min. The reaction was terminated by adding 1 ml of methanol to the reaction mixture, and the products were extracted by the method of Bligh and Dyer (12). The lower phase was washed with the theoretical upper phase, and dried with a N2 gas stream. The radioactivity in the extracted lipid was counted using TopCount (Packard Instrument, Meriden, CT).

Assay for Other Glycosphingolipid Synthase Activity

Galactosylceramide synthase activity was determined using the same method as for the LacCer synthase assay. The activity of the enzyme toward other GSLs was also determined by the same method, except that extraction of the product was carried out using Sep-Pak18 (13).

Assay for Glycopeptide beta -1,4-GalT Activity

Glycopeptide beta -1,4-GalT activity toward glycopeptide was determined as described previously (14) with a minor modification. The standard 0.1-ml incubation mixture contained 2.0 µmol of HEPES (pH 7.4), 1.0 µmol of MnCl2, 0.75 µmol of CDP-choline, 0.01 mg of Triton X-100, 1.9 µmol of dioleoyl phosphatidylcholine, 20 µg of ovalbumin, and 2.1 nmol (23 kBq) of UDP-[U-14C]galactose. Ovalbumin is a convenient substrate because it contains asparagine-linked oligosaccharides with terminal nonreducing GlcNAc residues (15, 16). Incubation was carried out at 37 °C for 30 min, and the reaction was terminated by cooling to 0 °C. Aliquots were removed, spotted on 1-inch squares of Whatman 3-MM paper and immediately placed in 10% trichloroacetic acid at room temperature. The assay papers were washed three times with 10% trichloroacetic acid (10 min each wash), the excess trichloroacetic acid was removed by washing with 95% ethanol (10 min), and the papers were air-dried. The amount of acid-precipitated [14C]galactose was quantified using liquid scintillation counter.

Purification Procedure

All purification steps were performed at 0-4 °C. Rat brains, which had been stored frozen at -80 °C were thawed, minced with scissors, and homogenized with nine volumes of the homogenizing solution (0.32 M sucrose, 0.25 mM DTT, 1 mM EDTA, pH 7.4) using a Potter-Elvehjem homogenizer with a mechanically driven Teflon pestle at 1200 rpm. The membrane fraction was prepared by successive centrifugation of the homogenate. The supernatant obtained by centrifugation of the homogenate at 800 × g for 10 min was then centrifuged at 10,000 × g for 30 min. The precipitate from this second centrifugation, designated the brain membrane fraction, was resuspended in 50 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM DTT, 0.02% NaN3, and 150 µM phenylmethylsulfonyl fluoride (buffer S), and stored at -135 °C. Since the enzyme activity of the membrane fraction obtained from the second centrifugation was greater, in terms of both total and specific activity, than that obtained by centrifugation at 100,000 × g for 60 min, the former was used as the membrane fraction.

Solubilization of the Enzyme with Detergent

The brain membrane fraction was suspended in buffer S, and the detergent Triton X-100 was added to 1.0%. The protein concentration of this mixture was adjusted to 4 mg/ml. The enzyme was extracted at 0 °C for 2 h without stirring. The mixture was then centrifuged at 100,000 × g for 60 min. The supernatant, designated the solubilized enzyme, was used for purification.

Step 1: Affinity Chromatography on WGA-agarose-- A WGA-agarose column (5.2 mg of WGA/ml of gel, 50 × 76 mm) was equilibrated with 10 mM Tris-HCl buffer (pH 7.4) containing 500 mM NaCl, 1% Triton X-100, 1 mM DTT, 0.02% NaN3 and 100 µM Pefabloc (buffer B). The NaCl concentration of the solubilized enzyme was adjusted to 0.5 M by adding <FR><NU>1</NU><DE>10</DE></FR> volume of 10 mM Tris-HCl buffer (pH 7.4) containing 5 M NaCl, 1% Triton X-100, 1 mM DTT, 0.02% NaN3, and 100 µM Pefabloc SC. The sample was then loaded onto the column and washed with buffer B. The protein adsorbed to the column was then eluted with the same solution containing 200 mM GlcNAc (buffer C). The column was operated at a flow rate of 5 ml/min.

Step 2: Affinity Chromatography on UDP-hexanolamine-Agarose-- The active fractions from the WGA-agarose column were supplemented with MnCl2 to achieve a final concentration of 10 mM, and the resulting sample was loaded to a column (32 × 50 mm) of UDP-hexanolamine-agarose (4.7 µmol/ml gel) previously equilibrated with 10 mM Tris-HCl buffer, pH 7.4, containing 500 mM NaCl, 10 mM MnCl2, 1 mM DTT, 1% Triton X-100, 0.02% NaN3, and 100 µM Pefabloc SC (buffer D). The column was washed with the same buffer, and the adsorbed protein was eluted with buffer D containing 1 mM UDP (buffer E). The column was operated at a flow rate of 2 ml/min.

Step 3: Hydroxylapatite Column Chromatography-- The active fractions from step 2 were subjected to gel filtration on a PD-10 column to substitute the buffer for 10 mM phosphate, pH 6.8, containing 1 mM DTT, 0.02% NaN3, 1% Triton X-100, and 100 µM Pefabloc SC (buffer F), thereby enabling the next chromatography on a hydroxylapatite column. The enzyme solution obtained was applied to a small column (0.5 ml) of hydroxylapatite equilibrated with buffer F. The column was washed with the same buffer. The adsorbed fraction was eluted by increasing the phosphate concentration of the buffer F to 600 mM.

Step 4: Ion Exchange Chromatography on a Mini Q Column-- The flow-through fraction was loaded directly onto a Mini Q column equilibrated with buffer F. The column was washed with the same buffer, and the adsorbed fraction was eluted with a linear gradient of 0-2.0 M NaCl in buffer F. The Smart system (Amersham Pharmacia Biotech) was used to operate the column.

Amino Acid Sequence Analysis of Lactosylceramide Synthase

The active fractions from the Mini Q column (fractions 7-13) were collected, concentrated, and subjected to SDS-PAGE. Coomassie-stained bands of 61 kDa were excised and treated with 0.3 µg of Achromobacter protease I (a gift from Dr. Masaki, Ibaraki University (17)) at 37 °C for 17 h in 100 mM Tris-HCl (pH 9.0) containing 0.1% SDS and 2 mM EDTA. The peptides thus generated were extracted from the gel and separated on columns of DEAE-5PW (2 × 20 mm; Tosoh, Tokyo, Japan) and Mightysil RP-18 (2 × 50 mm; Kanto Chemical, Tokyo, Japan) connected in series with a model 1090M (Hewlett Packard) liquid chromatography system. The peptides were eluted at a flow rate of 0.2 ml/min with a linear gradient of 0-60% solvent B, where solvents A and B were 0.09% (v/v) aqueous trifluoroacetic acid and 0.075% (v/v) trifluoroacetic acid in 80% acetonitrile, respectively. Selected peptides were subjected to Edman degradation on a model 477A automated protein sequencer (Applied Biosystems, Inc.) connected on-line to a model 120A phenylthiohydantoin analyzer (Perkin-Elmer) using an in house-generated gas phase program and to matrix-assisted laser desorption ionization time of flight mass spectrometry on a Reflex MALD-TOF (Bruker-Franzen Analytik, Bremen, Germany) in linear mode, using alpha -cyano-4-hydroxycinnamic acid as a matrix.

cDNA Cloning of Lactosylceramide Synthase

The degenerated oligonucleotides GGNYTNACNGTNGAACAAT and TTNARNCCRTCDATGAACTG were synthesized, based on the peptide sequences GLTVEQF and QFIDGLN, respectively. PCR amplification was performed on the Marathon-ReadyTM rat brain cDNA library (CLONTECH). The resulting PCR fragments were cloned into the pCR2.1 vector using a TA cloning kit (Invitrogen), and their DNA sequences were determined. 5'-rapid amplification of cDNA ends was performed using a Marathon cDNA amplification kit (CLONTECH). Screening of the cDNA library was performed using GeneTrapperTM cDNA Positive Selection System (Life Technologies) with the SuperScriptTM rat brain cDNA library (Life Technologies). The probes were biotinylated in accordance with the supplier's instructions. Plasmid cDNA was digested with GeneII followed by ExoIII. The resulting single-stranded DNA was hybridized with a biotinylated probe, and hybrids were selected with streptavidin-coated paramagnetic beads. The selected single-stranded DNA was repaired and transfected into E. coli. Positive clones were detected by PCR amplification. Probes for hybridization and PCR primers for the detection of positive clones were synthesized on the basis of the nucleotide sequence of the PCR fragment described above or the cDNA sequence obtained using the GeneTrapperTM cDNA Positive Selection System. The sequences of these oligonucleotide probes were as follows. The probes for hybridization were TCTATTCCCCATCACCATCG and TACAAGCTAGAGGCATCATG; the primers for PCR were TTGTGTGAAATGAAGGGACTG, GAGGCACAAGATCCCTGACAC, AGCTAGAGGCATCATGCTGAGAG, and AGTTCTGCGGAAGATACGTTGTTG.

DNA Sequencing

The DNA sequence was determined by the dideoxynucleotide chain termination method on an ALFexpress DNA sequencer (Amersham Pharmacia Biotech). M13 universal and reverse primers and synthetic oligonucleotides were used as sequencing primers.

Northern Blot Analysis

Rat multiple tissue northern blot (CLONTECH) was used for rat mRNA analysis. A 796-bp segment of lactosylceramide synthase cDNA (nucleotide positions 145-940, Fig. 3B) was labeled with [alpha -32P]dCTP using the Rediprime DNA labeling system (Amersham Pharmacia Biotech). Hybridization was performed in 6× SSC, 1.0% SDS at 65 °C for 17 h, and final washes were performed in 0.1× SSC, 0.1% SDS at 55 °C for 20 min.

Expression of LacCer Synthase in Sf9 Cells

cDNA encoding the coding and noncoding regions of LacCer synthase (nucleotide positions -50 to 1593, Fig. 3B) was inserted into the XhoI and EcoRI sites of the baculovirus transfer vector pFASTBAC1 (Life Technologies). The plasmid thus obtained was designated LacCer synthase pFASTBAC1. A recombinant virus was prepared using the BAC-TO-BAC Baculovirus Expression system (Life Technologies) according to the manufacturer's protocol. Cells (6 × 105 cells/ml) were mixed with recombinant or wild-type Autographa californica nuclear polyhedrosis virus (multiplicity of infection = 10) and incubated for 72 h at 27 °C. The cells were harvested, and LacCer synthase activity was determined as described above.

Other Methods

The protein concentration was determined as described by Bradford (18), using the Bio-Rad protein assay with bovine serum albumin as a standard. SDS-PAGE was performed by the method of Laemmli (19). N-Glycanase digestion was performed according to protocols provided by Genzyme (Cambridge, MA).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Purification of Lactosylceramide Synthase-- LacCer synthase was purified by the sequential application of two types of affinity chromatography, hydroxylapatite chromatography, and ion exchange chromatography. The typical elution profile of activity from the Mini Q column and the SDS-PAGE pattern of the fractions are shown in Fig. 1. LacCer synthase activity was eluted with 250 mM NaCl as a single peak (Fig. 1A), and the intensity of the broad band appearing at 61 kDa correlated well with the activity, suggesting that the protein band at 61 kDa corresponds to LacCer synthase. Table I shows a summary of the purification of LacCer synthase. LacCer synthase was successfully purified 62,000-fold from the rat brain membrane fraction extract to apparent homogeneity, with a 29.7% yield. LacCer synthase appeared to be a glycoprotein because it was adsorbed onto WGA-agarose during the purification step. To confirm the glycoprotein nature of LacCer synthase, the fraction (fraction 9) obtained from the final step was subjected to N-glycanase digestion. After N-glycanase digestion, the molecular mass of the enzyme decreased to 51 kDa, indicating that it is a glycoprotein containing about 15% N-linked carbohydrate moieties (Fig. 2).


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Fig. 1.   Elution profiles of rat brain LacCer synthase on Mini Q column chromatography (A) and SDS-PAGE analysis of active fractions (B). A, the flow-through fraction from the hydroxylapatite column was applied to a Mini Q column equilibrated with buffer F. After washing the column with buffer F for 12 min, elution was carried out with a 4.8-ml linear gradient of NaCl (0-2 M). Fractions (60 µl) were collected and examined for LacCer synthase activity. B, Mini Q fractions were subjected to 9% SDS-PAGE. The gel was silver-stained. Numbers above the gel correspond to Mini Q gradient fractions. Positions of molecular mass markers are shown on the left.

                              
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Table I
Purification of rat brain LacCer synthase


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Fig. 2.   N-Glycanase digestion of LacCer synthase. Purified LacCer synthase was digested with N-glycanase according to the method described under "Experimental Procedures." Positions of molecular mass markers are shown on the left. Lane 1, purified LacCer synthase; lane 2, purified LacCer synthase, deglycosylated with N-glycanase.

Properties of LacCer Synthase-- Maximal activity was observed when the pH of the cacodylate buffer was 7.2 (data not shown). Fig. 3A shows the effect of divalent metal cations. No enzyme activity was detected in the presence of EDTA. The enzyme was activated by Mn2+ and to a lesser extent by Mg2+ and Ca2+. Substrate specificity is shown in Fig. 3B. GlcCer was the best substrate among the GLSs examined. We also investigated whether the enzyme transferred Gal to a glycoprotein, and found that it exhibited low activity toward ovalbumin, a model glycoprotein substrate (14). The apparent Km value of LacCer synthase was 80.6 µM for UDP-galactose and 23.7 µM for glucosylceramide (data not shown).


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Fig. 3.   Properties of purified rat brain LacCer synthase. A, divalent cation requirement. Divalent metal cations and EDTA were present at 10 mM final concentration. B, substrate specificity. LacCer synthase activity was shown as relative activity. The reaction mixtures contained 27 nmol of the indicated GSL substrate or 20 µg of ovalbumin. Maximal activity was expressed as 100%.

Partial Amino Acid Sequence and cDNA Cloning of LacCer Synthase-- The partial amino acid sequence of rat brain LacCer synthase was determined by digestion of the purified enzyme with Achromobacter protease I, a lysine-specific protease. The results are summarized in Table II. On the basis of these sequences, degenerate primers were synthesized and used for PCR with a rat brain cDNA library as a template. One set of primers yielded a 203-bp product containing a sequence corresponding to peptide 3 (Fig. 4A). A cDNA clone that was extended 90 bp toward the 5'-end was obtained by 5'-rapid amplification of cDNA ends (Fig. 4A). This clone contained sequences corresponding to peptides 1, 3, and 4 and part of peptide 2. From these results, we concluded that these amplified products were generated from LacCer synthase cDNA. To obtain a clone containing full-length cDNA, a 20-bp primer was synthesized based on the sequence obtained and was used to screen a rat brain cDNA library. Using the GeneTrapper cDNA Positive Selection System (Life Technologies), a 1.6-kb cDNA (clone A1-84, Fig. 4A) was obtained. This contained a sequence corresponding to 398 amino acid residues, which included all the amino acid sequences obtained during the partial amino acid sequence analysis. Since we had no information about the N-terminal amino acid sequence of LacCer synthase, it was not clear whether clone A1-84 contained the entire coding region. In order to isolate clones containing additional sequences to the 5'-end, the cDNA library was rescreened. As a result, we obtained a 5.7-kb cDNA (clone G4-35, Fig. 4A). Fig. 4B shows the nucleotide sequence and deduced amino acid sequence of LacCer synthase. There are three possible initiation codons, ATG (Met1, Met7 and Met8, Fig. 3B). The nucleotide sequences surrounding the first and third ATGs agree with Kozak's rules (20). Met1 is more likely to be the initiation codon for the following reasons: 1) more than 90% of eukaryotic mRNAs have their initiation site at the 5'-proximal ATG and 2) the sequence between the 5'-end and the presumed initiating ATG codon appears to be a termination codon, TGA, which is in frame with the downstream open reading frame (position -267) (21). The open reading frame containing this initiation codon predicted a protein of 382 amino acids with a molecular weight of 44,776 and eight possible N-glycosylation sites. Hydropathy plot analysis revealed a considerable hydrophobic segment near the N terminus; this is the putative signal anchor sequence (Fig. 5). Analysis using the TMpred program (22) predicted that LacCer synthase would be a membrane-bound protein with one transmembrane helix composed of 20 amino acid residues (Leu15-Val34) at the N terminus, extending the N-terminal segment into the cytoplasmic surface.

                              
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Table II
Amino acid sequence and mass values of API peptides of rat brain LacCer synthase.
Purified rat LacCer synthase was cleaved with Achromobacter protease I, and the digested peptides were separated by reversed-phase high pressure liquid chromatography. Selected peptides were subjected to amino acid sequence analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry. Capital letters signify clearly identified residues; lowercase letters and X represent ambiguous and not identified residues, respectively. Calculated mass values were obtained from the sequence obtained; those in parentheses were from the deduced sequences (Fig. 4).


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Fig. 4.   cDNA and deduced amino acid sequences of rat brain LacCer synthase. A, restriction enzyme map and clones obtained from PCR, 5'-rapid amplification of cDNA ends (5'-RACE), and the pCMV-SPORT 2 library. The open box indicates the open reading frame. Solid lines indicate the 5'- and 3'-noncoding regions. The numbers correspond to the position in the cDNA sequence in B. B, cDNA and amino acid sequence of rat brain LacCer synthase. The first and second lines indicate the nucleotide and the deduced amino acid sequences, respectively. Nucleotide and amino acid positions are shown at the right. Position 1 refers to the first nucleotide and amino acid residue of the predicted LacCer synthase coding region. N-Glycosylation site consensus motifs are underlined. Amino acid sequences determined by Edman degradation of purified peptide fragments released by lysylpeptidase are double-underlined.


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Fig. 5.   Hydropathy plot of the deduced amino acid sequence of rat brain LacCer synthase. Hydropathy plot of the deduced amino acid sequence analyzed by the method of Kyte and Doolittle with a window of 10 (34). Positive values represent increased hydrophobicity.

Expression of LacCer Synthase-- To confirm that the cDNA obtained during our experiments encoded LacCer synthase, a recombinant baculovirus was prepared and used to infect Sf9 insect cells. Fig. 6 shows that a homogenate of Sf9 cells infected with this recombinant baculovirus exhibited significant LacCer synthase activity, whereas a homogenate of Sf9 cells infected with wild-type virus did not.


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Fig. 6.   Expression of LacCer synthase in Sf9 cells. LacCer synthase (shaded bar) and glycopeptide beta -1,4-GalT (open bar) activity in Sf9 cells infected with either recombinant LacCer synthase baculovirus or wild-type virus was measured as described in "Experimental Procedures."

Analysis of the Deduced Amino Acid Sequence of LacCer Synthase-- We searched the protein data base SWISS-PROT for similar sequences using the DNASIS (version 3.6) program. Mouse, bovine, and human glycopeptide beta -1,4-GalT showed significant homology with LacCer synthase (Fig. 7). The enzyme purified in the present study showed 39% identity with mouse glycopeptide beta -1,4-GalT, and higher identity was observed in the C-terminal domain. There was no significant homology with other GalTs, including galactosylceramide synthase (23) and GD1b/GM1/GA1 synthase (24). No sequence identity was observed between rat LacCer synthase and the short sequence reported for rat glycopeptide beta -1,4-GalT (25), indicating that the enzymes responsible for galactosylation of GlcCer and galactosylation of glycoproteins are different proteins.


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Fig. 7.   Sequence comparison of LacCer synthase predicted from the novel clone with glycopeptide beta -1,4-GalTs. Amino acids which are identical in LacCer synthase and three glycopeptide beta -1,4-GalTs are shaded. Gaps introduced to optimize the alignment are indicated by bars. Asterisks indicate cysteine residues that are predicted to form a disulfide linkage in glycopeptide beta -1,4-GalT. Open and closed triangles indicate amino acid residues involved in substrate binding in glycopeptide beta -1,4-GalT. The dotted line indicates a hexapeptide suggested to be the UDP-Gal binding site from a comparison with alpha -1,3-GalT. A, rat brain LacCer synthase; B-E, glycopeptide beta -1,4-GalT of rat, mouse, bovine, and human, respectively. The glycopeptide beta -1,4-GalT sequences are taken from Bendiak et al. (25) (rat), Shaper et al. (28) (mouse), Masri et al. (35) (bovine), and D'Agostaro, G. et al. (11) (human).

Comparison of the locations of cysteines in the predicted amino acid sequences of LacCer synthase (6 cysteines) and glycopeptide beta -1,4-GalT (7 cysteines) showed the conservation of 5 cysteines, including 2 cysteine residues (Cys108 and Cys223, marked with asterisks in Fig. 7) which are predicted to form a disulfide linkage in glycopeptide beta -1,4-GalT (26, 27).

Northern Blot Analysis-- The expression of LacCer synthase mRNA in rat tissues was analyzed by Northern blotting (Fig. 8). A major band of 6.5 kb and a minor band of 2.7 kb were detected in poly(A) RNA from the lungs, heart, skeletal muscle, kidney, and testis, with the highest level in the brain and the lowest level in the liver and spleen. The high expression of LacCer synthase mRNA in the brain may explain the large amount of GSLs in the brain.


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Fig. 8.   Northern blot analysis of poly(A)+ RNAs from rat tissues. Rat multiple tissue Northern blot (CLONTECH) was used for the analysis. Each lane contains 2 µg of poly(A)+ RNA. A 795-bp segment of LacCer synthase cDNA was labeled with [alpha -32P]dCTP using a Rediprime DNA labeling system (Amersham Pharmacia Biotech) and used as a probe. The position of LacCer synthase is 6.5 kb (A). Hybridization with rat glyceraldehyde-3-phosphate dehydrogenase cDNA was carried out as a control experiment (B). Size markers are indicated on the left. Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this study, we purified LacCer synthase from rat brain and obtained its cDNA. The deduced amino acid sequence of the LacCer synthase was different from that of glycopeptide beta -1,4-GalT previously reported (28), although there is a significant homology between these two enzymes especially in their C-terminal region (Fig. 7). The highest sequence identity (68.3%) was found between amino acids Leu260-Arg300 of the LacCer synthase sequence (boxed in Fig. 7).

Information on the active sites of glycopeptide beta -1,4-GalT is gradually accumulating. Aoki et al. (29) demonstrated that the peptide segment between Asp276 and Met328 in human glycopeptide beta -1,4-GalT (underlined in Fig. 7) may participate in UDP-Gal binding, using photoaffinity labeling. This region includes the area that showed the highest homology with LacCer synthase. They also identified the amino acid residues participating in UDP-Gal and GlcNAc binding in the C-terminal domain of human glycopeptide beta -1,4-GalT by site-directed mutagenesis. They showed that Tyr284, Tyr309, and Trp310 (marked with closed triangles in Fig. 7) were critically important for acceptor (GlcNAc) binding, and that Tyr309 was involved in donor (UDP-Gal) binding (29). Although the carbohydrate residue of the LacCer synthase acceptor is glucose, the amino acids corresponding to Tyr284 and Trp310 are well conserved. Tyr309, which is concerned with UDP-Gal binding, was substituted into Phe in LacCer synthase. Zu et al. (30) reported that Phe305, Pro306, Asn307, and Asn308 (marked with open triangles in Fig. 7) of human glycopeptide beta -1,4-GalT were involved in UDP-Gal binding. In LacCer synthase, three of these four amino acids, Phe305, Pro306, and Asn307, were conserved. These observations suggest that LacCer synthase has its catalytic site in the C-terminal domain, as is generally the case with other glycosyltransferases (31) and that the region that shows high homology with glycopeptide beta -1,4-GalT is involved in substrate binding.

Bakker et al. (32) reported the cloning of UDP-GlcNAc:GlcNAc beta -1,4-N-acetylglucosaminyltransferase from Lymnaea stagnalis. Although this enzyme was cloned by heterologous hybridization using the coding region of bovine glycopeptide beta -1,4-GalT cDNA as a probe, it transferred GlcNAc rather than Gal to GlcNAc with a beta -1,4-glycosidic linkage. This enzyme shows a significant homology with both glycopeptide beta -1,4-GalT and LacCer synthase in the boxed area of the C-terminal domain (Fig. 7). These three enzymes have a common property in that they catalyze the transfer of sugars with a beta -1,4-glycosidic linkage from UDP-sugar. It might therefore be speculated that this region is concerned with the substrate binding site and/or formation of beta -1,4-glycosidic linkages. A hexapeptide (RDKKNE, indicated by a dotted line in Fig. 7) in the C-terminal domain of glycopeptide beta -1,4-GalT has been suggested to be the UDP-Gal binding site from a comparison with alpha -1,3-GalT (33). However, there is no identical or similar peptide in the LacCer synthase amino acid sequence.

In summary, we have purified LacCer synthase from rat brain, and cloned its cDNA for the first time. We have also shown that LacCer synthase is different from glycopeptide beta -1,4-GalT. This information regarding the cDNA and amino acid sequence of LacCer synthase opens the door to creating knockout mice and LacCer synthase-deficient cells and to immunological studies. Since LacCer is a precursor for most GSLs, these would be useful tools for studying GSL functions.

    ACKNOWLEDGEMENT

We thank Hatsue Kato for the preparation of rat brain membrane fraction.

    FOOTNOTES

* This work was performed as part of a research and development project within the Industrial Science and Technology Frontier Program and was supported by the New Energy and Industrial Technology Development Organization.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF048687.

§ To whom correspondence should be addressed: Biological Science Laboratories, Kao Corporation, 2606, Akabane, Ichikaimachi, Haga, Tochigi 321-3497, Japan. Tel.: 81-285-68-7459; Fax: 81-285-68-7452; E-mail: 387533{at}kastanet.kao.co.jp.

1 The abbreviations used are: GSL, glycosphingolipids; GlcCer, glucosylceramide; LacCer, Galbeta 1-4GlcCer; LacCer synthase, UDP-galactose:glucosylceramide beta -1,4-galactosyltransferase; GalT, galactosyltransferase; glycopeptide beta -1,4-GalT, UDP-galactose:N-acetylglucosamine beta -1,4-galactosyltransferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; DTT, dithiothreitol; WGA, wheat germ agglutinin; bp, base pair(s); kb, kilobase pair(s); GM2, GalNAcbeta 1-4(NeuAcalpha 2-3)Galbeta 1-4GlcCer; GA2, GalNAcbeta 1-4Galbeta 1-4GlcCer; GA1, Galbeta 1-3GalNAcbeta 1-4Galbeta 1-4GlcCer; GM1, Galbeta 1-3GalNAcbeta 1-4(NeuAcalpha 2-3)Galbeta 1-4GlcCer; GD1b, Galbeta 1-3GalNAcbeta 1-4(NeuAcalpha 2-8NeuAcalpha 2-3)Galbeta 1-4GlcCer.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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