From the Biological Science Laboratories, Kao
Corporation, 2606, Akabane, Ichikaimachi, Haga, Tochigi 321-3497, Japan, the
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
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ABSTRACT |
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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 -1,4-galactosyltransferase (EC 2.4.1.38), which
catalyzes the transfer of Gal to
-1,4-GlcNAc in glycoproteins.
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INTRODUCTION |
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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 -1,4-galactosyltransferase) transfers galactose from UDP-Gal to glucosylceramide (GlcCer), generating a
-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).
-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
-1,4-galactosyltransferase (
-1,4-GalT) had little activity toward GlcCer, indicating that LacCer synthase and glycopeptide
-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 -1,4-GalT, although there was some
homology between the C-terminal domains of both enzymes.
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EXPERIMENTAL PROCEDURES |
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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),
[-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 -1,4-GalT Activity
Glycopeptide -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 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 -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 [-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).
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RESULTS |
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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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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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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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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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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 -1,4-GalT showed significant homology with LacCer
synthase (Fig. 7). The enzyme purified in
the present study showed 39% identity with mouse glycopeptide
-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
-1,4-GalT (25),
indicating that the enzymes responsible for galactosylation of GlcCer
and galactosylation of glycoproteins are different proteins.
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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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DISCUSSION |
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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 -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 -1,4-GalT is
gradually accumulating. Aoki et al. (29) demonstrated that the peptide segment between Asp276 and Met328
in human glycopeptide
-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
-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
-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
-1,4-GalT is
involved in substrate binding.
Bakker et al. (32) reported the cloning of UDP-GlcNAc:GlcNAc
-1,4-N-acetylglucosaminyltransferase from Lymnaea
stagnalis. Although this enzyme was cloned by heterologous
hybridization using the coding region of bovine glycopeptide
-1,4-GalT cDNA as a probe, it transferred GlcNAc rather than Gal
to GlcNAc with a
-1,4-glycosidic linkage. This enzyme shows a
significant homology with both glycopeptide
-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
-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
-1,4-glycosidic linkages. A hexapeptide (RDKKNE, indicated by a
dotted line in Fig. 7) in the C-terminal domain of
glycopeptide
-1,4-GalT has been suggested to be the UDP-Gal binding
site from a comparison with
-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 -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.
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ACKNOWLEDGEMENT |
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We thank Hatsue Kato for the preparation of rat brain membrane fraction.
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FOOTNOTES |
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* 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, Gal1-4GlcCer;
LacCer synthase, UDP-galactose:glucosylceramide
-1,4-galactosyltransferase; GalT, galactosyltransferase;
glycopeptide
-1,4-GalT, UDP-galactose:N-acetylglucosamine
-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, GalNAc
1-4(NeuAc
2-3)Gal
1-4GlcCer; GA2,
GalNAc
1-4Gal
1-4GlcCer; GA1,
Gal
1-3GalNAc
1-4Gal
1-4GlcCer; GM1,
Gal
1-3GalNAc
1-4(NeuAc
2-3)Gal
1-4GlcCer; GD1b,
Gal
1-3GalNAc
1-4(NeuAc
2-8NeuAc
2-3)Gal
1-4GlcCer.
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REFERENCES |
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