From the Division of Cell Biology, Institute of Life
Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan, the § Laboratory of Cancer Biology and Molecular
Immunology, Graduate School of Pharmaceutical Sciences, Tokyo
University, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the
¶ Laboratory of Animal Resources, Faculty of Bioindustry, Tokyo
University of Agriculture, 196 Aza-Yasaka, Abashiri, Hokkaido 099-2422, Japan, the ** Department of Biomembrane and Biofunctional Chemistry,
Graduate school of Pharmaceutical Sciences, Hokkaido University,
North-12, West-6, Kita-ku, Sapporo 060-0812, Japan, the
Tokyo
Research Laboratories, Kyowa Hakko Kogyo Company Limited, 3-6-6 Asahi-machi, Machida, Tokyo 194-8582, Japan, and the
Laboratory of Gene Function Analysis,
Institute of Molecular and Cell Biology (IMCB), National Institutes of
Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan
Received for publication, December 18, 2000, and in revised form, March 29, 2001
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ABSTRACT |
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A new member of the
UDP-N-acetylglucosamine: To date, three members of the human
The monoclonal antibody HNK-1 reacts to a sulfoglucuronyl
carbohydrate epitope, SO43-GlcA Carbohydrate chains on SGGLs are extended by stepwise reactions
catalyzed by glycosyltransferases. Chou et al. (19-22)
reported developmental changes in the activity of each enzyme in
correlation with the SGGL expression in rat brain. It was demonstrated
that Lc3Cer synthase, which catalyzes the transfer of
GlcNAc to the Gal residue of lactosylceramide (LacCer;
Gal The expression of the Lewis x (Lex; CD15) carbohydrate
structure, Lc3Cer synthase is an important enzyme with respect to
hematopoietic cell differentiation (35). HL-60 cells, a human
promyelocytic leukemic cell line, are capable of bidirectional
differentiation into a monocytoid or granulocytoid lineage (35). The
Lc3Cer synthase and GM3 synthase
(lactosylceramide: Stults and Macher (36) concluded that mature myeloid cells exhibited
Lc3Cer synthase is the enzyme controlling the expression of
neolacto-series GSLs and so plays important roles in many cells. In the
present study, we cloned and characterized a fifth member of the
Tumor Cell Lines and Monoclonal Antibodies (MAbs)--
The tumor
cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc.,
Rockville, MD) supplemented with 10% fetal bovine serum.
The cell lines were donated or purchased from American Type Culture
Collection (ATCC, Manassas, VA), Riken Cell Bank (RCB, Tsukuba,
Japan), Japan Cell Research Bank (JCRB, Tokyo, Japan), Immunobiological
laboratory (IBL, Fujioka, Japan), or Dai-Nippon Pharmaceutical Co.,
Ltd. (DNP, Osaka, Japan).
MAb 1B2 (anti-N-acetyllactosamine) which reacts to
nLc4Cer and nLc6Cer (neolactohexaosylceramide)
(IgM) (36, 37), were used for flow cytometry and immuno-TLC assays,
respectively. The 1B2 hybridoma cells were cultured in RPMI 1640 medium
(Life Technologies, Inc., Rockville, MD) supplemented with 15% fetal
bovine serum. The supernatant of 1B2 hybridoma culture medium was used
for experiments. The supernatant of 1B2 cells was kindly provided by
Seikagaku Kogyo Co. Ltd (Tokyo, Japan).
Isolation of Rat and Human Quantitative Analysis of the Four
Regarding the Transfection Experiments to Express Each of the Four Human
Separation of Glycolipids by Thin Layer Chromatography (TLC) and
Immuno-TLC with Anti-nLc4Cer (Anti-paragloboside)
MAb--
For glycolipid analysis, 1.0 × 108 of the
Namalwa transfectant cells with each Construction and Purification of
We prepared recombinant viruses as described previously (1).
Sf21 insect cells (Pharmingen) were infected with each
individual recombinant virus at a multiplicity of infection of 10 and
incubated at 27 °C for 72 h to yield conditioned media
containing recombinant
Bacu3GnT proteins separated by 10% SDS-polyacrylamide gel
electrophoresis were transferred to an Immobilon PVDF membrane
(Millipore, Bedford, MA). The membrane was probed with anti-FLAG
monoclonal antibody, and stained with the ECL Western blotting
detection reagents (Amersham Pharmacia Biotech). The intensity of
positive bands on Western blotting was measured by densitometer (43) to
determine the relative amounts of each Bacu3GnT protein.
Assaying of
The
We conducted an assay of the incorporation of radioactive sugar into
glycolipid acceptor substrates. The
The reaction mixture was centrifuged at 15,000 rpm for 5 min.
Radioactive glycolipid products were separated from the free radioactive UDP-[14C]GlcNAc using a Sep-Pak Plus
C18 Cartridge (Waters). For conditioning, a Sep-Pak Plus
C18 Cartridge was washed with 10 ml of 100% methanol, and
then washed twice with 10 ml of water. The supernatant of the reaction
mixture was loaded on the equilibrated cartridge. Elution of the
radioactive products was achieved using 5 ml of 100% methanol. Eluted
products were dried with evaporator, and then the residues were
dissolved in an adequate volume of 100% methanol. The dissolved
residues were separated on a HPTLC plate with a solvent system of
chloroform/methanol/0.2% CaCl2 (65:35:8, v/v/v).
The radioactive intensities of the bands were measured with a BAS 2000 Imaging Analyzer (Fuji Film, Tokyo, Japan).
Differentiation of HL-60 Cells in in Vitro Culture--
Human
myelogenous leukemia HL-60 cells were seeded at an initial density of
2 × 105 cells/ml and incubated with either 1 µM RA or 8 nM TPA (35). At various points
over the course of the culture, the cells were harvested, and the total
RNA in the cells was recovered for competitive RT-PCR.
Measurement of the Rat
The cDNA (738 bp; GenBankTM accession number AB045279)
encoding the partial ORF of rat A Novel cDNA Homologous to the Cloned
The human
On a phylogenetic tree (Fig. 2), the four
members of the Quantitative Analysis of Relative Activities of Four Recombinant
We observed no galactosyltransferase activity of the four
Bacu3GnT2 exhibited relative activity toward the following
pyridylaminated substrates: LNnT-PA, 100%; LNT-PA, 3.3%; LNFP-III-PA, 4.2%; and LNFP-V-PA, 4%; and exhibited no activity toward LNFP-II-PA or LNDFH-II-PA. Bacu3GnT3 exhibited relative activity toward LNnT-PA, 1.0%; LNT-PA, 0.8%; and LNFP-V-PA, 0.8%, and exhibited no activity toward LNFP-II-PA, LNFP-III-PA, or LNDFH-II-PA. Bacu3GnT4 exhibited relative activity toward only LNFP-V-PA, 0.2%, and exhibited no activity toward LNnT-PA, LNT-PA, LNFP-II-PA, LNFP-III-PA, or
LNDFH-II-PA. Bacu3GnT5 exhibited relative activity toward
pyridylaminated substrates, LNnT-PA, 18.2%; LNT-PA, 1.0%;
LNFP-III-PA, 0.9%; and LNFP-V-PA, 0.9%. It exhibited no activity
toward LNFP-II-PA or LNDFH-II-PA.
The
The oligosaccharide substrates having the polylactosamine structures,
repeats of units of lactosamine (Gal Relative Activities of Four
Leading to measurement of
As seen in Fig. 4, the homogenates of
Namalwa-3GnT5 cells exhibited strong activity for the synthesis of both
Lc3Cer and nLc5Cer. The homogenates of HL-60
cells also showed positive activities toward both acceptors, LacCer and
nLc4Cer. Relative activities were obtained by measurement
of the positive bands in Fig. 4. The activity of Namalwa-3GnT5 toward
LacCer is presented as 100%, and all other activities are given as
relative values. Namalwa-3GnT5 exhibited strong activity toward LacCer
resulting in the synthesis of Lc3Cer (100%), however, the
other three
Second, we measured the relative activities of the four Bacu3GnTs,
three of which, Bacu3Gn-T2, -T3, and -T4, were the same recombinant
enzymes used in the previous experiments (1) toward glycolipid
acceptors. Bacu3GnT5 was prepared in this study. As summarized in Table
III, the activity of Bacu3GnT5 toward
LacCer is presented as 100%. Bacu3GnT5 exhibited very strong
activities toward both LacCer and nLc4Cer in comparison
with the other enzymes. Interestingly, Bacu3GnT5 preferred
nLc4Cer over LacCer as an acceptor, and transferred GlcNAc
to nLc4Cer 4.67-fold more efficiently than to LacCer.
Bacu3GnT5 did not utilize galactosylceramide (GalCer) as an acceptor.
Bacu3GnT2 exhibited considerable activity toward nLc4Cer,
21.8%, but not toward LacCer. Bacu3GnT3 showed very faint activity
toward nLc4Cer, 0.8%. Again, Bacu3GnT4 did not show any activity toward the glycolipid acceptors.
Immuno-TLC Analysis of Glycoshingolipids (GSLs) Extracted from
Namalwa Cells Transfected with Each Correlation of Lc3Cer Synthesizing Activity with the
Amount of
On the other hand, Colo205 cells, exhibiting weaker activity than
KATOIII cells, expressed larger amounts of
Expression of the Human Change of the Transcript Level of Lc3Cer synthase plays a key role in the control of
carbohydrate synthesis in GSLs during cell differentiation and
development. As demonstrated in the present study, The truncated enzyme expressed in the insect cells preferred
nLc4Cer which has a longer carbohydrate chain than LacCer,
while the membrane-bound form exhibited almost the same activity toward both substrates. The difference in activity between the two forms may
be related to structural difference or the presence of detergent in the
reaction mixture. Glycosyltransferases are the Golgi enzymes bound to
the Golgi membrane by the transmembrane domain. We assume that the
truncated form, which is soluble due to the absence of transmembrane
domain, may easily access GSLs with a long carbohydrate chain, which
are more hydrophilic than GSLs with shorter carbohydrate chains. The
membrane-bound form expressed in Namalwa cells probably has more
physiological activity than the truncated form. In the reaction mixture
for Lc3Cer synthase has been detected in many tissues,
including the developing rat brain (14, 15, 20, 46), hematopoietic cells (35, 36, 47-49), and colorectal tissues (50-52). In all tissues
examined, the Lc3Cer synthase was identified to be a key enzyme in the extension of the carbohydrate chain on neolacto-series GSLs.
The homogenates of Namalwa-3GnT5 cells exhibited strong activity for
both GSL substrates, LacCer and nLc4Cer, as well as toward LNnT-2AB and the two shorter polylactosamine chains, 2LN-2AB and 3LN-2AB. This indicated that The Lc3Cer synthase is a key enzyme in the expression of a
series of neolactoglycolipids, i.e. nLc4Cer and
its derivatives. In particular, the expression level of two SGGLs,
SGGL-1 and SGGL-2, carrying the HNK-1 epitope is determined by the
Lc3Cer synthase (15, 19-22, 46). The change in the level
of Lc3Cer synthase is also an important enzyme in
hematopoietic cell differentiation. The changes in the level of
Holmes (50) reported the Regarding the other The The results of the present study strongly indicated that -galactose
1,3-N-acetylglucosaminyltransferase (
3Gn-T) family
having the
3Gn-T motifs was cloned from rat and human cDNA
libraries and named
3Gn-T5 based on its position in a phylogenetic
tree. We concluded that
3Gn-T5 is the most feasible candidate for
lactotriaosylceramide (Lc3Cer) synthase, an important
enzyme which plays a key role in the synthesis of lacto- or
neolacto-series carbohydrate chains on glycolipids.
3Gn-T5 exhibited
strong activity to transfer GlcNAc to glycolipid substrates, such as
lactosylceramide (LacCer) and neolactotetraosylceramide
(nLc4Cer; paragloboside), resulting in the synthesis of
Lc3Cer and neolactopentaosylceramide (nLc5Cer), respectively. A marked decrease in LacCer and increase in
nLc4Cer was detected in Namalwa cells stably expressing
3Gn-T5. This indicated that
3Gn-T5 exerted activity to synthesize
Lc3Cer and decrease LacCer, followed by conversion to
nLc4Cer via endogenous galactosylation. The following four
findings further supported that
3Gn-T5 is Lc3Cer
synthase. 1) The
3Gn-T5 transcript levels in various cells were
consistent with the activity levels of Lc3Cer synthase in
those cells. 2) The
3Gn-T5 transcript was presented in various
tissues and cultured cells. 3) The
3Gn-T5 expression was
up-regulated by stimulation with retinoic acid and down-regulated with
12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in
3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the
Lc3Cer synthase activity reported previously.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3-N-acetylglucosaminyltransferase
(
3Gn-T)1 family (
3Gn-T2,
-T3, and -T4) (1, 2) and five members of the human
1,3-galactosyltransferase (
3Gal-T) family (
3Gal-T1, -T2, -T3,
-T4, and -T5) have been identified (3-6). All of them share amino acid
motifs (
3Gn-T motifs or
3Gal-T motifs) in three regions of the
catalytic domain. The first,
3Gn-T, was cloned by an expression
cloning method using an anti-i antibody (7). However, this enzyme is
unique in that it does not have the
3Gn-T motifs although it
transfers GlcNAc to Gal with an
1,3-linkage, resulting in the
synthesis of polylactosamine chains. It was named iGn-T (7).
Thereafter,
3Gn-T1 was isolated based on structural similarity with
the
3Gal-T family (2). We previously reported three additional
3Gn-Ts,
3Gn-T2, -T3, and -T4, which are also structurally related
to the
3Gn-T family (1). However, the cDNA sequence of
3Gn-T1
was recently corrected by Zhou et al. (see Ref. 2). The
corrected sequence of
3Gn-T1 was identical to that of
3Gn-T2
which was isolated and reported by us (1). So, a total of four
3Gn-Ts, i.e. iGn-T,
3Gn-T2(-T1), -T3, and -T4, have
been described to date. To avoid further confusion, we note this fact,
but do not change the enzyme names used in this study. By transfection
experiments and in vitro enzymatic analysis, these four
3Gn-Ts were found to exhibit
3Gn-T activity that catalyzes the
synthesis of polylactosamine chains, but not
3Gal-T activity.
1-3Gal
1-4GlcNAc-R,
which is expressed in several glycoproteins involved in neural cell
recognition, and in two neolactoglycolipids, named
sulfoglucuronylglycolipid (SGGL)-1 and -2 (SGGL-1,
SO43-GlcA
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc
1-1ceramide; and SGGL-2,
SO43-GlcA
1-3Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc
1-1ceramide) (8, 9). Neurobiologists have suggested in a series of studies that the
HNK-1 epitope functions as a cell-cell interaction molecule during
the development of the brain to complete the nervous system (10-14).
The expression is developmentally and spatially regulated in the
nervous system. In rat cerebellum, SGGLs are expressed in a biphasic
manner, with the initial peak at around postnatal days (PD) 1-3, and
the second peak at PD20 (15). In the adult rat cerebellum, the
expression of SGGLs is kept constant (16, 17). Immunohistochemical
studies localized SGGLs in specific cells during the development of the
cerebellum, and suggested some biological functions for neuron guidance
(14, 15, 17, 18). In rat cerebral cortex, the expression of SGGLs
peaked at embryonic day (ED) 19 and then decreased until postnatal PD 5, and had almost completely disappeared by PD 20.
1-4Glc
1-1Cer) with a
1-3-linkage resulting in the
synthesis of Lc3Cer (GlcNAc
1-3Gal
1-4Glc
1-1Cer), is the key enzyme in the expression of SGGLs in the developing rat
brain, because only the activity of this synthase was well correlated
with the developmental change in SGGL expression.
1,3-fucosyl-N-acetyllactosamine,
Gal
1-4(Fuc
1-3)GlcNAc
1-R, is also regulated developmentally
and region specifically in the brain (23-31). The Lex also
functions as a cell-cell recognition molecule in the highly organized
structures of the central nervous system (30, 32, 33). The
Lex expression on GSLs in brain is also regulated by the
Lc3Cer synthase which synthesizes a root structure of the
GSLs carrying the Lex epitope (34). Thus, SGGLs and GSLs
carrying the Lex epitope are of interest in terms of their
biosynthetic regulation.
2,3-sialyltransferase), which share the acceptor
substrate lactosylceramide (LacCer); Gal
1-4Glc
1-1Cer, are key
enzymes in determining the biosynthetic flow of GSLs in the two
different directions. During the monocytic differentiation of HL-60
cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA)
treatment, ganglioside GM3 was markedly increased by up-regulation of
GM3 synthase and down-regulation of Lc3Cer synthase. In
contrast, during the granulocytic differentiation of HL-60 cells
induced by all-trans-retinoic acid (RA), neolacto-series
GSLs were markedly increased by up-regulation of Lc3Cer synthase.
3Gn-T activity toward both acceptor substrates, leading to the
expression of several types of neolacto-neutral GSLs in the cells,
while lymphoid cells exhibited activity for nLc4Cer, but
not LacCer. The lack of Lc3Cer synthesizing activity in
lymphoid cells explains the absence of neolacto-neutral GSLs.
3Gn-T family (
3Gn-T5), and identified
3Gn-T5 as the most
likely candidate for Lc3Cer synthase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3Gn-T5 cDNAs--
We
constructed a rat shank cDNA library for a random sequencing
project. During the sequencing, we found a rat cDNA encoding a
partial sequence of
3Gn-T5. This novel sequence (738 bp) did not
encode the full ORF. But it had the
3Gal-T (
3Gn-T) motifs which
are shared by the known
3Gal-Ts and
3Gn-Ts. The cDNA library of Colo205 cells constructed in a previous study (6) was screened with
the rat partial
3Gn-T5 cDNA as a probe to isolate the
full-length human
3Gn-T5 cDNA which possessed a 3.7-kilobase
pair insert DNA. We did not clone a rat full-length
3Gn-T5 in
this study.
3Gn-T Transcripts in Human
Tissues and Tumor Cell Lines by Competitive RT-PCR--
The principle
behind the competitive RT-PCR method for quantification of transcripts
was described in detail in our previous studies (6, 38). Total cellular
RNAs of various human tissues were purchased from
CLONTECH. Those of various tumor cell lines were
extracted and purified in our laboratory. As to the measurement of the
transcripts for three cloned
3Gn-T genes,
3Gn-T2, -T3, and -T4, the primers
and the PCR conditions were also reported previously (1).
3Gn-T5 gene, a standard DNA
plasmid was constructed by subcloning a full-length ORF cDNA into a
pBluescript SK(
) vector. A competitor DNA plasmid carrying a small
deletion (245 base pairs) within the ORF of the
3Gn-T5 cDNA was
constructed as follows. The standard DNA plasmid was double digested
with appropriate restriction endonucleases, MscI and
BglII, and was blunt-ended with T4 DNA polymerase. This was
followed by self-ligation of the deleted DNA plasmid. As to the
measurement of the
3Gn-T5 transcripts by competitive RT-PCR, we used
the following primer set: forward primer,
5'-TCTTATGACTGCTGATGATGACAT-3', and reverse primer,
5'-CTTTAGGATCTGTAGCATTCTTCC-3'.
3Gn-T Genes in Namalwa Cells--
Expression of glycosyltransferase
genes subcloned in pAMo was described in detail in a series of previous
papers (6, 7, 39-42). The construction of pAMo vector inserted with
each of three cloned
3Gn-T genes,
3Gn-T2, -T3, and -T4,
was also reported previously (1). Regarding the
3Gn-T5 gene, the
3Gn-T5 ORF fragment was
excised from the standard
3Gn-T5 DNA in pBluescript SK(
), and then
blunt-ended with T4 DNA polymerase. The blunt-ended fragment was
flanked with SfiI linker and inserted into the
SfiI site of pAMo vector. Each of the four
3Gn-T genes subcloned into a pAMo vector was
transfected by electroporation into Namalwa cells. These cells were
selected in the presence of geneticin (G418) (Life Technologies, Inc.)
at a concentration of 0.8 mg/ml. Stable transfectant cells were
obtained after 25 days exposure to geneticin. The levels of the
transcripts expressed in the Namalwa transfectant cells were measured
by means of competitive RT-PCR.
3Gn-T
gene were collected, washed twice with phosphate-buffered saline, and
then lyophilized. Total glycolipids were extracted from the lyophilized
cells. Crude glycolipids were extracted twice from the transfectant
cells, first with chloroform/methanol (2:1, v/v), and then with
chloroform/methanol/water (30:60:8, v/v/v). Samples dried with the
N2 evaporator were dissolved in methanol, then subjected to
mild alkaline treatment in 0.1 N KOH/methanol at 40 °C
for 2 h, and neutralized with 1 N acetic acid. After the free fatty acids had been removed with n-hexane, the
remaining fractions were dried with the N2 evaporator and
subjected to Folch's partition. The lower neutral glycolipid fractions
were dried with the N2 evaporator and subjected to
immuno-TLC analysis. Neutral glycolipid equivalent to 1.0 × 107 cells was applied to each lane of TLC. Neutral
glycolipids were separated by TLC (HPTLC Kieselgel 60, 5641; MERK,
Germany) with mixtures of chloroform/methanol/water (60:35:8, v/v/v)
and immuno-TLC analysis was performed with the antibody 1B2 as
described (36).
3Gn-T Proteins Fused with
FLAG Peptide--
The putative catalytic domain of each of
3Gn-T2,
-T3, -T4 had been expressed as a secreted protein fused with FLAG
peptide in insect cells as described in a previous study (1). In the present study, a 1.1-kilobase pair DNA fragment encoding a
COOH-terminal portion of
3Gn-T5 (amino acids 39 to 378) was
amplified by PCR. The PCR was performed with Platinum Pfx DNA
Polymerase (Life Technologies, Inc.), according to the supplier's
manual. The 5' and 3' primer sequences were flanked with
BamHI and XbaI sequences, respectively, to create
the restriction sites. Those sequences were as follows: forward primer,
5'-CGGGATCCATTGTGAGCCATATGAAGTCATAT-3', and reverse primer,
5'-GCTCTAGATGACAGTGAAAACATACAACATTC-3'. The amplified fragment was
first inserted between the BamHI and XbaI sites
of the pBluescript SK(
) vector. Subsequently, the insert DNA was excised by BamHI and NotI, and was inserted
between the BamHI and NotI sites of pVL1393-F2 to
yield pVL1393-F2G5. pVL1393-F2 is an expression vector derived from
pVL1393 (Pharmingen) and contains a fragment encoding the signal
peptide of human immunoglobulin (MHFQVQIFSFLLISASVIMSRG) and FLAG
peptide (DYKDDDDK).
3Gn-T proteins fused with FLAG peptide.
Bacu3GnT proteins were readily purified by anti-FLAG M1 antibody resin
(Sigma) and eluted with 50 mM TBS (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl), 4 mM
CaCl2 buffer (pH 7.4). The recombinant proteins obtained in
this system were named Bacu3GnT2, Bacu3GnT3, Bacu3GnT4, and Bacu3GnT5.
3Gn-T Activity--
Two types of each recombinant
3Gn-T, the soluble enzyme produced in the baculoexpression system
and the membrane-bound form expressed in the cell homogenates of
Namalwa transfectant cells and other cultured cell lines, were used to
determine the relative activities of each
3Gn-T toward various
substrates. Each soluble Bacu3Gn-T purified was adjusted to the same
amount as described above and used for the assay. The various cultured
cells were solubilized in a 20 mM HEPES buffer (pH 7.2)
containing 2% Triton X-100, and 20 µg of total protein in the cell
homogenates was used for the enzyme reaction. Various oligosaccharides
were fluorescently labeled with 2-aminobenzamide (2AB) or
pyridylaminated (PA) (1, 44) and used for acceptors.
3Gn-T activity was assayed in a 20-µl reaction mixture
containing 150 mM sodium cacodylate buffer (pH 7.2), 50 mM UDP-GlcNAc, 10 mM MnCl2, 0.4%
Triton CF-54, 1 µM 2AB-acceptor or PA-acceptor substrate,
and the enzyme source. After incubation at 37 °C for 16 h, the
reaction was terminated by boiling for 3 min, and the mixture was
diluted with 80 µl of water. After centrifugation at 15,000 rpm for 5 min, the supernatant was filtered using an Ultrafree-MC column
(Millipore). A 10-µl aliquot of each supernatant was subjected to
high performance liquid chromatography on a TSK-gel ODS-80TS QA column (4.6 × 300 mm; Tosoh, Tokyo,
Japan). The reaction products were eluted with 20 mM
ammonium acetate buffer (pH 4.0) containing 7% methanol at a flow rate
of 1.0 ml/min at 50 °C and monitored with a fluorescence
spectrophotometer, JASCO FP-920 (JASCO, Tokyo, Japan) (1, 44).
3Gn-T activity for glycolipids
was assayed in a 25-µl reaction mixture containing 150 mM
sodium cacodylate buffer (pH 7.2), 480 µM UDP-GlcNAc, 175 nCi of UDP-[14C]GlcNAc (Amersham Pharmacia Biotech), 10 mM MnCl2, 0.4% Triton CF-54, 10 nmol of
glycolipid acceptor substrate, and the enzyme source. After incubation
at 37 °C for 16 h, the reaction was terminated by boiling for 3 min, and 200 µl of water containing 0.1 M KCl was added.
3Gn-T5 Transcript in Developing
Brain--
Brains of SD rats (Charles River Japan, Tokyo, Japan) were
separated into cerebral cortex and cerebellum, and the total RNA recovered from each tissue was subjected to competitive RT-PCR assay.
Total RNA was extracted from a mixture of 4-6 rat brains at each stage
of development, and was used for cDNA synthesis.
3Gn-T5 was used as a standard, from which a competitor DNA was generated by deletion of a BsmBI
fragment (185 bp). A primer set, a forward primer,
5'-CAAGATTTCACTGATTCTTTCCAC-3' and a reverse primer,
5'-GTCCTGTAGGTCTTGTGAGTGTCC-3', was used for the competitive RT-PCR
assay. The competitive RT-PCR experiment was performed twice on each sample.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3Gal-Ts or
3Gn-Ts--
A novel cDNA sequence encoding
3Gn-T motifs
was found during the random sequencing of a rat shank cDNA library.
We cloned a full-length human cDNA homologous (AB045278) to the rat
cDNA (AB045279), and named it
3Gn-T5. The amino acid sequence
homology in the corresponding region between human and rat was 82.9%.
This indicated that the two cDNAs are orthologous.
3Gn-T5 cDNA contains a full-length ORF
encoding a protein of 378 amino acids, as shown in Fig.
1.
3Gn-T5 is predicted to be a typical
type II membrane protein consisting of a NH2-terminal cytoplasmic domain of 12 residues, a transmembrane segment of 20 residues, and a stem region and catalytic domain of 346 residues.
3Gn-T5 had the three motifs typical of members of the
3Gal-T and
3Gn-T families. On ClustalW analysis (Fig. 1), four cysteine residues were found to be conserved in the four
3Gn-Ts which indicates that some of these cysteines are essential for maintenance of
the tertiary structure of
3Gn-Ts. In the second
3Gn-T motif, a
triplet of aspartic acid residues, DDD, which may be a dication binding
site as proposed in a crystallization study of other
glycosyltransferases (45), was conserved. Four possible
N-glycosylation sites were found in the primary sequence of
3Gn-T5. One of them was conserved in all
3Gn-Ts. The
3Gn-T5 gene was found to be localized to a
draft genome sequence (GenBankTM accession number AC025833)
and the 3.7-kilobase pair cDNA containing the ORF is composed of a
single exon.
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Fig. 1.
ClustalW alignment for comparison of
3Gn-T5 with the other four
3Gn-Ts. Multiple sequence analysis (ClustalW)
of the four members of the
3Gn-T family. Introduced gaps are shown
with hyphens. The three
3Gn-T motifs are
boxed. The putative transmembrane domains are
underlined. Asterisks indicate the amino acids identical
among all proteins. Conserved amino acids are shown by dots.
Cysteine residues conserved in the four
3Gn-Ts,
3Gn-T2, -T3, -T4,
and -T5, are indicated by open arrows. Possible
N-glycosylation sites in the
3Gn-T5 sequence are
double underlined. A possible N-glycosylation
site conserved in all proteins is indicated by a closed
arrow.
3Gn-T family apparently formed a cluster which is
separated from
3Gal-T members.
3Gn-T5 is positioned in the
3Gn-T family branch, however, it is in an outer branch away from the
cluster of the other members. Three enzymes,
3Gn-T2, -T3, and -T4,
form a subfamily in the phylogenetic tree and
3Gal-T4 and iGn-T also
form a subfamily. The divergence of
3Gn-T5 occurred earlier than
that of any other
3Gn-T member.
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Fig. 2.
A phylogenetic tree of
3Gal-Ts and
3Gn-Ts. A
phylogenetic tree of
3Gal-Ts and
3Gn-Ts was constructed by means
of the neighbor joining method (53) based on the amino acid sequences.
The branch length indicates the evolutionary distance between each
member.
3Gn-T5 Transcripts in Human Tissues and
Various Cell Lines--
As summarized in Fig.
3 and Table I,
this gene was expressed in almost all tissues and cell lines with very
few exceptions, although the expression level was different depending
on the tissue and cell line. The tissues expressing
3Gn-T5 at a considerably high level were lung,
colon, placenta, testis, pituitary gland, and cerebellum. Brain, liver
(very low), spleen, lymph node, and thymus expressed
3Gn-T5 at a low level. Colonic adenocarcinoma, Colo205, SW620, lung cancer cell lines, EBC-1, HAL8, LX-1, PC-7, and
RERF-LC-MS, and stomach cancer cell lines, KATOIII, MKN7, and HSC43
expressed the
3Gn-T5 transcripts at a high
level. All neuroblastoma cells examined, except for NAGAI and GOTO
cells, expressed the
3Gn-T5 transcript at a
relatively high level. On the other hand, all leukemic cells derived
from lymphocytes, except for NALL-1 (lymphoblastic leukemia), expressed
3Gn-T5 almost at an undetectable level. U937
(monocyte-like) and HL-60 (promyelocytic leukemia) cells expressed it
at a considerable level. HepG2 cells did not express it at all. The
expression levels in the cultured cells reflected those in the original
tissues derived therefrom.
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Fig. 3.
Quantitative analysis of transcripts of
3Gn-Ts in various human tissues by competitive
RT-PCR. The transcript levels of
3Gn-T5
in 36 kinds of human tissue were determined by competitive RT-PCR. The
single stranded cDNA for the
3Gn-T5
transcript was amplified together with 200 ag/µl of the
respective competitor DNA. The human
-actin
transcripts in the respective tissues were also quantified with
200 fg/µl of the
-actin competitor DNA. The value for the
3Gn-T5 transcript divided by that for the
respective
-actin transcript is shown as a
bar chart.
Quantitative analysis of 3Gn-T transcripts in various human cell
lines by competitive RT-PCR
3Gn-Ts, Which Were
Produced as Truncated Forms of Fusion Protein with the FLAG Peptide in
a Baculoexpression System, Toward Oligosaccharides--
The
recombinant enzymes of four
3Gn-Ts,
3Gn-T2, -T3, -T4, and -T5,
produced as fusion proteins with a FLAG peptide in the baculoexpression
system were named Bacu3GnT2, -3GnT3, -3GnT4, and -3GnT5, respectively.
The amounts of each enzyme were made equal for assaying the
3Gn-T activity.
3Gn-Ts
toward LNnT-PA and agalacto-LNnT-PA (data not shown). The
3Gn-T
activity of Bacu3GnT2 toward LNnT-PA was strongest among all
combinations of the enzyme and the substrate. Thus, the activity of
Bacu3GnT2 toward LNnT-PA is presented as 100%, and all other activities are given as relative values.
3Gn-T activity of Bacu3GnT5 toward LNnT-PA was almost one-fifth
the activity of Bacu3GnT2. The three Bacu3GnTs, -3GnT2, -3GnT3, and
-3GnT5, exhibited weak activity for LNT-PA and LNFP-V-PA. Bacu3GnT2 and
-3GnT5 also exhibited weak activity for LNFP-III-PA. LNFP-II-PA and
LNDFH-II-PA could not be utilized as acceptor substrate for any
Bacu3GnT examined. Bacu3GnT4 activity was almost undetectable for all
substrates except a very faint activity for LNFP-V-PA. In a previous
study, we confirmed that Bacu3GnT3 and Bacu3GnT4 apparently exhibited
detectable levels of
3Gn-T activity toward LNnT-PA and LNT-PA
because we used an excess of recombinant enzyme for the reaction
(1).
1-4GlcNAc; LN), were labeled
with 2AB and used as acceptor substrates (44). The LNnT-2AB
oligosaccharide was used as a control. 2LN, 3LN, 4LN, and 5LN in Table
II indicate that each oligosaccharide has 2-, 3-, 4-, or 5-repeating
lactosamine (LN) units, respectively. The
3Gn-T activity toward
LNnT-2AB of Bacu3GnT2 was again the strongest among all combinations of
enzyme and substrate, therefore its activity is expressed as 100%, and
the activities of the other combinations are expressed relative to this
value in Table II. Bacu3GnT2 transferred
a GlcNAc with almost the same level of activity to all polylactosamine
substrates regardless of the number of LN units. Bacu3GnT3 exhibited
low, but apparently positive activity for all lengths of
polylactosamine substrate. The activity of Bacu3GnT4 was again hardly
detected with the amount of recombinant protein used in the present
study. Interestingly, Bacu3GnT5 preferred the shorter substrates,
i.e. 2LN-2AB and 3LN-2AB. The activities of Bacu3GnT5 for
the longer polylactosamine chains, 4LN-2AB and 5LN-2AB, were almost
one-tenth of those for the shorter chains.
Specific activity of recombinant 3Gn-Ts expressed in the baculo
system toward polylactosamine chains
3Gn-Ts, Which Were Produced in Two
Different Types of Recombinant Enzymes, Toward Glycolipid
Substrates--
To elucidate the relative activities toward glycolipid
acceptors, two recombinant
3Gn-Ts were compared. First, the
homogenates of Namalwa cells stably expressing each
3Gn-T gene were used as a source of
recombinant enzyme.
3Gn-T activity in Namalwa transfectant
cells, we determined the transcript level of each
3Gn-T gene expressed in each Namalwa
transfectant cell. The wild-type Namalwa cells expressed substantial
amounts of
3Gn-T2 endogenously, 3.3 units, but
not the other
3Gn-Ts (
3Gn-T3, -T4, and -T5). The amounts of
transcript in cells transfected with the
3Gn-T2,
3Gn-T3,
3Gn-T4, or
3Gn-T5 gene, were 21.2, 15.4, 24.4, and 10.0 units,
respectively. The amount of each transcript in cells mock-transfected
with the pAMo vector alone was just as much as that in Namalwa wild
cells. We used HL-60 cells as a control for this experiment. HL-60
expressed substantial amounts of two
3Gn-T transcripts,
i.e.
3Gn-T2; 5.3 units and
3Gn-T5; 0.9 units, but possessed no transcript of
3Gn-T3 and
3Gn-T4.
3Gn-Ts showed no activity toward LacCer. The wild-type
Namalwa cells and the mock-transfected Namalwa cells showed faint
activity, 2.6 and 2.5%, respectively, for the synthesis of
nLc5Cer. The other transfectant cells did not show an
increase in either activity, although the transcript for the
transfected gene was apparently overexpressed in the cells. Their
activities were 3.2, 2.8, and 2.4%, respectively. In contrast, Namalwa-3GnT5 exhibited strong activity toward nLc4Cer. The
relative activity of Namalwa-3GnT5 for nLc5Cer synthesis
was almost the same, 105.2%, as that for Lc3Cer
synthesis.
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Fig. 4.
Assaying of Lc3Cer and
nLc5Cer synthesizing activities for the acceptors, LacCer
and nLc4Cer, respectively, in the homogenates of Namalwa
cells stably expressing each 3Gn-T
gene. The homogenates of Namalwa wild-type cells, Namalwa
mock-transfected cells, and Namalwa cells stably expressing each
3Gn-T gene were used as enzyme sources. The
Lc3Cer synthesizing activity (left panel) and
the nLc5Cer synthesizing activity (right panel)
were determined from [14C]GlcNAc incorporation into
LacCer (left panel) and nLc4Cer (right
panel). Following the enzyme reaction, reaction products were
separated by HPTLC and the radioactivity in the positive bands was
measured by BAS 2000.
Specific activity of recombinant 3Gn-Ts expressed in the baculo
system toward glycolipid acceptors
3Gn-T Gene--
As seen from
the orcinol staining results of neutral GSLs (Fig.
5A), among multiple bands of GSLs,
the band intensity of LacCer of Namalwa-3GnT5 apparently decreased as
compared with that of the other transfectants. By immunostaining using
the 1B2 mAb (Fig. 5B), positive bands were detected in all
transfectants including the wild-type and mock transfectant cells.
However, the nLc4Cer band of Namalwa-3GnT5 cells showed a
very strong intensity as compared with that of the other transfectants.
The positive band faintly detected below that of nLc4Cer of
Namalwa-3GnT5 cells corresponded to nLc6Cer. It is known
that mAb 1B2 reacts with both nLc4cer and
nLc6Cer (37). The above results are interpreted as follows.
The overexpressed
3Gn-T5 in the cells consumed the substrate,
LacCer, and thereafter, the product Lc3Cer was
galactosylated by (an) endogenous
1,4-galactosyltransferase(s) to
produce nLc4Cer, because Namalwa cells are known to
endogenously possess an excess amount of
4Gal-T1. Some of the
nLc4Cer produced in the cells was further converted to
nLc5Cer by the overexpressed
3Gn-T5 and again
galactosylated to produce nLc6Cer by endogenous
4Gal-T(s).
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Fig. 5.
Orcinol staining and HPTLC immunostaining of
neutral glycolipids extracted from Namalwa cells and
transfectants. A, neutral glycolipids were visualized
by orcinol staining. Total glycolipids were extracted from lyophilized
cells, equivalent to 1.0 × 108 cells. Standard
glycolipids, GlcCer, LacCer, Lc3Cer, and
nLc4Cer (from top to bottom), are presented in the
left lane. B, the neutral GSL bands were
immunostained with mAb 1B2 (anti-nLc4Cer and
anti-nLc6Cer).
3Gn-T5 Transcript in Various Cultured Tumor
Cells--
The Lc3Cer synthesizing activity was highest in
KATOIII cells among various cultured cancer cells examined in this
study (Table IV). Thus, the activity of
KATOIII was set as 100%, and the values for Lc3Cer
synthesizing activity of the other cells were calculated relative to
this (see Table IV). The level of Lc3Cer synthesizing activity was almost correlated with the amount of
3Gn-T5 transcript (the mathematical correlation coefficient is 0.83), but not with the
levels of the other
3Gn-T transcripts (the mathematical correlation coefficients of other
3Gn-Ts are all under 0.45). KATOIII cells possessed the most
3Gn-T5, followed by Colo205, EBC-1, LS180, and
AOI cells in decreasing order both for activity and the amount of
transcript. However, SK-N-SH cells are an exception which exhibited low
activity, despite the relatively high expression of
3Gn-T5 transcripts. We do not know why the Lc3Cer synthesizing
activity of SK-N-SH cells conflicted with the amount of
3Gn-T5
transcript. Some cancer cells, HepG2, Jurkat, Daudi, Ramos, and GOTO
cells, did not express the
3Gn-T5 gene at all,
and exhibited no activity of Lc3Cer synthase.
Correlation of Lc3Cer synthase activity with the transcript
level of 3Gn-T5 in various tumor cells
3Gn-T2 transcript than KATOIII cells. HepG2
cells expressed considerable amounts of
3Gn-T2
and -T3 transcripts, but they exhibited no activity of
Lc3Cer synthase.
3Gn-T4 was
expressed at a very low or undetectable level in all cell lines
examined. The above results indicated that the Lc3Cer
synthesizing activity is mainly directed by
3Gn-T5 in these cell lines.
3Gn-T5 Transcripts during
Differentiation in the Human Promyelocytic Cell Line HL-60--
As
seen in Fig. 6, the expression level of the
3Gn-T5 transcript during the granulocytic
differentiation by RA treatment apparently increased in comparison to
that of the wild-type HL-60 cell. On the other hand, it markedly
declined during the monocytic differentiation induced by TPA
treatment.
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Fig. 6.
Change of 3Gn-T5
transcript levels during differentiation of HL-60 cells by induction
with RA or TPA. The expression level of the
3Gn-T5 transcript in HL-60 cells was
quantified by competitive RT-PCR. The value was divided by that for the
respective
-actin transcript. Non-treated HL-60 cells (
) are
indicated at 0 days. HL-60 cells were cultured in the presence of 1 µM RA (
) or 8 nM TPA (
) for up to 4 or
2 days, respectively.
3Gn-T5 in the Developing Rat
Brain--
As shown in Fig. 7, in the rat
cerebral cortex, the expression level of the
3Gn-T5 transcript peaked at around ED19, and had almost completely disappeared by PD14. The adult cerebral cortex
expressed no
3Gn-T5 transcript. In contrast,
cerebellum showed biphasic changes in the transcript level. The level
of
3Gn-T5 transcript decreased from ED19 to
PD3, and then increased until PD8. Subsequently, the level again
decreased till PD14. In the adult cerebellum, the expression level was
comparatively high.
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Fig. 7.
Change of 3Gn-T5
transcript levels during rat brain development. Total RNA was
extracted from a mixture of 5-7 rat brains at each developmentally
stage, fetal (ED19), neonatal (PD3, PD8, and PD14), and adult, and
subjected to the measurement of
3Gn-T5
transcript by competitive RT-PCR. The value for the transcript in
cerebellum (
) and cerebral cortex (
) was divided by that for the
respective rat
-actin transcript for normalization. The competitive
RT-PCR experiments were performed twice on each sample. The values of
two experiments are expressed as the mean ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3Gn-T2, -T3, and
-T4 are not the Lc3Cer synthase. We did not examine
activities of the recombinant enzymes of iGn-T. However, the transcript
levels of iGn-T in various tumor cell lines were not
correlated with the Lc3Cer and nLc5Cer
synthesizing activities in the respective cell lysates (data not
shown). So, we can rule out iGn-T as a candidate for the
Lc3Cer synthase. We concluded that
3Gn-T5 is the most feasible candidate for the following reasons. 1) Bacu3GnT5 and the
homogenates of Namalwa-3GnT5 cells exhibited strong activity to
synthesize Lc3Cer in vitro. 2) LacCer was
consumed to be converted to neolacto-series GSLs in the Namalwa-3GnT5
cells. 3) The transcript levels of
3Gn-T5 in
various tissues and cell lines were consistent with the
Lc3Cer synthesizing activity as reported previously. 4) The
expression level of the
3Gn-T5 transcript in
various cultured cancer cells was well correlated with the
Lc3Cer synthesizing activity. 5) The changes in the
3Gn-T5 transcript level during HL-60
differentiation and during rat brain development were consistent with
those of the Lc3Cer synthesizing activity as reported by others (15, 20, 35, 46).
3Gn-T assay, the membrane-bound form probably exists as a
micellar penetrating the Golgi membrane. It is likely that the
membrane-bound enzyme rather than the truncated soluble form interacts
with glycolipid substrates.
3Gn-T5 effectively recognizes a polylactosamine structure within two units of lactosamine. The marked
reduction in the
3Gn-T5 activity for the longer polylactosamine chain would suggest that GSLs with a long polylactosamine chain, such
as nLc6Cer, are not good substrates for
3Gn-T5. In
previous studies (15, 20, 46), the activity of
3Gn-T toward LacCer and nLc4Cer was measured using tissue homogenates of rat
brain during development. Both activities showed not only very similar profiles of change during the development, but almost the same level.
This may be consistent with the present results showing that the two
activities are directed by a single enzyme,
3Gn-T5. The wild-type
and mock-transfected Namalwa cells showed weak
3Gn-T activity for
nLc4Cer, but no activity for LacCer. We could not identify
which enzyme directs this activity in the wild-type Namalwa cells. The
activity may be directed by endogenous
3Gn-T2 or there may be some
unknown
3Gn-T(s) in the Namalwa cells.
3Gn-T5 transcript in developing rat brain
almost paralleled that in Lc3Cer synthesizing activity
reported previously (15, 20, 46). This strongly indicated that
3Gn-T5 is the Lc3Cer synthase. To confirm this, we will
examine whether or not
3Gn-T5 is co-localized with HNK-1 on SGGLs
and CD15 on neolacto-series GSLs in a future study.
3Gn-T5 transcript during HL-60 differentiation
shown in Fig. 6 are consistent with the results of Nakamura et
al. (35). Stults et al. (36) reported that lymphoid
cell lines lack Lc3Cer synthesizing activity, but possess
nLc5Cer synthesizing activity, whereas myeloid cell lines express both activities. Almost all lymphoid cell lines we examined in
this study showed very low or undetectable levels of
3Gn-T5 transcript, while HL-60 cells (promyelocytic leukemia) and U937 cells
(monocyte-like) expressed substantial amounts of
3Gn-T5. This again
supported that
3Gn-T5 is responsible for the synthesis of
Lc3Cer in hematopoietic cells.
3Gn-T activities for the synthesis of GSL
in the cells homogenates of a human colon cancer cell line, SW403. The
SW403 cell homogenates showed almost the same level of
3Gn-T
activity toward two GSL acceptors, LacCer and nLc4Cer. This
could be interpreted to mean that both activities were directed by a
single enzyme,
3Gn-T5, as in the case of rat brain. In the present
study, we demonstrated that recombinant
3Gn-T5 effectively catalyzes
the biosynthesis of both Lc3Cer and nLc5Cer at
almost the same efficiency. There is some controversy (52) over whether
a single enzyme,
3Gn-T5, synthesizes both Lc3Cer and
nLc5Cer in colon tissue. Based on the Basu study (52), there appear to be unknown
3Gn-Ts in colon tissue, which
differentially catalyze the synthesis of Lc3Cer or
nLc5Cer. Another controversy with the present study relates
to the report by Holmes et al. (51) in which significantly
high activity of Lc3Cer synthase was detected in colonic
adenocarcinoma tissues of patients and cell lines derived therefrom,
but in contrast, the activity was undetectable in normal colonic
epithelial cells. In the present study, all colonic adenocarcinoma cell
lines expressed substantial amounts of
3Gn-T5 transcript. This is
consistent with the results of Holmes et al. (51).
However, normal colon tissue also expressed substantial amounts of the
transcript. We will examine whether or not the
3Gn-T transcripts are
markedly up-regulated in the colorectal cancer tissues of patients.
3Gn-Ts examined in this study,
3Gn-T2 was
most active toward polylactosamine acceptors, and it effectively extended the polylactosamine chain even on the longer chain acceptors. Thus,
3Gn-T2 is the most probable candidate for the enzyme which functions to extend the polylactosamine chain.
3Gn-T5 is involved only in the synthesis of short polylactosamine chains or initiation of
polylactosamine synthesis.
3Gn-T3 and
3Gn-T4 exhibited very little activity, almost undetectable, toward all substrates examined. They apparently showed positive
3Gn-T activity in a previous study
(1), because excess amounts of enzyme were used for the assay. The
native acceptor substrates for
3Gn-T3 and -T4 may be different from
the Gal residue acceptor, and some unknown native acceptors may exist
for
3Gn-T3 and -T4.
3Gn-T (
3Gal-T) family is very large with 10 members, 1Gn-T,
3Gal-T1-5, and
3Gn-T2-5, having been cloned and analyzed to date.
The possibility therefore exists that some as yet unidentified
3Gn-T(s) exhibits Lc3Cer synthase activity.
3Gn-T5 is
the most feasible candidate for Lc3Cer synthase. In the future, we will assess the biological functions of this synthase, the
key enzyme determining the expression of biologically functional epitopes on GSLs.
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ACKNOWLEDGEMENT |
---|
We thank Kazuya Kabayama, Graduate School of Pharmaceutical Sciences, Hokkaido University, for help with immuno-TLC analysis. We also thank Dr. Satoshi Nakagawa and Sachiko Yokokawa, Kyowa Hakko Co. Ltd., for homology search and excellent technical assistance, respectively.
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FOOTNOTES |
---|
* 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 DDBJ/GenBankTM/EBI Data Bank
with the accession numbers AB045278 for human 3Gn-T5 and
AB045279 for rat
3Gn-T5.
§§ To whom all correspondence should be addressed: Laboratory of Gene Function Analysis, Institute of Molecular and Cell Biology (IMCB), National Institutes of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan. Tel.: 81-298-61-3200; Fax: 81-298-61-3201; E-mail: h.narimatsu@aist.go.jp.
Published, JBC Papers in Press, March 3, 2001, DOI 10.1074/jbc.M011369200
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ABBREVIATIONS |
---|
The abbreviations used are:
3Gn-T, UDP-GlcNAc:
-galactose
1,3-N-acetylglucosaminyltransferase;
3Gal-T, UDP-galactose:
-N-acetylglucosamine
1,3-galactosyltransferase;
UDP-GlcNAc, uridine
diphosphate-N-acetylglucosamine;
mAb, monoclonal antibody;
bp, base pair(s);
PCR, polymerase chain reaction;
ORF, open reading
frame;
HPTLC, high performance thin layer chromatography;
SGGL, sulfoglucuronylglycolipid;
GSL, glycosphingolipid;
Lea, Lewis a;
Leb, Lewis b;
Lex, Lewis x;
SSEA-1, stage-specific embryonal antigen-1;
LacCer, lactosylceramide
(Gal
1-4Glc
1-1Cer);
Lc3Cer, lactotriaosylceramide
(GlcNAc
1-3Gal
1-4Glc
1-1Cer);
nLc4Cer, neolactotetraosylceramide (paragloboside:
Gal
1-4GlcNAc
1-3Gal
1-4Glc
1-1Cer);
nLc5Cer, GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc
1-1Cer;
nLc6Cer, Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc
1-1Cer;
PA, pyridylaminated;
2AB, 2-aminobenzamide;
LNnT, lacto-N-neotetraose
(Gal
1-4GlcNAc
1-3Gal
1-4Glc);
LNT, lacto-N-tetraose
(Gal
1-3GlcNAc
1-3Gal
1-4Glc);
LNFP-II, lacto-N-fucopentaose-II
(Gal
1-3(Fuc
1-4)GlcNAc
1-3Gal
1-4Glc);
LNFP-III, lacto-N-fucopentaose-III
(Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4Glc);
LNFP-V, lacto-N-fucopentaose-V
(Gal
1-3GlcNAc
1-3Gal
1-4(Fuc
1-3)Glc);
LNDFH-II, lacto-N-difucohexaose-II
(Gal
1-3(Fuc
1-4)GlcNAc
1-3Gal
1-4(Fuc
1-3)Glc);
LN, N-acetyllactosamine (Gal
1-4GlcNAc);
2LN, Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc;
3LN, (Gal
1-4GlcNAc
1-3)2Gal
1-4GlcNAc;
4LN, (Gal
1-4GlcNAc
1-3)3Gal
1-4GlcNAc;
5LN, (Gal
1-4GlcNAc
1-3)4Gal
1-4GlcNAc;
RA, retinoic
acid;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
RT-PCR, reverse transcriptase-polymerase chain reaction;
GM3 synthase, lactosylceramide:
2,3-sialyltransferase.
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REFERENCES |
---|
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