From the Tokyo Research Laboratories, Kyowa Hakko
Kogyo Company, Limited, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, the ¶ Division of Cell Biology, Institute of Life Science, Soka
University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, the
Laboratory of Cancer Biology and Molecular Immunology, Graduate
School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113, and the ** Laboratory of Animal Resources, Faculty
of Bioindustry, Tokyo University of Agriculture, 196 Aza-Yasaka,
Abashiri-shi, Hokkaido 099-2422, Japan
Received for publication, June 2, 2000, and in revised form, September 28, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have isolated three types of cDNAs
encoding novel A family of human A PCR cloning approach using degenerate primers corresponding to
conserved regions in the Very interestingly, a During the course of study to isolate Nomenclature of Cell Lines--
Namalwa KJM-1, a subline of the human Burkitt
lymphoma cell line Namalwa, was cultivated in serum-free RPMI 1640 medium as described (9, 10). Cell lines SK-N-MC and Colo205 were
obtained from the American Type Culture Collection. These cell lines
were cultured in RPMI 1640 medium containing 10% fetal calf serum. Sf9 and Sf21 insect cells were cultured at
27 °C in TNM-FH insect medium (Pharmingen) as described previously
(11).
Preparation of cDNA Libraries and Single Strand
cDNAs--
cDNA libraries of human gastric mucosa and human
placenta were constructed as described previously (12). Single strand
cDNAs were synthesized from total RNA prepared from the
neuroblastoma cell line SK-N-MC.
Isolation of Human DNA Sequencing--
DNA sequences were determined by the
dideoxynucleotide chain termination method using an ABI
PRISMTM 377 DNA sequencer (Applied Biosystems, Inc.).
Construction of Plasmids for Expressing Expression of Flow Cytometric Analysis--
Transfected Namalwa KJM-1 cells
(5 × 106 cells) were incubated in 100 µl of
phosphate-buffered saline for 60 min at 37 °C in the presence or
absence of 20 milliunits of Clostridium perfringens neuraminidase (N2133, Sigma). These cells were stained with human anti-i-antigen serum (Den) (13), followed by fluorescein
isothiocyanate-conjugated goat anti-human IgM, and were analyzed on a
FACSCalibur apparatus (Becton Dickinson) as described (8). For lectin
staining, cells were stained with 10 µg/ml fluorescein
isothiocyanate-labeled Lycopersicon esculentum pokeweed
mitogen (LEA) or agglutinin (PWM; both from EY Laboratories).
Construction and Purification of
Sf9 insect cells were cotransfected with BaculoGold viral DNA
(Pharmingen) according to the manufacturer's instruction and each of
plasmids pVL1393-F2G2, pVL1393-F2G3, and pVL1393-F2G4 and were
incubated for 3 days at 27 °C to produce individual recombinant viruses. These viruses were amplified three times to reach titers of
~109 plaque-forming units/ml. Sf21 insect cells
(4 × 107 cells; Pharmingen) were infected at a
multiplicity of 10 and incubated in 30 ml of TNM-FH insect medium at
27 °C for 72 h to yield conditioned medium including
recombinant Silver Staining and Western Blot Analysis--
The enzymes
purified above (3 µl) were subjected to SDS-polyacrylamide gel
electrophoresis, followed by silver staining or Western blot analysis.
Silver staining was performed using a silver staining kit (Wako
Bioproducts). Proteins separated on 6% SDS-polyacrylamide gel were
transferred to an polyvinylidene difluoride membrane (Immobilon,
Millipore Corp.) in a Trans-Blot SD cell (Bio-Rad). The membrane
was blocked with phosphate-buffered saline containing 5% skim milk at
4 °C overnight and then incubated with 10 µg/ml M2 antibody
(Sigma). The membrane was stained with ECL Western blot detection
reagents (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Glycosyltransferase Assays and Product Characterization--
The
N-acetylglucosaminyltransferase activities of the purified
proteins (15 µl) were assayed in 50 mM MOPS (pH 7.5), 5 mM MgCl2, 5 mM UDP-GlcNAc, and 10 mM unlabeled acceptors (a total volume of 40 µl). The
following oligosaccharides were used as acceptors: lactose
(Gal
To further confirm the structure of the reaction product derived from
lactose, it was digested with endo-
Endo-
Since the amount of the purified proteins was not enough for accurate
quantification, enzymatic activity is defined as picomoles of acceptor
substrate N-acetylglucosaminylated per ml of culture medium/h. The amounts of reaction products were determined from their
absorbance intensities using individual standards.
Alternatively, the N-acetylglucosaminyltransferase
activities of the purified proteins (15 µl) were assayed in 200 mM MOPS (pH 7.5), 20 mM MgCl2, 20 mM UDP-GlcNAc, and 50 µM pyridylaminated acceptors (total volume of 30 µl). As acceptors, the following pyridylaminated oligosaccharides were used: LNnT,
lacto-N-fucosylpentaose (LNFP) III
(Gal
The reaction product derived from pyridylaminated LNnT was identified
by comparison of the retention time on HPLC with that of the
pyridylaminated standard oligosaccharide
GlcNAc Preparation and Fractionation of Blood Leukocytes--
Human
polymorphonuclear leukocytes, monocyte-enriched population, and
lymphocyte-enriched population were obtained as described previously (10).
Quantitative Analysis of the Three
Single strand cDNAs were synthesized with an oligo(dT) primer from
6 µg of DNase I-treated total RNA from human tissues (colon, jejunum,
stomach body, stomach antrum, and esophagus) and cell lines (HL-60 and
Colo205) as described previously (4). Single strand cDNAs from
human leukocytes were prepared as described (10). In addition, single
strand cDNAs were synthesized with an oligo(dT) primer from 1 µg
of poly(A)+ RNAs from 35 human tissues
(CLONTECH) using a SuperscriptTM
pre-amplification system for first strand cDNA synthesis (Life Technologies, Inc.) according to manufacturer's instructions. After
cDNA synthesis, the reaction mixture was diluted 50-fold with
H2O and then stored at
Competitive RT-PCR was performed with AmpliTaq GoldTM
(PerkinElmer Life Sciences). The annealing temperatures and specific
primers used are listed in Table II. The amount of each of the Determination of Chromosomal Localization--
The chromosomal
localizations of the Identification and Isolation of
A Kyte-Doolittle hydropathy analysis (17) revealed that
Fig. 1A shows a multiple alignment of the amino acid
sequences of Production of Secreted Recombinant Proteins Fused to the FLAG
Peptide--
To examine the enzymatic activities of
On the other hand, Substrate Specificity of
Since Den, LEA, and PWM are likely to recognize non-sialylated
poly-N-acetyllactosamines more preferentially than
sialylated ones, the transfected cells were treated with neuraminidase
before staining. As shown in Fig. 4,
expression of Expression Levels of the
In this study, we identified three novel
We constructed the secreted recombinant proteins for All of the recombinant proteins showed Gal
To date, Analysis of substrate specificity revealed that The phylogenetic analysis using the amino acid sequences of the
putative catalytic domains indicated that the members of the In this study, we isolated three types of novel Recently, Amado et al. (76) have reported the
existence of four additional members of the 1,3-N-acetylglucosaminyltransferases
(designated
3Gn-T2, -T3, and -T4) from human gastric mucosa and the
neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type
2 transmembrane proteins of 397, 372, and 378 amino acids,
respectively. They share motifs conserved among members of the
1,3-galactosyltransferase family and a
1,3-N-acetylglucosaminyltransferase (designated
3Gn-T1), but show no structural similarity to another type of
1,3-N-acetylglucosaminyltransferase (iGnT). Each
of the enzymes expressed by insect cells as a secreted protein fused to
the FLAG peptide showed
1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not
1,3-galactosyltransferase activity. These
enzymes exhibited different substrate specificity. Transfection of
Namalwa KJM-1 cells with
3Gn-T2, -T3, or -T4 cDNA led to an
increase in poly-N-acetyllactosamines recognized by an
anti-i-antigen antibody or specific lectins. The expression profiles of
these
3Gn-Ts were different among 35 human tissues.
3Gn-T2 was
ubiquitously expressed, whereas expression of
3Gn-T3 and -T4 was
relatively restricted.
3Gn-T3 was expressed in colon, jejunum,
stomach, esophagus, placenta, and trachea.
3Gn-T4 was mainly
expressed in brain. These results have revealed that several
1,3-N-acetylglucosaminyltransferases form a family with
structural similarity to the
1,3-galactosyltransferase family.
Considering the differences in substrate specificity and distribution,
each
1,3-N-acetylglucosaminyltransferase may play
different roles.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3-galactosyltransferases
(
3Gal-Ts)1 consisting of
five members (
3Gal-T1, -T2, -T3, -T4, and -T5) was recently
identified (1-4). The first
1,3-galactosyltransferase (
3Gal-T1),
which catalyzes the formation of type 1 oligosaccharides, was
isolated by us using an expression cloning approach (1). Expression
patterns of
3Gal-T1 and type 1 oligosaccharides strongly suggested
the existence of
3Gal-T1 homologs. For instance, type 1-derived
oligosaccharides such as sialyl-Lea were known to be
expressed in colon and pancreatic cancer cell lines, whereas expression
of
3Gal-T1 was detected in brain, but not in cancer cells. Our early
approach using Southern hybridization failed to detect the existence of
3Gal-T1 homologous genes. However, recent accumulation of nucleotide
sequence information on human cDNAs and genes such as
expressed sequence tags (ESTs) enabled us to search homologous
genes that do not have high similarity as detected by hybridization,
but show significant similarity. A homology search based on the
nucleotide or amino acid sequence of
3Gal-T1 led to the isolation of
3Gal-T2, -T3, and -T4, indicating that
3Gal-Ts form a family
(1-3).
3Gal-T2 catalyzed a similar reaction, but showed different substrate
specificity compared with
3Gal-T1. The activity of
3Gal-T3 has
not been detected, whereas the corresponding mouse enzyme exhibits weak
3Gal-T activity for both GlcNAc and GalNAc (5). On the other hand,
3Gal-T4 transfers galactose to the GalNAc residue of
asialo-GM2 or GM2 to catalyze the formation of
asialo-GM1 or GM1, respectively (3).
3Gal-T4
may be a human homolog of rat GM1 and GD1
synthases (6) since these enzymes shows 79.4% identity at the amino
acid level.
3Gal-T family has enabled us to isolate a
fifth member (
3Gal-T5) of this family, which catalyzes the synthesis
of type 1 oligosaccharides and is the most probable candidate involved
in the biosynthesis of a cancer-associated sugar antigen,
sialyl-Lea, in gastrointestinal and pancreatic cancer cells
(4).
1,3-N-acetylglucosaminyltransferase
(designated
3Gn-T1) has been recently isolated based on the
structural similarity to the
3Gal-Ts (7).
3Gn-T1 shows
significant overall similarity to
3Gal-Ts (15-19%) and shares
motifs conserved among the
3Gal-Ts, but is structurally distinct
from another type of
1,3-N-acetylglucosaminyltransferase
(iGnT) that was isolated by expression cloning using an
anti-i-antigen antibody (8).
3Gn-T1 exhibits
1,3-N-acetylglucosaminyltransferase activity instead of
1,3-galactosyltransferase activity. This result provides an
exception that a glycosyltransferase structurally related to the
3Gal-T family uses distinct donor (GlcNAc versus Gal) and acceptor (Gal versus GlcNAc) substrates, maintaining the
same linkage specificity (
1,3-linkage).
3Gal-T1 homologs, we have
identified three additional types of putative members of the
3Gal-T
family. In this study, we show additional examples that
glycosyltransferases structurally related to the
3Gal-T family
exhibit
1,3-N-acetylglucosaminyltransferase activity, but
not
1,3-galactosyltransferase activity. These results indicate that
1,3-N-acetylglucosaminyltransferases (
3Gn-Ts) form a
family having structural similarity to the
3Gal-T family. Alignment of primary sequences of all members of the
3Gn-T and
3Gal-T families revealed that the members are clustered into four subgroups, probably reflecting enzymatic activity and substrate specificity. Transfection experiments and in vitro enzymatic analysis
have demonstrated that
3Gn-T2, -T3, and -T4 are able to catalyze the initiation and elongation of poly-N-acetyllactosamine sugar
chains; however, they exhibit different substrate specificity. These
results, taken together with the different distributions of these
enzymes, indicate that
3Gn-T2, -T3, and -T4 each exert distinct
roles in physiological and pathological processes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3-Galactosyltransferases and
1,3-N-Acetylglucosaminyltransferases--
To simplify discussion,
five members of the cloned human
3Gal-Ts will be called
3Gal-T1,
-T2, -T3, -T4, and -T5 according to the designation of Kolbinger
et al. (2), Clausen and co-workers (3), and Narimatsu and
co-workers (4). Five types of
3Gn-Ts cloned to date will be referred
to tentatively as follows. A
3Gn-T and newly isolated
3Gn-Ts in
this study, which show structural similarity to the
3Gal-T family,
will be called
3Gn-T1, -T2, -T3, and -T4. Another type of
3Gn-T,
which was isolated by expression cloning using anti-i-antigen antibody
(8) and showed no structural similarity to the
3Gal-T family, will
be called iGnT according to Fukuda et al. (8).
Gn-T2, -T3, and -T4 cDNAs--
EST
fragments encoding amino acid sequences similar to the human
3Gal-T1
sequence were retrieved from the EST division of the
GenBankTM/EBI Data Bank using the FrameSearch algorithm
(Compugen) (Table I). The human
3Gn-T2
cDNA fragment (600 bp) was isolated from a human gastric mucosa
cDNA library by PCR using primers 5'-CCGGACAGATTTAAAGACTTTCTGC-3' and 5'-GTAGAGGCCAGAGTAAACAACTTCT-3'. The human
3Gn-T3 cDNA
fragment (200 bp) was isolated from a human placenta cDNA library
by PCR using primers 5'-CGTGGGGCAACTGATCCAAAACG-3' and
5'-ACCCAGGAAGACATCATCAATGGG-3'. The PCR conditions were 99 °C
for 10 min; followed by 30 cycles of 95 °C for 1 min, 65 °C for 1 min, and 72 °C for 2 min; and 72 °C for 10 min of incubation. The
3Gn-T2 and -T3 cDNA fragments were cloned into pT7Blue (Novagen)
and sequenced. Digoxigenin-labeled probes were prepared from the
above-mentioned 600- and 200-bp fragments using a PCR DIG probe
synthesis kit (Roche Molecular Biochemicals) and used to isolate the
full-length cDNAs for
3Gn-T2 and -T3 by plaque hybridization.
The full-length
3Gn-T2 cDNA (1.9 kb) was prepared from phage DNA
by SalI and XbaI digestion and subcloned into
pBluescript II SK(+) to yield pBS-
3Gn T2. A plasmid (pBS-
3Gn T3)
containing the full-length
3Gn T3 cDNA in pBluescript SK(
) was
recovered by in vivo excision. The full-length
3Gn T4
cDNA (1.3 kb) was amplified from single strand cDNAs derived from the neuroblastoma cell line SK-N-MC by PCR using primers 5'-CACAGCCTGAGACTCATCTCGCT-3' and 5'-AGGCATCAATTTCGCATCACGATAG-3' and
was inserted into the pT7Blue-T vector to make pT7B-
3Gn T4.
List of the ESTs used to isolate 3Gn-T2, -T3, and -T4 cDNAs
3Gn T2, -T3, and -T4
in Animal Cells--
A
3Gn T2 cDNA fragment (prepared from
pBS-
3Gn T2 by SalI and XbaI digestion,
followed by blunting and addition of the SfiI linker
(5'-CTTTAGAGCA-3' and 5'-CTCTAAAG-3')) was inserted between the
SfiI sites of pAMo to yield pAMo-
3Gn T2. A
3Gn T3
cDNA fragment prepared from pBS-
3Gn T3 by HindIII and
NotI digestion was inserted between the HindIII
and NotI sites of pAMo to yield pAMo-
3Gn T3. A
3Gn T4
cDNA fragment (prepared from pT7B-
3Gn T4 by SmaI and
HincII digestion, followed by addition of the
SfiI linker) was inserted between the SfiI sites
of pAMo to yield pAMo-
3Gn T4.
3Gn T2, -T3, and -T4 in Namalwa KJM-1
Cells--
Namalwa KJM-1 cells were transfected with pAMo-
3Gn T2,
pAMo-
3Gn T3, or pAMo-
3Gn T4 by electroporation as described (9, 10) and grown for 24 h. Stably transfected cells were selected by
cultivation for >14 days in the presence of G418 (0.5 mg/ml).
3Gn-T Proteins Fused to the
FLAG Peptide--
The putative catalytic domain of each
3Gn-T2,
-T3, and -T4 was expressed as a secreted protein fused to the FLAG
peptide in insect cells. A 1.1-kb DNA fragment encoding a COOH-terminal portion of
3Gn-T2 (amino acids 31-397) was amplified by PCR using primers 5'-CACGGATCCAGCCAAGAAAAAAATGGAAAAGGGGA-3' and
5'-ATCCGATAGCGGCCGCTTAGCATTTTAAATGAGCACTCTGCAAC-3', digested with
BamHI and NotI, and inserted between the
BamHI and NotI sites of pVL1393-F2 to yield
pVL1393-F2G2. pVL1393-F2 is an expression vector derived from pVL1393
(Pharmingen) and contains a fragment encoding the signal peptide of
human immunoglobulin
(MHFQVQIFSFLLISASVIMSRG) and the FLAG peptide
(DYKDDDDK). Joining in-frame a cDNA fragment with a unique
BamHI site of pVL1393-F2 just downstream of the COOH
terminus of the FLAG peptide enables the cDNA product to be
secreted as a protein fused to the FLAG peptide. A 1.0-kb DNA fragment
encoding a COOH-terminal portion of
3Gn-T3 (amino acids 38-372) was
amplified by PCR using primers 5'-CGCGGATCCTCCCCACGGTCCGTGGACCAG-3' and
5'-ATAGTTTAGCGGCCGCGGAAGGGCTCAGCAGCGTCG-3', digested with
BamHI and NotI, and inserted between the
BamHI and NotI sites of pVL1393-F2 to yield
pVL1393-F2G3. A 0.9-kb DNA fragment encoding a COOH-terminal portion of
3Gn-T4 (amino acids 56-378) was amplified by PCR using primers
5'-ATAAGATCTGCAGGAGACCCCACGGCCCACC-3' and
5'-ATAGTTATGCGGCCGCCTCAGGCTGTTGCCCAACCCAC-3', digested with BglII and NotI, inserted between the
BamHI and NotI sites of pVL1393-F2 to yield
pVL1393-F2G4. The PCR-amplified portions of pVL1393-F2G2, pVL1393-F2G3,
and pVL1393-F2G4 were sequenced to confirm the absence of possible PCR errors.
3Gn-T proteins fused to the FLAG peptide, which were
readily purified by anti-FLAG M1 antibody resin (Sigma) according to
the protocol of the manufacturer. Briefly, the culture medium (30 ml)
was collected by centrifugation and added to NaCl (150 mM
final concentration), NaN3 (0.1% final concentration), and M1 antibody resin (30 µl) to adsorb the
recombinant
3Gn-T proteins on the resin. The resin was recovered by
centrifugation and washed three times with buffer (1 ml) consisting of
50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM CaCl2. The recombinant
3Gn-T proteins
were eluted with buffer (90 µl) consisting of 50 mM
Tris-HCl (pH 7.4), 150 mM NaCl, and 2 mM EDTA,
followed by the addition of CaCl2 (4 mM final
concentration), and stored at 4 °C until use. The amount of the
purified proteins was not enough for accurate quantification.
1-4Glc), N-acetyllactosamine (Gal
1-4GlcNAc), lacto-N-neotetraose (LNnT;
Gal
1-4GlcNAc
1-3Gal
1-4Glc),
p-lacto-N-neohexaose (p-LNnH;
Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc), and
lacto-N-tetraose (LNT;
Gal
1-3GlcNAc
1-3Gal
1-4Glc). LNnT and LNT were purchased from
Oxford Glycosystems. Lactose and p-LNnH were purchased from Sigma. Parallel reactions were done in the absence of UDP-GlcNAc to
identify products. After incubation at 37 °C for the appropriate times (2 h for
3Gn-T2 and 16 h for
3Gn-T3 and -T4), the
reactions were terminated by boiling. After centrifugation, the
reaction mixtures were analyzed by high-pH anion-exchange
chromatography with pulsed amperometric detection (HPAE/PAD, Dionex
Corp.). CarboPac PA10 was used as a column, and elution was performed
with a gradient of 40-125 mM NaOH in 30 min at a flow rate
of 1 ml/min. The structures of the reaction products derived from
lactose and LNnT were confirmed by comparison of their retention times
on HPLC with those of the standard oligosaccharides
GlcNAc
1-3Gal
1-4Glc and
GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc, which were prepared
from LNnT and p-LNnH by digestion with jack bean
-galactosidase.
-galactosidase or modified by
1,4-galactosyltransferase. To avoid the inhibitory effect of MOPS on
endo-
-galactosidase, an N-acetylglucosaminyltransferase assay was done without MOPS using lactose as an acceptor. The reaction
was stopped by boiling, and the reaction mixture was recovered by
centrifugation. The reaction mixture (15 µl) was added to acetate
buffer (50 mM final concentration; pH 5.8) and incubated
with 250 milliunits/ml Escherichia freundii
endo-
-galactosidase (Seikagaku Kogyo) (14) in a total volume of 41 µl at 37 °C for 16 h, followed by analysis with HPAE/PAD as
described above. Alternatively, the reaction mixture (15 µl) was
added to Tris-HCl (50 mM final concentration; pH 8.0) and
incubated with 750 milliunits/ml bovine milk
1,4-galactosyltransferase (Sigma) in a total volume of 40 µl at
37 °C for 16 h, followed by analysis with HPAE/PAD as described above.
-galactosidase digestion of the product yielded two peaks that
comigrated with the standard oligosaccharides GlcNAc
1-3Gal
and
glucose at a 1:1 molar ratio. These results clearly indicated that the
product derived from lactose was GlcNAc
1-3Gal
1-4Glc.
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4Glc), LNT, LNFP-II (Gal
1-3(Fuc
1-4)GlcNAc
1-3Gal
1-4Glc), LNFP-V
(Gal
1-3GlcNAc
1-3Gal
1-4(Fuc
1-3)Glc), and
lacto-N-difucosylhexaose II
(Gal
1-3(Fuc
1-4)GlcNAc
1-3Gal
1-4(Fuc
1-3)Glc). After incubation at 37 °C for the appropriate times (2 h for
3Gn-T2 and 15 h for
3Gn-T3 and -T4), the reactions were
terminated by boiling and analyzed by HPLC as described previously,
with exception that HPLC was performed at 50 °C with a flow rate of
0.5 ml/min (9, 10). Parallel reactions were done in the absence of
UDP-GlcNAc to identify products and to check hydrolysis of substrate
and product. The oligosaccharides were purchased from Oxford
Glycosystems and pyridylaminated according to the method of Kondo
et al. (15). The amounts of products were determined
from their fluorescence intensities using pyridylaminated lactose as a standard.
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc, which was prepared
from pyridylaminated p-LNnH by digestion with jack bean
-galactosidase. To further confirm the structure of the reaction
product, the reaction product was modified by
1,4-galactosyltransferase to examine whether p-LNnH was
produced or not. The reaction mixtures (20 µl) was incubated with 20 milliunits of bovine milk
1,4-galactosyltransferase in the presence
or absence of UDP-Gal (20 mM) in a total volume of 30 µl
at 37 °C for 15 h according to manufacturer's recommendations. The product further modified by
1,4-galactosyltransferase comigrated with pyridylaminated p-LNnH on HPLC. These results indicated
that the product was pyridylaminated
GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc. The
galactosyltransferase activities of the purified proteins (15 µl)
were assayed using pyridylaminated oligosaccharides
(GlcNAc
1-3Gal
1-4Glc and LNnT) as substrates as described
previously (4).
3Gn-T Transcripts in Human
Tumor Cell Lines and Human Tissues by Competitive RT-PCR--
The
levels of the
3Gn-T2, -T3, and -T4 transcripts were measured by
competitive RT-PCR as described in detail previously (4, 16).
Competitor DNA plasmids carrying a small deletion within the respective
cDNA were constructed by appropriate restriction endonuclease
digestion as shown in Table II. For
instance, a competitor DNA plasmid for measuring
3Gn-T2 transcripts
was prepared by deleting the 227-bp Eco81I-PflMI
fragment in
3Gn-T2 cDNA from the standard DNA plasmid pBS-
3Gn
T2.
Oligonucleotide primers and conditions used for competitive RT-PCR
analysis
80 °C until use.
3Gn-T transcripts was normalized by the amount of
-actin transcripts (4,
16).
3Gn-T2, -T3, and -T4 genes were determined
using 3'-EST mapping data (NCBI Protein Database). The chromosomal
localization of the
3Gal-T1 gene was determined by PCR analysis
using a series of genomic DNAs from hamster-human somatic hybrids
(BIOSMAPTM Somatic Cell Hybrid PCRableTM DNAs,
BIOS Laboratories) and specific primers (5'-TTCAGCCACCTAACAGTTGCCAGG-3' and 5'-ATACCTTCTTCGTGGCTTGGTGGAG-3'). The predicted fragment of 495 bp
was amplified only when genomic DNA from a hybrid containing human
chromosome 2 (hybrid 852) was used, indicating that this gene is
located on chromosome 2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3Gn-T2, -T3, and -T4--
A
homology search in the EST division of the GenBankTM/EBI
Data Bank using the FrameSearch algorithm revealed the existence of six
types of cDNAs encoding proteins with low but significant similarity to
3Gal-T1, three of which have been reported recently to
be
3Gal-T2, -T3, and -T4 (2, 3). Based on the nucleotide sequence of
the ESTs shown in Table I, we prepared specific probes and isolated
three types of full-length cDNAs encoding novel proteins (designated
3Gn-T2, -T3, and -T4) of 397, 372, and 378 amino acid
residues, respectively, with structural similarity to
3Gal-T1.
3Gn-T2 and -T3 cDNAs were obtained from human gastric mucosa, and
3Gn-T4 cDNA was from the neuroblastoma cell line
SK-N-MC.
3Gn-T2, -T3,
and -T4 show type 2 transmembrane topology typical of most
glycosyltransferases.
3Gn-T2 is predicted to consist of an
N-terminal cytoplasmic domain of 9 residues, a transmembrane segment of
19 residues, and a stem region and catalytic domain of 369 residues.
3Gn-T3 is predicted to consist of an N-terminal cytoplasmic domain
of 11 residues, a transmembrane segment of 21 residues, and a stem
region and catalytic domain of 340 residues. The predicted coding
region of
3Gn-T4 has two potential initiation codons, both of which
are in agreement with Kozak's rule (18). Therefore, it is
predicted that
3Gn-T4 is composed of two different N-terminal
cytoplasmic domains of 29 and 4 residues, a transmembrane segment of 20 residues, and 329 residues containing the stem region and catalytic
domain (Fig. 1a).
View larger version (64K):
[in a new window]
Fig. 1.
Comparison of novel
3Gn-Ts with
3Gn-T1 and
the
3Gal-T family. A, multiple
sequence analysis (ClustalX) of the
3Gn-T family (
3Gn-T1, -T2,
-T3, and -T4) and the
3Gal-T family (
3Gal-T1, -T2, -T3, -T4, and
-T5). Introduced gaps are shown by hyphens. The putative
transmembrane domains are underlined. Asterisks indicate
identical amino acids in all proteins. Conserved amino acids are shown
by colons. Cysteine residues conserved in all the proteins
or subgroups are shaded. The white arrows
indicate cysteine residues conserved in all the proteins except for
3Gn-T1. The black arrow indicates the conserved possible
N-glycosylation site. B, phylogenetic tree of the
3Gn-T and
3Gal-T families. The phylogenetic tree was produced
with ClustalX presented in A using amino acid sequences of
the predicted catalytic domains. The chromosomal localizations of the
respective genes, except for the
3Gn-T1 gene, are indicated in
parentheses (Refs. 4 and 49 and this study).
3Gn-T2, -T3, and -T4 as well as
3Gn-T1 and five
members of the
3Gal-T family.
3Gn-T2, -T3, and -T4 show 19-24,
22-26, and 22-25% identities, respectively, to the
3Gal-T family (
3Gal-T1, -T2, -T3, -T5, and -T5), whereas they show 15, 18, and
15% identities to
3Gn-T1.
3Gn-T2, -T3, and -T4 show 40-45% identity one another. The sequence similarities are limited to the
putative catalytic regions. Several sequence motifs conserved in the
3Gal-T family are also shared by
3Gn-T2, -T3, and -T4 as well as
3Gn-T1. Twenty-five amino acid residues located separately in the
putative catalytic regions are identical among all the proteins. Three
cysteine residues conserved in all members of the
3Gal-T family are
also maintained in
3Gn-T2, -T3, and -T4, whereas two of these are
not conserved in
3Gn-T1 (Fig. 1A, white
arrows), indicating that
3Gn-T1 is relatively distinct from
other members, especially in the context of the three-dimensional structure. There are five potential N-linked glycosylation
sites in
3Gn-T2, three in
3Gn-T3, and three in
3Gal-T4. One
site in a highly conserved motif is maintained among all the proteins (Fig. 1A, black arrow). The phylogenetic tree of
these proteins generated using the amino acid sequences of the putative
catalytic domains demonstrates that
3Gn-T2, -T3, and -T4 form a
subgroup, indicating that they have similar enzymatic activity (Fig.
1B).
3Gn-T2, -T3,
and -T4, we expressed the putative catalytic domain of each enzyme
(amino acids 31-397 of
3Gn-T2, amino acids 38-372 of
3Gn-T3,
and amino acids 56-378 of
3Gn-T4) as a secreted protein fused to
the FLAG peptide in Sf21 insect cells. The FLAG-fused
recombinant proteins were partially purified using anti-FLAG M1
antibody resin and analyzed by SDS-polyacrylamide gel electrophoresis,
followed by silver staining (Fig.
2A) or Western blotting using
anti-FLAG monoclonal antibody (Fig. 2B). Two major bands
with apparent molecular masses of 45.5 and 48 kDa, broad bands of
42-45 kDa, and two major bands of 37.6 and 40 kDa were observed
specifically for
3Gn-T2, -T3, and -T4, respectively. The FLAG-fused
recombinant proteins for
3Gn-T2, -T3, and -T4 have predicted
molecular masses of 43,674, 39,507, and 37,608 Da for the respective
polypeptides, indicating glycosylation of the recombinant proteins
produced by insect cells.
View larger version (74K):
[in a new window]
Fig. 2.
SDS-polyacrylamide gel electrophoresis
analysis of the secreted recombinant enzymes. The FLAG-fused
secreted proteins for 3Gal-T2 (lanes 1), -T3 (lanes
2), and -T3 (lanes 3) were purified by anti-FLAG
antibody resin and subjected to SDS-polyacrylamide gel electrophoresis,
followed by silver staining (A) or Western blotting using
anti-FLAG antibody M2 (B).
3Gn-T2, -T3, and -T4 Are
1,3-N-Acetylglucosaminyltransferases--
The glycosyltransferase
activities of the partially purified FLAG-fused recombinant proteins
were examined. When lactose was used as an acceptor,
3Gn-T2, -T3,
and -T4 showed a significant amount of
N-acetylglucosaminyltransferase activity, whereas no activity was detected in a sample prepared from the conditioned medium
of insect cells infected with empty vector virus. The structure of the
product was estimated to be GlcNAc
1-3Gal
1-4Glc by comparing the
retention time on HPLC with that of the standard oligosaccharide (Fig.
3A). To further confirm the
structure of the product, it was digested with endo-
-galactosidase
or modified by
1,4-galactosyltransferase. Digestion of the product
by E. freundii endo-
-galactosidase yielded two peaks
comigrating with the standard oligosaccharides GlcNAc
1-3Gal and
glucose at a 1:1 molar ratio (Fig. 3, compare A and
B). Modification of the product by bovine milk
1,4-galactosyltransferase yielded a peak comigrating with LNnT (Fig.
3, compare A and C). These results clearly
indicated that the product was GlcNAc
1-3Gal
1-4Glc.
View larger version (19K):
[in a new window]
Fig. 3.
HPLC analysis of the reaction product
generated from lactose by recombinant
3Gn-T2. A, the
N-acetylglucosaminyltransferase activity of the purified
3Gn-T2 protein was assayed using lactose (Gal
1-4Glc) as an
acceptor. The reaction mixture was analyzed using high-pH
anion-exchange chromatography with pulsed amperometric detection. The
peaks for substrate lactose and the generated product are labeled
S and P, respectively. Arrow 1 indicates the elution position of the standard oligosaccharide
GlcNAc
1-3Gal
1-4Glc. Based on the elution position, the peak
indicated by the asterisk seems to be GlcNAc, which may be a
degradation product of UDP-GlcNAc. The peak with a retention time of
2-3 min may be glycerol, which appeared in the absence of UDP-GlcNAc.
B, the reaction mixture described in A was
analyzed after digestion with endo-
-galactosidase. Arrows
2 and 3 indicate the elution positions of the standard
oligosaccharides GlcNAc
1-3Gal and glucose, respectively.
C, the reaction mixture described in A was
analyzed after galactosylation with
1,4-galactosyltransferase.
Arrow 4 indicates the elution position of the standard
oligosaccharide LNnT (Gal
1-4GlcNAc
1-3Gal
1-4Glc). Based on
the elution position, the peak indicated by the double
asterisks seems to be Gal
1-4GlcNAc, which would be a
galactosylation product of GlcNAc indicated by the asterisk
in A.
3Gn-T2, -T3, and -T4 showed no
1,3-galactosyltransferase activity for GlcNAc
1-3Gal
1-4Glc or
LNnT. Taken together,
3Gn-T2, -T3, and -T4 were demonstrated to be novel
1,3-N-acetylglucosaminyltransferases.
3Gn-T2, -T3, and -T4--
Analysis of
the substrate specificity of
3Gn-T2, -T3, and -T4 revealed that
these enzymes utilized common oligosaccharides as substrates, but
the substrate preference was
significantly different (Tables III and
IV).
3Gn-T2 and -T4 showed more
preferential activity for LNnT than for LNT, which is consistent with
the nature of
3Gn-T1 and iGnT (7, 8). In contrast,
3Gn-T3
utilized LNT as a substrate comparable to LNnT. In common, fucosylation at the penultimate GlcNAc residue in LNnT or LNT yielded poor substrates (LNFP-III, LNFP-II, and lacto-N-difucosylhexaose
II). LNFP-V, which is an LNT derivative fucosylated at the reducing terminal glucose residue, was a relatively good substrate for
3Gn-T2
compared with LNT.
Analysis of substrate specificity of 3Gn-T2, -T3, and -T4 using
pyridylaminated oligosaccharides as substrates
3Gn-T2, -T3, and -T4 for
pyridylaminated LNnT were 330, 7.1, and 1.9 pmol/ml of medium/h,
respectively. LNDFH-II, lacto-N-difucosylhexaose II.
Analysis of substrate specificity of 3Gn-T2, -T3, and -T4 using
unlabeled oligosaccharides as substrates
3Gn-T2, -T3, and -T4 for LNnT were 4900, 230, and 420 pmol/ml of medium/h, respectively. The activity of
3Gn-T1 was quoted
from Ref. 7. LacNAc, N-acetyllactosamine; ND, not
determined.
3Gn-T2 transferred GlcNAc efficiently to both lactose and
p-LNnH, as well as LNnT, whereas the relative activity
N-acetyllactosamine was 21% compared with that of LNnT.
3Gn-T3 preferred lactose (235% relative activity) as a substrate,
followed by LNnT (100%) and p-LNnH (45%), whereas it
showed no activity for N-acetyllactosamine.
3Gn-T4 showed
33, 9, and 0% relative activities for lactose, N-acetyllactosamine, and p-LNnH, respectively,
compared with LNnT (100%).
3Gn-T1 was reported to efficiently
utilize N-acetyllactosamine as well as lactose (Table III),
showing a significant difference in substrate specificity compared with
3Gn-T2, -T3, and -T4.
3Gn-T2, -T3, and -T4 Each Direct Biosynthesis of
Poly-N-acetyllactosamines in Namalwa KJM-1 Cells--
To examine the
in vivo enzymatic activities of
3Gn-T2, -T3, and -T4,
Namalwa KJM-1 cells were transfected with pAMo-
3Gn T2, pAMo-
3Gn
T3, pAMo-
3Gn T4, or the empty vector pAMo and examined for changes
in the surface expression of the poly-N-acetyllactosamine sugar chains by flow cytometric analyses using the anti-i-antigen antibody (Den) and specific lectins (LEA and PWM).
3Gn-T2, -T3, or -T4 increased the levels of
poly-N-acetyllactosamines recognized by Den, LEA, or
PWM compared with the vector (pAMo) transfectant, consistent with
in vitro enzymatic activity. In particular, expression of
3Gn-T3 or -T4 led to a remarkable increase in reactivity to Den, in
contrast to the slight increase in the
3Gn-T2 transfectant. On the
other hand, reactivity to LEA or PWM was increased in the
3Gn-T2 transfectant more clearly than in the other two
transfectants. These results indicate that
3Gn-T2, -T3, and -T4 each
are involved in the biosynthesis of poly-N-acetyllactosamine
sugar chains in transfected cells.
View larger version (26K):
[in a new window]
Fig. 4.
Flow cytometric analysis of Namalwa KJM-1
cells stably transfected with 3Gn-T2, -T3, or
-T4 cDNA. Namalwa KJM-1 cells were stably transfected with
plasmid pAMo-
3Gn T2, pAMo-
3Gn T3, or pAMo-
3Gn T4, which
directs expression of
3Gn-T2, -T3, or -T4, respectively, or with the
empty vector pAMo. These cells were stained with anti-i-antigen
antiserum or poly-N-acetyllactosamine-recognizing lectins
(LEA or PWM) and subjected to flow cytometric analysis as described
under "Experimental Procedures" (thick lines). As
controls, the transfectants were stained with phosphate-buffered saline
(thin lines).
3Gn-T2, -T3, and -T4
Transcripts--
The expression levels of the
3Gn-T2, -T3, and -T4
transcripts were examined by competitive RT-PCR. These genes were
differentially expressed in human tissues and cells (Table
V).
3Gn-T2 was ubiquitously expressed
in the tissues and cells tested, but expression of
3Gn-T3 and -T4
was relatively restricted.
3Gn-T3 was expressed in colon, jejunum,
stomach (body and antrum), esophagus, placenta, and trachea.
3Gn-T4
was mainly expressed in brain tissues such as whole brain, hippocampus,
amygdala, cerebellum, and caudate nucleus, as well as in colon,
esophagus, and kidney.
Quantitative analysis of transcripts of 3Gn-T2, -T3, and -T4 in
various human tissues and cells by competitive RT-PCR
1,3-N-Acetylglucosaminyltransferase activities were
detected in several tissues, cells, and sera, some of which were
characterized using partially purified enzymes (19-28).
Poly-N-acetyllactosamines are known to serve as backbone
oligosaccharides for presenting the sialyl-LeX and
sialyl-Lea determinants, which function as selectin ligands
in leukocytes and several cancer cells such as colon cancer cells
(29-40). The human promyelocytic leukemia cell line HL-60 and the
human colon adenocarcinoma cell line Colo205 are known to express
1,3-N-acetylglucosaminyltransferase activities as well as
poly-N-acetyllactosamines presenting the sialyl-Lea and sialyl-LeX determinants.
Therefore, it was of significant interest to examine the expression
levels of
3Gn-T2, -T3, and -T4 in leukocytes and cancer cells such
as HL-60 and Colo205.
3Gn-T2, but not
3Gn-T3 and -T4, was
significantly expressed in HL-60 and Namalwa KJM-1 cells (Table V) as
well as in human peripheral polymorphonuclear cells and lymphocytes
(data not shown). On the other hand,
3Gn-T2 and -T3, but not
3Gn-T4, were highly expressed in the colon cancer cell line Colo205
(Table V).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3-N-acetylglucosaminyltransferases (
3Gn-T2, -T3, and
-T4) that show structural similarity to
3Gn-T1 as well as the
3Gal-T family, including five members (
3Gal-T1, -T2, -T3, -T4,
and T5), demonstrating the existence of a
3Gn-T family now
consisting of four members (
3Gn-T1, -T2, -T3, and -T4). The
existence of the multiple enzymes showing similar activity is a common
feature of glycosyltransferases, which was demonstrated for Gal
2,3-sialyltransferases I-VI (9, 41-47, 49-52), GalNAc
2,6-sialyltransferases I-VI (53-58), NeuAc
2,8-sialyltransferases I-V (59-69),
1,3-fucosyltransferases
III-VII and IX (10, 70-75, 77, 78)), core 2
1,6-N-acetylglucosaminyltransferases 1-3 (79-82), large
I
1,6-N-acetylglucosaminyltransferases (83), polypeptide
N-acetylgalactosaminyltransferases (84-91),
1,4-galactosyltransferases 1-7 (92-100), and
1,3-galactosyltransferases 1-5 (1-4). Discovery of the
3Gn-T
family in this study has clearly demonstrated a new feature of
glycosyltransferases, that the
3Gn-T and
3Gal-T families show
structural similarity despite of differences in both the transfer sugar
(GlcNAc versus Gal) and the acceptor sugar (Gal
versus GlcNAc).
3Gn-T2, -T3,
and -T4 fused to the FLAG peptide. Western blot analysis using
anti-FLAG antibody revealed that the secreted enzymes were successfully
produced by insect cells and were readily recovered by anti-FLAG M1
antibody resin. The FLAG-fused proteins adsorbed to the resin were
eluted under mild conditions using buffer containing 2 mM
EDTA. Since the eluted proteins showed activity comparative to that of
the adsorbed proteins, it was confirmed that EDTA treatment did not
damage the enzymes (data not shown). The molecular masses of the
recovered proteins were equal to or larger than the predicted ones for
their polypeptides, indicating some glycosylation and no significant
degradation of the recovered proteins.
1,3-N-acetylgalactosaminyltransferase activity for common
oligosaccharides, whereas their substrate preference was significantly
different. Since the amount of the recombinant proteins used in this
study was not enough for determination of the protein concentration, we could not precisely compare the relative activities of the enzymes. However, the relative activities of
3Gn-T2 for LNnT, lactose, Gal
1-4GlcNAc, and p-LNnH seemed to be higher than those
of other enzymes (Tables III and IV). Considering the variety of
acceptor substrates and the different reactivities of the transfected
cells to anti-i-antigen antibody or PWM and LEA lectins, the higher activity of
3Gn-T2 for these oligosaccharides may reflect substrate specificity.
3Gn-T activities have been detected in several tissues,
cells, and sera, some of which were characterized using partially
purified enzymes (19-28). Based on the substrate specificity,
3Gn-T1, but not
3Gn-T2, -T3, and -T4, may correspond to a
3Gn-T partially purified from calf serum.
3Gn-T2 and -T4 showed
more preferential activity for LNnT than for LNT, which was similar to
the nature of the calf serum enzyme as well as
3Gn-T1 and iGnT (7,
8, 26); however,
3Gn-T2 and -T4 were distinguished from the calf
serum enzyme and
3Gn-T1 by the activities for lactose (Gal
1-4Glc) and N-acetyllactosamine (Gal
1-4GlcNAc)
(Table IV). On the other hand,
3Gn-T3 is quite unique since it
showed activity for LNT comparable to LNnT. It has been reported that
human colon cancer tissues and the colon cancer cell line Colo205
contain cancer-associated glycosphingolipids with dimeric
Lea antigens
(Gal
1-3(Fuc
1-4)GlcNAc
1-3Gal
1-3(Fuc
1-4)GlcNAc) (101). Considering the substrate specificity and expression in colon
tissues and Colo205 cells,
3Gn-T3 is likely to be the most probable
candidate involved in the biosynthesis of the backbone structure of
dimeric Lea (Gal
1-3GlcNAc
1-3Gal
1-3GlcNAc). On
the other hand, it is difficult to ascribe the
3Gn-T activities
detected in crude samples to the isolated
3Gn-Ts (
3Gn-T1, -T2,
-T3, and -T4 or iGnT) because of the following reasons: differences in
experimental conditions such as substrates used, the possibility of the
existence of multiple
3Gn-Ts in the crude samples, and the
possibility of the existence of additional unidentified
3Gn-Ts.
3Gn-T2, -T3, and -T4
each could be involved in the initiation and elongation of
poly-N-acetyllactosamine synthesis by itself, which was
demonstrated by increased expression of
poly-N-acetyllactosamines in the transfected cells. The
different reactivities of the respective transfectants to the
anti-i-antigen antibody or LEA and PWM lectins may reflect the
preference of the antibody and lectins as well as substrate specificity
of these enzymes. On the other hand, expression of two or more
3Gn-Ts in the same cell, which was clearly demonstrated for Colo205
cells, indicates that poly-N-acetyllactosamine sugar chains
might be synthesized by the concerted action of multiple
3Gn-Ts.
Since Namalwa KJM-1 cells express endogenous
3Gn-T2, at least two
3Gn-Ts may be involved in the biosynthesis of
poly-N-acetyllactosamines in the
3Gn-T3 or
3Gn-T4 transfectants.
3Gal-T
and
3Gn-T families are clustered into the following four subgroups:
3Gal-T1, -T2, -T3, and -T5;
3Gn-T2, -T3, and -T4;
3Gal-T4; and
3Gn-T1 (Fig. 1b). Whereas
3Gal-T1, -T2, -T3, and -T5
catalyze the formation of the Gal
1-3GlcNAc structure,
3Gal-T4
forms the Gal
1-3GalNAc structure, indicating that enzymatic
activity may reflect structural similarity.
3Gn-T1 was reported to
have
3Gn-T activity (7); however,
3Gn-T2, -T3, and -T4 resemble
the
3Gal-T family rather than
3Gn-T1. In addition,
3Gn-T1 is
structurally distinct from the other members of the
3Gn-T and
3Gal-T families because it does not have 2 of the 3 cysteine
residues conserved by all the other members. There was no direct
relationship between the subgroups and chromosomal localizations of the
genes (Fig. 1B).
3Gn-T1, -T2, -T3, and -T4 exhibited no
structural similarity to another type of
3Gn-T (named iGnT) that was
isolated by expression cloning using the anti-i-antigen antibody,
suggesting that the in vivo substrate specificity of iGnT
might be quite different from that of other
3Gn-Ts.
3Gn-T genes, which
enabled us to discriminate the respective enzymes at the molecular
level. Considering the enzymatic activities in vitro and
in vivo as well as the expression patterns of the
3Gn-Ts, the respective enzymes are likely to play different roles. The poly-N-acetyllactosamine or GlcNAc
1-3Gal structure
appears in glycolipids, keratan sulfate proteoglycans, and human milk oligosaccharides, in addition to N- and O-glycans
of glycoproteins. Therefore, the existence of multiple
3Gn-Ts is not
strange. It remains to be determined which
3Gn-T makes which types
of sugar chains. Poly-N-acetyllactosamines are known to be
synthesized at various positions by the concerted action of several
glycosyltransferases required for the elongation or formation of
specific sugar branches preferred by elongation enzymes. For example,
poly-N-acetyllactosamines are preferentially formed in the
specific branch in complex type N-glycans, which are formed
by
1,6-N-acetylglucosaminyltransferase V (102). In
addition, core 2
1,6-N-acetylglucosaminyltransferases 1 and 2 and large I
1,6-N-acetylglucosaminyltransferases
are branching enzymes critical for elongation with
poly-N-acetyllactosamines (48, 80, 81, 103-105). Discovery
of the multiple
3Gn-Ts in addition to other multiple
glycosyltransferases involved in the biosynthesis of
poly-N-acetyllactosamines (e.g.
1,4-galactosyltransferases,
3Gal-Ts,
1,6-N-acetylglucosaminyltransferase V, core 2
1,6-N-acetylglucosaminyltransferases, and large I
1,6-N-acetylglucosaminyltransferases) indicates that regulation of poly-N-acetyllactosamine synthesis may be more
complex than previously recognized. Definitive determination of the
enzymatic activities and expression patterns of the
3Gn-Ts as well
as experiments using knockout mice may provide insight into their
functions in physiological and pathological processes.
3Gal-T family, although
their enzymatic activities were not determined. Based on the
characteristics of the primary structures and chromosomal
localizations, three of them may correspond to
3Gal-T2, -T3, and
-T4. Additional members of the
3Gn-T and
3Gal-T families
remain to be investigated.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Minoru Fukuda (Burnham Institute) for the generous gift of the anti-i-antigen antiserum. We thank Drs. Shoko Nishihara and Takashi Kudo and Hiroko Iwasaki (Soka University) for great help in and discussion of this study. We also thank Sachiko Kodama for excellent technical assistance, Reiko Koda for DNA sequencing, and Mayumi Ibonai and Dr. Atsuhiro Hasegawa for providing the synthetic oligonucleotides. We thank Kazumi Kurata-Miura for advice and suggestions throughout this work.
![]() |
Note Added in Proof |
---|
Recently, Zhou et al. (7) have
corrected the nucleotide sequence and deduced amino acid sequence of
the 1,3-N-acetylglucosaminyltransferase (
3Gn-T1) cDNA
that they had previously published (see "Corrections" in
Proc. Natl. Acad. Sci. U.S.A. (2000) 97, 11673-11975). This developed from an unfortunate cDNA clone
substitution in their laboratory. The corrected sequence of
3Gn-T1
was identical to that of
3Gn-T2. Consequently, besides iGnT, there
are three types of
3Gn-Ts described to date, not four. The sequence
of
3Gn-T1 used in this paper is that of the corrected, substituted cDNA clone. To try to prevent further confusion, we point out this fact
but do not change the enzyme names used in this paper.
![]() |
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 sequences reported in this paper have been
submitted to the DDBJ/GenBankTM/EBI Data Bank
with accession numbers AB049584 (3Gn-T2), AB049585 (
3Gn-T3), and AB049586 (
3Gn-T4).
§ These authors contributed equally to this work and should be considered as first authors.
To whom correspondence should be addressed. Tel.:
81-427-25-2555; Fax: 81-427-26-8330; E-mail:
ksasaki@kyowa.co.jp.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M004800200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
3Gal-Ts, UDP-galactose:
-N-acetylglucosamine
1,3-galactosyltransferases;
EST, expressed sequence tag;
GM1, Gal
1-3GalNAc
1-4(NeuAc
2-3)Gal
1-4Glc-Cer;
GM2, GalNAc
1-4(NeuAc
2-3)Gal
1-4Glc-Cer;
GD1, Gal
1-3GalNAc
1-4(NeuAc
2-8NeuAc
2-3)Gal
1-4Glc-Cer;
PCR, polymerase chain reaction;
3Gn-T, UDP-GlcNAc:
-galactose
1,3-N-acetylglucosaminyltransferase;
bp, base pair(s);
kb, kilobase pair(s);
LEA, L. esculentum agglutinin;
PWM, pokeweed mitogen;
MOPS, 4-morpholinepropanesulfonic acid;
LNnT, lacto-N-neotetraose;
p-LNnH, p-lacto-N-neohexaose;
LNT, lacto-N-tetraose;
LNFP, lacto-N-fucosylpentaose;
HPLC, high performance liquid chromatography;
RT-PCR, reverse
transcription-polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sasaki, K., Sasaki, E., Kawashima, K., Hanai, N., Nishi, T., and Hasegawa, M. (July 5, 1994) Japanese Patent 0618759 A 940705 |
2. |
Kolbinger, F.,
Streiff, M. B.,
and Katopodis, A. G.
(1998)
J. Biol. Chem.
273,
433-440 |
3. |
Amado, K.,
Almeida, R.,
Carneiro, F.,
Leverly, S. B.,
Holmes, E. H.,
Nomoto, M.,
Hollingsworth, M. A.,
Hassan, H.,
Schwientek, T.,
Nielsen, P. A.,
Bennett, E. P.,
and Clausen, H.
(1998)
J. Biol. Chem.
273,
12770-12778 |
4. |
Isshiki, S.,
Togayachi, A.,
Kudo, T.,
Nishihara, S.,
Watanabe, M.,
Kubota, T.,
Kitajima, M.,
Shiraishi, N.,
Sasaki, K.,
Andoh, T.,
and Narimatsu, H.
(1999)
J. Biol. Chem.
274,
12499-12507 |
5. |
Hennet, T.,
Dinter, A.,
Kuhnert, P.,
Mattu, T. S.,
Rudd, M. P.,
and Berger, E. G.
(1998)
J. Biol. Chem.
273,
58-65 |
6. |
Miyazaki, H.,
Fukumoto, S.,
Okada, M.,
Hasegawa, T.,
Furukawa, K.,
and Furukawa, K.
(1997)
J. Biol. Chem.
272,
24794-24799 |
7. |
Zhou, D.,
Dinter, A.,
Gutierrez Gallego, R.,
Kamerling, J. P.,
Vliegenthart, J. F.,
Berger, E. G.,
and Hennet, T.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
406-411 |
8. |
Sasaki, K.,
Kurata-Miura, K.,
Ujita, M.,
Angata, K.,
Nakagawa, S.,
Sekine, S.,
Nishi, T.,
and Fukuda, M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14294-14299 |
9. |
Sasaki, K.,
Watanabe, E.,
Kawashima, K.,
Sekine, S.,
Dohi, T.,
Oshima, M.,
Hanai, N.,
Nishi, T.,
and Hasegawa, M.
(1993)
J. Biol. Chem.
268,
22782-22787 |
10. |
Sasaki, K.,
Kurata, K.,
Funayama, K.,
Nagata, M.,
Watanabe, E.,
Ohta, S.,
Hanai, N.,
and Nishi, T.
(1994)
J. Biol. Chem.
269,
14730-14737 |
11. | Shinkai, A., Shinoda, K., Sasaki, K., Morishita, Y., Nishi, T., Matsuda, Y., Takahashi, I., and Anazawa, H. (1997) Protein Expression Purif. 10, 379-385[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Ikehara, Y.,
Kojima, N.,
Kurosawa, N.,
Kudo, T.,
Kono, M.,
Nishihara, S.,
Issiki, S.,
Morozumi, K.,
Itzkowitz, S.,
Tsuda, T.,
Nishimura, S. I.,
Tsuji, S.,
and Narimatsu, H.
(1999)
Glycobiology
9,
1213-1224 |
13. | Feizi, T., Childs, R. A., Watanabe, K., and Hakomori, S.-I. (1979) J. Exp. Med. 149, 975-980[Abstract] |
14. |
Fukuda, M. N.
(1981)
J. Biol. Chem.
256,
3900-3905 |
15. | Kondo, A., Suzuki, J., Kuraya, N., Hase, S., Kato, I., and Ikenaka, T. (1990) Agric. Biol. Chem. 54, 2169-2170[Medline] [Order article via Infotrieve] |
16. | Kudo, T., Ikehara, Y., Togayachi, A., Morozumi, K., Watanabe, M., Nakamura, M., Nishihara, S., and Narimatsu, H. (1998) Lab. Invest. 78, 797-811[Medline] [Order article via Infotrieve] |
17. | Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve] |
18. | Kozak, M. (1989) J. Cell Biol. 108, 229-241[Abstract] |
19. | Holmes, E. H. (1988) Arch. Biochem. Biophys. 260, 461-468[Medline] [Order article via Infotrieve] |
20. |
Piller, F.,
and Cartron, J. P.
(1983)
J. Biol. Chem.
258,
12293-12299 |
21. |
Holmes, E. H.,
Hakomori, S.,
and Ostrander, G. K.
(1987)
J. Biol. Chem.
262,
15649-15658 |
22. |
van den Eijnden, D. H.,
Koenderman, A. H.,
and Schiphorst, W. E.
(1988)
J. Biol. Chem.
263,
12461-12471 |
23. | Holmes, E. H., and Levery, S. B. (1989) Arch. Biochem. Biophys. 274, 14-25[Medline] [Order article via Infotrieve] |
24. | Basu, M., Khan, F. A., Das, K. K., and Zhang, B. J. (1991) Carbohydr. Res. 209, 261-277[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Gu, J.,
Nishikawa, A.,
Fujii, S.,
Gasa, S.,
and Taniguchi, N.
(1992)
J. Biol. Chem.
267,
2994-2999 |
26. | Kawashima, H., Sueyoshi, S., Li, H., Yamamoto, K., and Osawa, T. (1990) Glycoconjugate J. 7, 323-334[Medline] [Order article via Infotrieve] |
27. | Stults, C. L., and Macher, B. A. (1993) Arch. Biochem. Biophys. 303, 125-133[CrossRef][Medline] [Order article via Infotrieve] |
28. | Tsuji, Y., Urashima, T., and Matsuzawa, T. (1996) Biochim. Biophys. Acta 1289, 115-121[Medline] [Order article via Infotrieve] |
29. |
Fukuda, M.,
Spooncer, E.,
Oates, J. E.,
Dell, A.,
and Klock, J. C.
(1984)
J. Biol. Chem.
259,
10925-10935 |
30. | Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484[Medline] [Order article via Infotrieve] |
31. | Phillips, M. L., Nudelman, E., Gaeta, F. C. A., Perez, M., Singhai, A. K., Hakomori, S.-I., and Paulson, J. C. (1990) Science 250, 1130-1132[Medline] [Order article via Infotrieve] |
32. | Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. (1990) Science 250, 1132-1135[Medline] [Order article via Infotrieve] |
33. | Lowe, B. J. (1994) in Molecular Glycobiology (Fukuda, M. , and Hindsgaul, O., eds) , pp. 163-205, Oxford University Press, Oxford |
34. | Fukushima, K., Hirota, M., Terasaki, P. I., Wakisaka, A., Togashi, H., Chia, D., Suyama, N., Fukushi, Y., Nudelman, E., and Hakomori, S. (1984) Cancer Res. 44, 5279-5285[Abstract] |
35. | Nakamori, S., Kameyama, M., Imaoka, S., Furukawa, H., Ishikawa, O., Sasaki, Y., Kabuto, T., Iwanaga, T., Matsushita, Y., and Irimura, T. (1993) Cancer Res. 53, 3632-3637[Abstract] |
36. |
Sawada, R.,
Tsuboi, S.,
and Fukuda, M.
(1994)
J. Biol. Chem.
269,
1425-1431 |
37. |
Carlsson, S. R.,
Sasaki, H.,
and Fukuda, M.
(1986)
J. Biol. Chem.
261,
12787-12795 |
38. |
Maemura, K.,
and Fukuda, M.
(1992)
J. Biol. Chem.
267,
24379-24386 |
39. |
Lee, N.,
Wang, W. C.,
and Fukuda, M.
(1990)
J. Biol. Chem.
265,
20476-20487 |
40. |
Wilkins, P. P.,
McEver, R. P.,
and Cummings, R. D.
(1996)
J. Biol. Chem.
271,
18732-18742 |
41. |
Gillespie, W.,
Kelm, S.,
and Paulson, J. C.
(1992)
J. Biol. Chem.
267,
21004-21010 |
42. |
Kitagawa, H.,
and Paulson, J. C.
(1994)
J. Biol. Chem.
269,
17872-17878 |
43. |
Lee, Y. C.,
Kojima, N.,
Wada, E.,
Kurosawa, N.,
Nakaoka, T.,
Hamamoto, T.,
and Tsuji, S.
(1994)
J. Biol. Chem.
269,
10028-10033 |
44. | Kim, Y. J., Kim, K. S., Kim, S. H., Kim, C. H., Ko, J. H., Choe, I. S., Tsuji, S., and Lee, Y. C. (1996) Biochem. Biophys. Res. Commun. 228, 324-327[CrossRef][Medline] [Order article via Infotrieve] |
45. | Kitagawa, H., and Paulson, J. C. (1993) Biochem. Biophys. Res. Commun. 194, 375-382[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Wen, D. X.,
Livingston, B. D.,
Medzihradszky, K. F.,
Kelm, S.,
Burlingame, A. L.,
and Paulson, J. C.
(1992)
J. Biol. Chem.
267,
21011-21019 |
47. |
Kitagawa, H.,
and Paulson, J. C.
(1994)
J. Biol. Chem.
269,
1394-1401 |
48. | Brockhausen, I. (1999) Biochim. Biophys. Acta 1473, 67-95[Medline] [Order article via Infotrieve] |
49. | Kono, M., Takashima, S., Liu, H., Inoue, M., Kojima, N., Lee, Y. C., Hamamoto, T., and Tsuji, S. (1998) Biochem. Biophys. Res. Commun. 253, 170-175[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Ishii, A.,
Ohta, M.,
Watanabe, Y.,
Matsuda, K.,
Ishiyama, K.,
Sakoe, K.,
Nakamura, M.,
Inokuchi, J.,
Sanai, Y.,
and Saito, M.
(1998)
J. Biol. Chem.
273,
31652-31655 |
51. |
Fukumoto, S.,
Miyazaki, H.,
Goto, G.,
Urano, T.,
Furukawa, K.,
and Furukawa, K.
(1999)
J. Biol. Chem.
274,
9271-9276 |
52. |
Okajima, T.,
Fukumoto, S.,
Miyazaki, H.,
Ishida, H.,
Kiso, M.,
Furukawa, K.,
Urano, T.,
and Furukawa, K.
(1999)
J. Biol. Chem.
274,
11479-11486 |
53. |
Kurosawa, N.,
Hamamoto, T.,
Lee, Y. C.,
Nakaoka, T.,
Kojima, N.,
and Tsuji, S.
(1994)
J. Biol. Chem.
269,
1402-1409 |
54. |
Kurosawa, N.,
Inoue, M.,
Yoshida, Y.,
and Tsuji, S.
(1996)
J. Biol. Chem.
271,
15109-15116 |
55. |
Sjoberg, E. R.,
Kitagawa, H.,
Glushka, J.,
van Halbeek, H.,
and Paulson, J. C.
(1996)
J. Biol. Chem.
271,
7450-7459 |
56. |
Lee, Y. C.,
Kaufmann, M.,
Kitazume-Kawaguchi, S.,
Kono, M.,
Takashima, S.,
Kurosawa, N.,
Liu, H.,
Pircher, H.,
and Tsuji, S.
(1999)
J. Biol. Chem.
274,
11958-11967 |
57. |
Okajima, T.,
Fukumoto, S.,
Ito, H.,
Kiso, M.,
Hirabayashi, Y.,
Urano, T.,
Furukawa, K.,
and Furukawa, K.
(1999)
J. Biol. Chem.
274,
30557-30562 |
58. |
Okajima, T.,
Chen, H.-H.,
Ito, H.,
Kiso, M.,
Tai, T.,
Furukawa, K.,
Urano, T.,
and Furukawa, K.
(2000)
J. Biol. Chem.
275,
6717-6723 |
59. |
Sasaki, K.,
Kurata, K.,
Kojima, N.,
Kurosawa, N.,
Ohta, S.,
Hanai, N.,
Tsuji, S.,
and Nishi, T.
(1994)
J. Biol. Chem.
269,
15950-15956 |
60. | Nara, K., Watanabe, Y., Maruyama, K., Kasahara, K., Nagai, Y., and Sanai, Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7952-7956[Abstract] |
61. |
Haraguchi, M.,
Yamashiro, S.,
Yamamoto, A.,
Furukawa, K.,
Takamiya, K.,
Lloyd, K. O.,
Shiku, H.,
and Furukawa, K.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10455-10459 |
62. |
Nakayama, J.,
Fukuda, M. N.,
Hirabayashi, Y.,
Kanamori, A.,
Sasaki, K.,
Nishi, T.,
and Fukuda, M.
(1996)
J. Biol. Chem.
271,
3684-3691 |
63. |
Livingston, B. D.,
and Pauson, J. C.
(1993)
J. Biol. Chem.
268,
11504-11507 |
64. | Eckhardt, M., Mühlenhoff, M., Bethe, A., Koopman, J., Frosch, M., and Gerardy-Schahn, R. (1995) Nature 373, 715-718[CrossRef][Medline] [Order article via Infotrieve] |
65. |
Yoshida, Y.,
Kojima, N.,
Kurosawa, N.,
Hamamoto, T.,
and Tsuji, S.
(1995)
J. Biol. Chem.
270,
14628-14633 |
66. | Nakayama, J., Fukuda, M. N., Fredette, B., Ranscht, B., and Fukuda, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7031-7035[Abstract] |
67. |
Scheidegger, E. P.,
Sternberg, L. R.,
Roth, J.,
and Lowe, J. B.
(1995)
J. Biol. Chem.
270,
22685-22688 |
68. | Kojima, N., Yoshida, Y., and Tsuji, S. (1995) FEBS Lett. 373, 119-122[CrossRef][Medline] [Order article via Infotrieve] |
69. |
Kono, M.,
Yoshida, Y.,
Kojima, N.,
and Tsuji, S.
(1996)
J. Biol. Chem.
271,
29366-29371 |
70. | Kukowska-Latallo, J. F., Larsen, R. D., Nair, R. P., and Lowe, J. B. (1990) Genes Dev. 4, 1288-1303[Abstract] |
71. |
Lowe, J. B.,
Kukowska-Latallo, J. F.,
Nair, R. P.,
Larsen, R. D.,
Marks, R. M.,
Macher, B. A.,
Kelly, R. J.,
and Ernst, L. K.
(1991)
J. Biol. Chem.
266,
17467-17477 |
72. | Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) Cell 63, 1349-1356[Medline] [Order article via Infotrieve] |
73. |
Weston, B. W.,
Nair, R. P.,
Larsen, R. D.,
and Lowe, J. B.
(1992)
J. Biol. Chem.
267,
4152-4160 |
74. |
Weston, B. W.,
Smith, P. L.,
Kelly, R. J.,
and Lowe, J. B.
(1992)
J. Biol. Chem.
267,
24575-24584 |
75. | Nishihara, S., Nakazato, M., Kudo, T., Kimura, H., Ando, T., and Narimatsu, H. (1993) Biochem. Biophys. Res. Commun. 190, 42-46[CrossRef][Medline] [Order article via Infotrieve] |
76. | Amado, M., Almeida, R., Schwientek, T., and Clausen, H. (1999) Biochim. Biophys. Acta 1473, 35-53[Medline] [Order article via Infotrieve] |
77. |
Natsuka, S.,
Gersten, K. M.,
Zenita, K.,
Kannagi, R.,
and Lowe, J. B.
(1994)
J. Biol. Chem.
269,
16789-16794 |
78. |
Kudo, T.,
Ikehara, Y.,
Togayachi, A.,
Kaneko, M.,
Hiraga, T.,
Sasaki, K.,
and Narimatsu, H.
(1998)
J. Biol. Chem.
273,
26729-26738 |
79. | Bierhuizen, M. F., and Fukuda, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9326-9330[Abstract] |
80. |
Yeh, J.-C.,
Ong, E.,
and Fukuda, M.
(1999)
J. Biol. Chem.
274,
3215-3221 |
81. |
Schwientek, T.,
Nomoto, M.,
Levery, S. B.,
Merkx, G.,
van Kessel, A. G.,
Bennett, E. P.,
Hollingsworth, M. A.,
and Clausen, H.
(1999)
J. Biol. Chem.
274,
4504-4512 |
82. |
Schwientek, T.,
Yeh, J.-C.,
Levery, S. B.,
Keck, B.,
Merkx, G.,
van Kessel, A. G.,
Fukuda, M.,
and Clausen, H.
(2000)
J. Biol. Chem.
275,
11106-11113 |
83. | Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev. 7, 468-478[Abstract] |
84. |
Bennett, E. P.,
Hassan, H.,
and Clausen, H.
(1996)
J. Biol. Chem.
271,
17006-17012 |
85. | Bennett, E. P., Hassan, H., Hollingsworth, M. A., and Clausen, H. (1999) FEBS Lett. 460, 226-230[CrossRef][Medline] [Order article via Infotrieve] |
86. |
Bennett, E. P.,
Hassan, H.,
Mandel, U.,
Hollingsworth, M. A.,
Akisawa, N.,
Ikematsu, Y.,
Merkx, G.,
van Kessel, A. G.,
Olofsson, S.,
and Clausen, H.
(1999)
J. Biol. Chem.
274,
25362-25370 |
87. |
Bennett, E. P.,
Hassan, H.,
Mandel, U.,
Mirgorodskaya, E.,
Roepstorff, P.,
Burchell, J.,
Taylor-Papadimitriou, J.,
Hollingsworth, M. A.,
Merkx, G.,
van Kessel, A. G.,
Eiberg, H.,
Steffensen, R.,
and Clausen, H.
(1998)
J. Biol. Chem.
273,
30472-30481 |
88. |
Homa, F. L.,
Hollander, T.,
Lehman, D. J.,
Thomsen, D. R.,
and Elhammer, A. P.
(1993)
J. Biol. Chem.
268,
12609-12616 |
89. |
Ten Hagen, K. G.,
Hagen, F. K.,
Balys, M. M.,
Beres, T. M.,
Van Wuyckhuyse, B.,
and Tabak, L. A.
(1998)
J. Biol. Chem.
273,
27749-27754 |
90. |
White, T.,
Bennett, E. P.,
Takio, K.,
Sorensen, T.,
Bonding, N.,
and Clausen, H.
(1995)
J. Biol. Chem.
270,
24156-24165 |
91. | White, K. E., Lorenz, B., Evans, W. E., Meitinger, T., Strom, T. M., and Econs, M. J. (2000) Gene (Amst.) 246, 347-356[CrossRef][Medline] [Order article via Infotrieve] |
92. | Narimatsu, H., Sinha, S., Brew, K., Okayama, H., and Qasba, P. K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4720-4724[Abstract] |
93. |
Shaper, N. L.,
Hollis, G. F.,
Douglas, J. G.,
Kirsch, I. R.,
and Shaper, J. H.
(1988)
J. Biol. Chem.
263,
10420-10428 |
94. | Masri, K. A., Appert, H. E., and Fukuda, M. N. (1988) Biochem. Biophys. Res. Commun. 157, 657-663[Medline] [Order article via Infotrieve] |
95. |
Almeida, R.,
Amado, M.,
David, L.,
Levery, S. B.,
Holmes, E. H.,
Merkx, G.,
van Kessel, A. G.,
Hassan, H.,
Bennett, E. P.,
and Clausen, H.
(1997)
J. Biol. Chem.
272,
31979-31992 |
96. |
Sato, T.,
Furukawa, K.,
Bakker, H.,
van den Eijnden, D. H.,
and Van Die, I.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
472-477 |
97. |
Nomura, T.,
Takizawa, M.,
Aoki, J.,
Arai, H.,
Inoue, K.,
Wakisaka, E.,
Yoshizuka, N.,
Imokawa, G.,
Dohmae, N.,
Takio, K.,
Hattori, M.,
and Matsuo, N.
(1998)
J. Biol. Chem.
273,
13570-13577 |
98. |
Schwientek, T.,
Almeida, R.,
Levery, S. B.,
Holmes, E.,
Bennett, E. P.,
and Clausen, H.
(1998)
J. Biol. Chem.
273,
29331-29340 |
99. |
Okajima, T.,
Yoshida, K.,
Kondo, T.,
and Furukawa, K.
(1999)
J. Biol. Chem.
274,
22915-22918 |
100. |
Almeida, R.,
Levery, S. B.,
Mandel, U.,
Kresse, H.,
Schwientek, T.,
Bennett, E. P.,
and Clausen, H.
(1999)
J. Biol. Chem.
274,
26165-26171 |
101. |
Stroud, M. R.,
Levery, S. B.,
Nudelman, E. D.,
Salyan, M. E.,
Towell, J. A.,
Roberts, C. E.,
Watanabe, M.,
and Hakomori, S.
(1991)
J. Biol. Chem.
266,
8439-8446 |
102. | Dennis, J. W., Granovsky, M., and Warren, C. E. (1999) Biochim. Biophys. Acta 1473, 21-34[Medline] [Order article via Infotrieve] |
103. |
Ujita, M.,
McAuliffe, J.,
Suzuki, M.,
Hindsgaul, O.,
Clausen, H.,
Fukuda, M. N.,
and Fukuda, M.
(1999)
J. Biol. Chem.
274,
9296-9304 |
104. |
Ujita, M.,
McAuliffe, J.,
Hindsgaul, O.,
Sasaki, K.,
Fukuda, M. N.,
and Fukuda, M.
(1999)
J. Biol. Chem.
274,
16717-16726 |
105. |
Ujita, M.,
McAuliffe, J.,
Schwientek, T.,
Almeida, R.,
Hindsgaul, O.,
Clausen, H.,
and Fukuda, M.
(1998)
J. Biol. Chem.
273,
34843-34849 |