From the Department of Biochemistry, Sasaki
Institute, Kanda-Surugadai 2-2, and Core Research for Evolutional
Science and Technology (CREST) of the Japan Science and Technology
Corporation, Kanda-Surugadai 2-3, Chiyoda-ku, Tokyo 101-0062, and
§ Biomolecular Characterization Division, the Institute of
Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako,
Saitama 351-0198, Japan
Received for publication, November 11, 2002, and in revised form, January 2, 2003
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ABSTRACT |
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The Gal The Gal It has been clarified so far (18-29) that seven In this study, we first purified 6SGN-specific Materials--
UDP-[3H]galactose (1400 GBq/mmol)
was purchased from PerkinElmer Life Sciences. PAPS, UDP-Gal,
p-nitrophenyl(pNP)- Purification of 6SGN-specific Cloning of the cDNAs Encoding Expression of Synthesis of
Gal Assay of Determination of Protein Concentrations--
The protein
concentrations were estimated using the Bio-Rad Protein Assay dye
reagent with bovine serum albumin as a standard.
Amino Acid Sequence Analysis--
Coomassie-stained bands in
SDS-PAGE gels were excised and treated with 0.2 µg of
Achromobacter protease I (a gift from Dr. Masaki, Ibaraki
University) (43) at 37 °C for 12 h in 0.1 M Tris-HCl (pH 9.0) containing 0.1% SDS. Peptides generated were extracted from the gel and separated on columns of DEAE-5PW (2 × 20 mm; Tosoh, Tokyo) and Mightysil RP-18 (2 × 50 mm; Kanto
Chemical, Tokyo) connected in series with a model 1100 (Hewlett-Packard) liquid chromatography system. Peptides were eluted at
a flow rate of 0.1 ml/min using a linear gradient of 0-60% solvent B,
where solvents A and B were 0.09% (v/v) aqueous trifluoroacetic acid and 0.075% (v/v) trifluoroacetic acid in 80%(v/v) acetonitrile, respectively. Selected peptides were subjected to Edman degradation using a model 477A automated protein sequencer (Applied Biosystems, Inc.) connected on-line to a model 120A PTH analyzer (PerkinElmer Life
Sciences) and to a Reflex matrix-assisted laser desorption ionization
time of flight mass spectrometer (Bruker-Franzen Analytik, Bremen,
Germany) in linear mode using 2-mercaptobenzothiazole (44) as a matrix.
Northern Blot Analysis--
Human Multiple Tissue Northern blot
membranes (Clontech, Palo Alto, CA) were used
according to the manufacturer's instructions. The mRNA content in
each lane of the Northern blot membrane was normalized to the mRNA
expression level of Purification of 6SGN-specific
After asialo-agalacto-ovomucin-Sepharose chromatography, 6SGN-specific
Expression of Seven
The specific activities of
Results of kinetic analysis of Expression of General Discussion--
In this study, we showed the following.
(i) Porcine 6SGN-specific
KS is poly-6-O-sulfated poly-N-acetyllactosamine
(reviewed in Ref. 13). Several glyco- and sulfotransferases involved in KS synthesis have been reported. Five GlcNAc6ST genes have so far been
identified, and all the enzymes can act on non-reducing terminal GlcNAc
but not on internal GlcNAc such as Gal
On the other hand, 6-sulfosialyl-Lewis X is synthesized by sequential
reactions of GlcNAc6ST,
1
4(SO
6)GlcNAc
moiety is present in various N-linked and
O-linked glycans including keratan sulfate and
6-sulfosialyl-Lewis X, an L-selectin ligand. We previously found
1,4-galactosyltransferase (
4GalT) activity in human colonic mucosa, which prefers GlcNAc 6-O-sulfate (6SGN) as an
acceptor to non-substituted GlcNAc (Seko, A., Hara-Kuge, S., Nagata,
K., Yonezawa, S., and Yamashita, K. (1998) FEBS Lett. 440, 307-310). To identify the gene for this enzyme, we purified the enzyme
from porcine colonic mucosa. The purified enzyme had the characteristic requirement of basic lipids for catalytic activity. Analysis of the
partial amino acid sequence of the enzyme revealed that the purified
4GalT has a similar sequence to human
4GalT-IV. To confirm this
result, we prepared cDNA for each of the seven
4GalTs cloned to
date and examined substrate specificities using the membrane fractions
derived from
4GalT-transfected COS-7 cells. When using several
N-linked and O-linked glycans with or without 6SGN residues as acceptor substrates, only
4GalT-IV efficiently recognized 6SGN, keratan sulfate-related oligosaccharides, and Gal
1
3(SO
6GlcNAc
1
6)
GalNAc
1-O-pNP, a precursor for 6-sulfosialyl-Lewis X. These results suggested that
4GalT-IV is a 6SGN-specific
4GalT and may be involved in the biosynthesis of various
glycoproteins carrying a 6-O-sulfated N-acetyllactosamine moiety.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1
4(SO
6)GlcNAc structure has
been found in N-linked and O-linked glycans of
glycoproteins and is involved in various biological events (1, 2). The sulfated disaccharide unit has been believed to be biosynthesized by
6-O-sulfation of non-reducing terminal GlcNAc and, following
1,4-galactosylation, on the basis of the substrate specificities of
GlcNAc 6-O-sulfotransferases
(GlcNAc6STs)1 so far reported
(2-11). Although the galactosylation step had long been unclear, we
partially purified a GlcNAc 6-O-sulfate (6SGN)-specific
1,4-galactosyltransferase (
4GalT) from human colonic mucosa (12).
The enzyme preferred 6SGN and 6SGN-containing oligosaccharides to
non-sulfated GlcNAc residues as acceptor substrates, whereas bovine
milk
4GalT-I showed the opposite specificity. The
Neu5Ac
2
6GlcNAc
1
sequence was a poor substrate for the 6SGN-specific
4GalT, suggesting that a sulfate residue at the C-6 of
GlcNAc is essential for the acceptor recognition. This preference for
6SGN residues suggests that the enzyme is involved in the biosynthesis
of keratan sulfate (KS),
((SO
6)Gal
1
4(SO
6)GlcNAc
1
3)n (reviewed in Ref. 13), and 6-sulfosialyl-Lewis X, known as a L-selectin
ligand (14-17). However, molecular cloning of the enzyme has not yet
been performed.
4GalT genes exist.
All the
4GalTs are synthesized as type II membrane-bound proteins
and reside in the Golgi apparatus (30). These seven enzymes have been
characterized in terms of substrate specificity.
4GalT-I is abundant
in bovine and human milk in a soluble form and is the first
galactosyltransferase for which the corresponding cDNA has been
isolated (18-21).
4GalT-I acts on non-reducing terminal GlcNAc as
an acceptor, and whereas in the presence of
-lactalbumin, the
enzyme prefers Glc as a lactose synthase to GlcNAc (31, 32). This
enzyme is involved in the elongation of
poly-N-acetyllactosamine repeats (33).
4GalT-II
and -III act on GlcNAc residues in several glycoproteins and specific
glycolipids, and
4GalT-II is affected by
-lactalbumin in a
similar manner to
4GalT-I (22).
4GalT-IV acts on Lc3
(GlcNAc
1
3Gal
1
4Glc
1
Cer) (26) and
Gal
1
3(GlcNAc
1
6)GalNAc(core2); the latter galactosylation at
the C-4 of GlcNAc leads to the formation of the sialyl-Lewis X
structure (34).
4GalT-V has been shown to have strong activity for
core2 and core6(GlcNAc
1
6GalNAc) (35). The enzyme also
recognized
GlcNAc
1
2(GlcNAc
1
6)Man
1
6 moieties in N-linked tetra-antennary glycans (23, 36) and was suggested to be involved in the biosynthesis of tumor-associated N-glycans in concert with
N-acetylglucosaminyltransferase V (37). Lactosylceramide synthase has been purified from rat brain, and its
molecular cloning was performed based on partial amino acid sequences (25). The enzyme is an orthologue of human
4GalT-VI (24,
27).
4GalT-VII is a
-Xyl:
1,4GalT, equal to
galactosyltransferase-I which is involved in the synthesis of the
proximal sequence in various glycosaminoglycans,
Gal
1
3Gal
1
4Xyl
1
Ser/Thr (28, 29).
Although the substrate specificities of these seven
4GalTs have been
extensively studied, it remained unclear which enzymes can act on 6SGN
residues in KS, N-linked, and O-linked glycans, and whether or not 6SGN-specific
4GalT is encoded by a novel gene.
This issue is important for studying the biological roles of
6-sulfo-N-acetyllactosamine-containing glycans including KS and 6-sulfosialyl-Lewis X.
4GalT from porcine
colonic mucosa and partially determined its amino acid sequence. We
obtained a sequence similar to human
4GalT-IV. Moreover, we isolated
cDNA for each of the seven
4GalTs cloned to date, and we
introduced expression vectors containing these cDNAs individually into COS-7 cells and prepared membrane fractions to study their substrate specificities. As a result, we found that among the seven
4GalTs,
4GalT-IV is the only enzyme to efficiently act on
KS-related oligosaccharides and GlcNAc-6-O-sulfated core2, a
precursor of 6-sulfosialyl-Lewis X.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-xylose (Xyl-O-pNP), and glucosylceramide (GlcCer) were purchased
from Sigma. Gal
1
3(GlcNAc
1
6)GalNAc
1-O-pNP
(core2-O-pNP) was obtained from Funakoshi Co., Ltd. (Tokyo,
Japan).
GlcNAc
1
2Man
1
3(GlcNAc
1
2Man
1
6)Man
1
4GlcNAc
1
4GlcNAc (biGP) was prepared from egg yolk sialoglycopeptide (38) by hydrazinolysis re-N-acetylation (39), mild acid hydrolysis, and Streptococcus 6646K
-galactosidase (40) (Seikagaku
Corp., Tokyo, Japan) digestion. GlcNAc
1
3Gal
1
4Glc(GL) was
prepared from lacto-N-tetraose (41) by digestion with
Streptococcus 6646K
-galactosidase. Ricinus
communis agglutinin-I (RCA-I)-agarose(4 mg/ml gel) was
purchased from Hohnen Oil Co. (Tokyo, Japan). Keratan sulfate
(sodium salt and bovine cornea) was purchased from Seikagaku Corp.
Gal
1
4(SO
6)GlcNAc
1
3Gal
1
4(SO
6)GlcNAc(L2L2) and
Gal
1
4(SO
6)GlcNAc
1
3(SO
6)Gal
1
4(SO
6)GlcNAc(L2L4) were kindly provided by Seikagaku Corp.
SO
6GlcNAc
1
3Gal
1
4
(SO
6)GlcNAc(agL2L2) and SO
6GlcNAc
1
3(SO
6)Gal
1
4(SO
6)GlcNAc(agL2L4) were prepared by Streptococcus 6646K
-galactosidase
digestion from L2L2 and L2L4, respectively. GlcNAc
6-O-sulfate (6SGN),
Neu5Ac
2
6GlcNAc
1
3Gal
1
4Glc(agLST-b), and
SO
6GlcNAc
1
2Man
1
3(Man
1
6)Man
1
4GlcNAc
1
4(Fuc
1
6)GlcNAc(6S-biGP) were prepared as described previously (12).
L-
-Phosphatidic acid,
L-
-phosphatidylcholine,
L-
-lysophosphatidylcholine, and L-
-phosphatidylethanolamine were from egg yolk lecithin;
L-
-phosphatidylinositol was from soybean;
L-
-phosphatidyl-L-serine and sphingomyelin were from bovine brain; ceramides were from bovine brain sphingomyelin; D-sphingosine was from bovine brain cerebrosides;
N,N-dimethylsphingosine, N-acetyl-D-sphingosine, and
N-stearoyl-D-sphingosine prepared from
D-sphingosine, sphingosine 1-phosphate, and stearylamine were purchased from Sigma. Octylamine was purchased from Aldrich.
4GalT from Porcine Colonic
Mucosa--
The following procedures were performed at 4 °C. Six kg
of porcine colon was purchased from Tokyo Shibaura Zohki Co. Ltd. (Tokyo, Japan). The mucus layer was scraped off the colon; 3 liters of
PBS was added, and the mixture was homogenized with a Potter-Elvehjem type homogenizer and then centrifuged at 1,000 × g for
30 min. The extraction was performed once, and the two supernatant
fractions were mixed and ultracentrifuged at 100,000 × g for 1 h. The precipitated microsomes were washed once
with 0.5 M KCl and extracted twice with 1 liter of 20 mM HEPES-NaOH (pH 7.2), 1 M NaCl, 10 mM MnCl2, 1% (w/v) Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, and 10% (v/v) glycerol followed by ultracentrifugation. The
extract was dialyzed against 10 mM HEPES-NaOH (pH 7.2), 5 mM MnCl2, 1 mM dithiothreitol, and
20% glycerol (buffer A), and applied on a UDP-hexanolamine-Sepharose
column (7 µmol/ml of gel; 2 × 10 cm; equilibrated with 20 mM HEPES-NaOH (pH 7.2), 10 mM MnCl2, 0.1% (w/v) Triton X-100, 1 mM
dithiothreitol, and 10% glycerol (buffer B)) (12). After washing with
buffer B containing 0.15 M NaCl, the enzyme fractions were
eluted with buffer B containing 1 M NaCl and dialyzed
against buffer A. Next, the dialysate was reapplied on a
UDP-hexanolamine-Sepharose column (1.4 × 6.5 cm; equilibrated
with buffer B) and eluted with buffer B containing 1 mM
UDP. Each fraction was dialyzed against buffer A and assayed for
4GalT activity. The enzyme fractions were applied on an
asialo-agalacto-ovomucin-Sepharose column (6.7 mg mucin/ml of gel;
1 × 7.5 cm; equilibrated with buffer B) (12). The enzyme was
eluted with a linear gradient of NaCl (0-0.2 M) in buffer
B and then dialyzed. The enzyme fractions were concentrated and used
for biochemical analyses.
4GalTs--
cDNAs
encoding
4GalT-II, -III, -IV, -V, and -VII were amplified from
SuperScriptTM human testis cDNA library (Invitrogen) by
PCR, and cDNAs encoding
4GalT-I and -VI were amplified by PCR
from QUICK-CloneTM cDNA for human colon adenocarcinoma
and mouse brain (Clontech, Palo Alto, CA),
respectively. Oligonucleotide primers used for the PCRs were
5'-tttggatccTGTAGCCCACACC-3' (forward primer for
4GalT-I),
5'-tttgatatcAGGTCTCTTATCCGTG-3' (reverse primer for
4GalT-I),
5'-tttggatccGGATGAGCAGACTGCTG-3' (forward primer for
4GalT-II),
5'-tttgatatcGTCCATTAGTGTCAGCC-3' (reverse primer for
4GalT-II),
5'-tttggatccAGGATGTTGCGGAGGCT-3' (forward primer for
4GalT-III),
5'-tttgatatcGAGGAGTCAGTGTGAAC-3' (reverse primer for
4GalT-III),
5'-tttggatccACATGGGCTTCAACCTG-3' (forward primer for
4GalT-IV),
5'-tttgatatcATCCAGGGTCATGCACC-3' (reverse primer for
4GalT-IV),
5'-tttggatccTGGCTGCAGCATGC-3' (forward primer for
4GalT-V),
5'-tttgatatcTCAGTACTCGTTCAC-3' (reverse primer for
4GalT-V),
5'-tttggatccGGAAGATGTCTGTGCTC-3' (forward primer for
4GalT-VI),
5'-tttgatatcCAGCATTGTGGTCTA-3' (reverse primer for
4GalT-VI),
5'-tttggatccGCACGATGTTCCC-3' (forward primer for
4GalT-VII), and
5'-tttgatatcCAGCTCAGCTGAATG-3' (reverse primer for
4GalT-VII).
Sequences in lowercase letters contain appropriate restriction sites.
Amplified cDNAs were digested with BamHI and EcoRV and cloned between the respective sites of pcDNA3
(Invitrogen). The constructed plasmids were named
pcDNA3-
4GalT-I, -II, -III, -IV, -V, -VI, and -VII, respectively,
and these sequences were confirmed using an Applied Biosystems PRISM®
310 Genetic Analyzer (PE Biosystems).
4GalTs in COS-7 Cells--
The plasmids (1 µg) were transfected into COS-7 cells on 35-mm dishes using
Lipofectin Reagent (Invitrogen) according to the manufacturer's
instructions. After 48 h, the cells were washed twice with
phosphate-buffered saline, scraped off the dishes in 10 mM
HEPES-NaOH (pH 7.2) and 0.25 M sucrose, and homogenized. The homogenates were ultracentrifuged at 100,000 × g
for 1 h. The precipitated crude membranes were suspended in 20 mM HEPES-NaOH (pH 7.2) and kept at
80 °C until use.
1
3(SO
6GlcNAc
1
6)GalNAc
1-O-pNP(6S-core2-O-pNP)--
The
6-O-sulfation of the GlcNAc residue in
core2-O-pNP was performed by GlcNAc6ST-1 (42). Briefly, 2 ml
of reaction mixture containing 0.1 M sodium cacodylate (pH
6.8), 10 mM MnCl2, 0.1% (w/v) digitonin, 0.1 M NaF, 2 mM Na2-ATP, 0.2 mM PAPS, 0.75 mM core2-O-pNP, and a
membrane fraction prepared from GlcNAc6ST-1-expressing COS-7 cells (11)
(the specific activity was 8 nmol/h when using 1 mM
core2-O-pNP and 17 µM PAPS) was incubated at
37 °C for 24 h and subjected to paper electrophoresis. The
product fractions were cut off and extracted with water. The extract
was applied on a Sephadex G-25 column (1.4 × 68 cm, equilibrated
and eluted with EtOH/water, 5:95 (v/v)). The sulfated oligosaccharide
fractions were collected and concentrated. Finally, 150 nmol of
6S-core2-O-pNP was obtained.
4GalT Activity--
Twenty µl of reaction mixture
consisting of 50 mM HEPES-NaOH (pH 7.2), 10 mM
MnCl2, 0.5% (w/v) Triton X-100, 250 µM
UDP-Gal, 0.3 µM UDP-[3H]Gal (4.9 × 105 dpm), 0.5 mM acceptor substrate, and the
membrane fraction approximately diluted was incubated at 37 °C for
1 h. In the cases of purified porcine 6SGN-specific
4GalT,
0.5%(w/v) Triton X-100 was replaced by 0.001% (w/v)
D-sphingosine and 0.05% (w/v) Triton X-100. The 3H-labeled products derived from biGP, 6SGN, GL,
Xyl-O-pNP, core2-O-pNP, 6S-core2-O-pNP, 6S-biGP, and KS were purified by paper
electrophoresis (pyridine/acetic acid/water, 3:1:387, pH 5.4). The
reaction mixtures in the case of agL2L2, agL2L4, and agLST-b were
treated with 1 ml of 0.01 N HCl at 100 °C for 10 min,
neutralized with 1 N NaOH, concentrated, and subjected to
paper electrophoresis. The acid hydrolysis was necessary for the
destruction of excess UDP-[3H]Gal, which moves to a
similar position as 3H-galactosylated agL2L2 and agL2L4 and
for removal of sialic acid residue in the case of agLST-b. After
drying, in the cases of neutral substrates, the paper was further
developed with a solvent system, pyridine/ethyl acetate/acetic
acid/water (5:5:1:3). The paper was monitored for radioactivity with a
radiochromatogram scanner, and the 3H-labeled products were
extracted with water and applied on a RCA-I-agarose column (0.7 × 2.5 cm). Elution was started with 4 ml of 10 mM sodium
phosphate (pH 7.0), 0.15 M NaCl, followed by the same
buffer containing 10 mM lactose. The bound fractions (eluted with 10 mM lactose) or retarded fractions were
collected and measured for radioactivity. In the case of GlcCer, the
reaction mixtures were directly applied on a high performance TLC
silica gel plate (Kieselgel 60 F254) (Merck) and developed
with a solvent system (CHCl3/MeOH/0.2% CaCl2,
60:35:7). After drying, the plates were monitored for radioactivity,
and the 3H-labeled products were extracted with
CHCl3/MeOH (1:1) and counted. The membrane fraction derived
from pcDNA3-transfected COS-7 cells (C-MF) was used as a
control for intrinsic
4GalT activities. To calculate the amount of
exogenous
4GalT activities, values of activities in C-MF were
subtracted from those of apparent enzymatic activities obtained under
the same conditions. Enzymatic activity values presented here were
means of four independent experiments. Standard deviations were less
than 5% in all cases. The concentration of KS was presented as moles
of galactose.
-actin. 32P-Labeled probe was
prepared from the cDNA fragment (excised from pcDNA3-
4GalT-IV by BamHI and EcoRV
digestion) with a Random Primed DNA Labeling Kit (Roche Diagnostics)
using [
-32P]dCTP (PerkinElmer Life Sciences) according
to the manufacturer's instructions. The membranes were pre-hybridized
in ExpressHyb Solution (Clontech) at 68 °C for
2 h and then hybridized with the 32P-labeled probe in
the same solution at 68 °C for 16 h. The Northern blot
membranes were washed in 2× SSC, 0.05% SDS at room temperature and
then in 0.1× SSC, 0.1% SDS at 50 °C. The radioactivity was detected with FLA-2000 (Fuji Photo Film Co. Ltd., Tokyo).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
4GalT from Porcine Colonic Mucosa
and Its Basic Lipid Requirement for Activity--
We showed previously
(12) that a 6SGN-specific
4GalT exists in human colonic mucosa. To
purify this enzyme and to isolate a corresponding cDNA based on
partial amino acid sequences, we used porcine colonic mucosa as an
enzyme source. Porcine colonic mucosa also contained 6SGN-specific
4GalT activity. When the enzyme was purified 2,300-fold by a second
UDP-hexanolamine-Sepharose column chromatography (Table
I), the enzymatic activity disappeared (Fig. 1A). Because such a loss
of enzymatic activity has been reported for a glucuronyltransferase,
which requires sphingomyelin for activity in a highly purified state
(45), we added various lipid compounds to the enzyme reaction mixtures.
As shown in Table II, most of the
phospholipids and ceramides had no ability to restore the enzymatic
activity, whereas the activity appeared in the presence of
D-sphingosine and N,N-dimethylsphingosine. Stearylamine could also restore the activity, whereas octylamine could
not, suggesting that basic, lipidous compounds with long hydrophobic
chains are essential at least for the in vitro enzymatic activity. The minimum concentrations of D-sphingosine,
N,N-dimethylsphingosine, and stearylamine giving the maximal
activity were ~10, 20, and 10 µM, respectively (Fig.
2).
Purification of 6SGN-specific 4GaIT from porcine colonic mucosa
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Fig. 1.
A, second UDP-hexanolamine-Sepharose
column chromatography of the 1 M NaCl-eluted fractions on
first UDP-hexanolamine-Sepharose column chromatography. Each 30-ml
fraction was collected. The column was washed with buffer B followed by
buffer B containing 1 mM UDP (arrowheads).
6SGN-specific 4GalT activities (
) were assayed in the absence
(upper) or presence (lower) of 0.001%
D-sphingosine as described under "Experimental
Procedures." The fractions indicated by the bar were
collected. B, asialo-agalacto-ovomucin-Sepharose column
chromatography of the enzyme fractions obtained in A. Each
12-ml fraction was collected. The column was washed with buffer B
followed by a linear gradient of 0-0.2 M NaCl (- - -) in
buffer B. The enzymatic activities were assayed in the presence of
0.001% D-sphingosine. The fractions indicated by the
bar were collected and used for biochemical
analysis.
Effects of various lipids on purified 6SGN-specific 4GaIT activity
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Fig. 2.
Effects of D-sphingosine ( ),
N,N-dimethylsphingosine (
), and stearylamine (
)
on the purified 6SGN-specific
4GalT.
Indicated concentrations of the lipids were added to the enzyme
reaction mixtures. The concentration of Triton X-100 was 0.05% (w/v)
in these assays.
4GalT was purified 24,000-fold (Fig. 1B and Table I). We
tried further purification by various chromatographic methods but were
unsuccessful. The final enzyme fractions contained two major
(45kDa and 42kDa) and two minor (59kDa and 52kDa) proteins as
determined by SDS-PAGE analysis (Fig. 3).
Each protein band was digested with a lysine-specific protease, and the
peptide fragments were separated by reversed phase high pressure liquid chromatography, and their amino acid sequence was analyzed. One or two
peptide fragments from each band were sequenced (Table III), and the sequences were compared
with known protein sequences using the BLAST search system. The band
a contained one sequence that is equal to that of human
polypeptide:N-acetylgalactosaminyltransferase-2 (46). The
band b contained two sequences that are very similar to that
of human N-acetylglucosaminyltransferase-I (47, 48). Because
the two enzymes utilize UDP-sugars as donor substrates, they may be
co-purified with 6SGN-specific
4GalT by UDP-hexanolamine-Sepharose column chromatographies. On the other hand, the bands c and
d contained a common peptide sequence, which is similar to
that of human
4GalT-IV (24, 26). This result suggested that the purified 6SGN-specific
4GalT corresponds to
4GalT-IV.
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Fig. 3.
SDS-PAGE analysis of the enzyme fractions
obtained by asialo-agalacto-ovomucin-Sepharose column
chromatography. The enzyme fractions (10 µg as protein) were
subjected to SDS-PAGE (10% gel) under reducing conditions and
visualized with Coomassie Brilliant Blue.
Amino acid sequences of peptide fragments derived from the bands a-d
in Fig. 3
4GalTs in COS-7 Cells and Their Substrate
Specificities--
To confirm that
4GalT-IV is 6SGN-specific, and
the only 6SGN-specific
4GalT among the seven
4GalTs so far
identified, we prepared expression vectors containing each
4GalT
cDNA and analyzed substrate specificities using the membrane
fractions derived from vector-transfected COS-7 cells. Seven
4GalT
genes have been identified within the data bases provided by the human
genome project to date (18-29). The membrane fraction derived from
vacant pcDNA3-transfected COS-7 cells (C-MF) was used as a control
for intrinsic
4GalT activities. C-MF had weak
4GalT activity; the
specific activities using biGP, 6SGN, agL2L2, Xyl-O-pNP, GL,
core2-O-pNP, GlcCer, and 6S-core2-O-pNP as
acceptor substrates were 0.20, 0.34, 0.43, 0.20, 0.12, 0.47, 0, and
0.16 nmol/min/mg of protein, respectively. To calculate the amount of
exogenous
4GalT activities, values derived from C-MF were subtracted
from those of the apparent enzymatic activities obtained under the same conditions.
4GalT-I, -II, -III, -IV, -V, -VI, and
-VII were 6.6 (using biGP as an acceptor), 3.4 (core2-O-pNP), 2.6 (core2-O-pNP), 1.5 (6SGN), 1.5 (GlcCer), 0.32 (GlcCer), and 3.7 nmol/min/mg of protein
(Xyl-O-pNP), respectively. The relative activities of the
seven
4GalTs for eight acceptor substrates are summarized in Table
IV. Among the seven
4GalTs, only
4GalT-IV recognized 6SGN as a good acceptor in comparison with biGP
and GL, and this profile of
4GalT-IV was equal to the substrate
specificity of human colonic 6SGN-specific
4GalT previously reported
by us (12). The linkage position of [3H]Gal in
[3H]Gal
1
agL2L2 and
([3H]Gal
1
)6S-core2-O-pNP synthesized by
4GalT-IV was confirmed to be the C-4 of GlcNAc by RCA-I-agarose
affinity chromatography (Fig. 4), which
binds to Gal
1
4GlcNAc (49). [3H]Gal
1
agL2L2
(Fig. 4B, solid line) and
([3H]Gal
1
)6S-core2-O-pNP (Fig.
4C, solid line) bound to the column weaker than
([3H]Gal
1
)core2-O-pNP (Fig.
4A). To examine whether the weak binding is caused by the
presence of 6-O-sulfate, the sulfated products were treated
with mild methanolysis (0.05 N HCl/MeOH, 25 °C, 4 h) (50) and applied onto the lectin column. The digests bound to the
column and were eluted with 10 mM lactose (Fig. 4,
B and C, dotted lines), but the
digests flowed through the column by digestion with
Gal
1
4GlcNAc-specific diplococcal
-galactosidase (data not
shown), showing that [3H]Gal is attached to the C-4 of
GlcNAc in the sulfated products. 6SGN was a poor substrate at best for
the other
4GalTs (Table IV) even at the lower concentrations, 0.1 or
0.2 mM (data not shown), excluding the possibility that the
absence or low level of activity was due to the inhibitory effect of a
high concentration of 6SGN; such as has been found in several
4GalTs
(34, 51). These results indicated that
4GalT-IV is the only
6SGN-specific enzyme among the seven
4GalTs, consistent with the
amino acid sequence of the purified porcine 6SGN-specific
4GalT as
described above.
Substrate specificities of seven 4GalTs
View larger version (14K):
[in a new window]
Fig. 4.
RCA-I-agarose column chromatography of the
reaction products,
[3H]Gal core2-O-pNP
(A), [3H]Gal
agL2L2
(B, solid line), and
([3H]Gal
)6S-core2-O-pNP
(C, solid line). The reaction
products purified by paper electrophoresis and paper chromatography
were applied on a RCA-I-agarose column and eluted as described under
"Experimental Procedures." [3H]Gal
agL2L2 and
([3H]Gal
)6S-core2-O-pNP were treated with
mild methanolysis and applied on a RCA-I column (dotted
lines in B and C, respectively).
V0, void volume.
4GalT-III prefers O-linked type core2-O-pNP to
N-linked type biGP, whereas
4GalT-I and -II recognize
both biGP and core2-O-pNP as good acceptors. It has been
shown that
4GalT-V acts on core2 (35) and a specific branching
structure of N-linked glycans (36). As shown in Table IV,
4GalT-V could act on GlcCer, similar to
4GalT-VI. Recently, Lee
et al. (52) reported that
4GalT-VI-deficient CHO cells
have an ability to synthesize lactosylceramides, and they suggested the
existence of a lactosylceramide synthase other than
4GalT-VI.
4GalT-V may be the second lactosylceramide synthase.
4GalT-IV recognizes several 6SGN-containing oligosaccharides as good
acceptors (Table V). KS-related
oligosaccharides, agL2L2 and agL2L4, were good substrates for
4GalT-IV. AgL2L2 was a poor substrate at best for the other
4GalTs (Table IV). KS is also a substrate for
4GalT-IV with a
specific activity of 0.045 nmol/min/mg of protein at 1 mM
KS. To expose GlcNAc residues at the non-reducing termini of KS chains,
we treated KS by mild acid hydrolysis to remove sialic acid and
Streptococcus 6646K
-galactosidase digestion. The
enzymatic activity for the asialo-agalacto-KS was 3.3-fold higher than
that for intact KS. This result suggests that
4GalT-IV can also act
on KS long chains. Because agLST-b was a poor substrate for
4GalT-IV, sialic acid cannot replace sulfate at the C-6 of GlcNAc.
This substrate selectivity is the same as for human colonic 6SGN-specific
4GalT (12).
4GalT-IV also efficiently acts on 6S-biGP and 6S-core2-O-pNP; the 6SGN moiety is present in
N-linked and O-linked glycans in various
glycoproteins (1). The 6-sulfosialyl-Lewis X on core2 glycans, which is
synthesized from 6S-core2 by
1
4-galactosylation,
2
3-sialylation, and
1
3-fucosylation, functions as an
L-selectin ligand moiety in the early step of lymphocyte homing in
lymph nodes (14-17).
4GalT-IV is the only enzyme recognizing
6S-core2-O-pNP as a good substrate among the seven
4GalTs
(Table IV), suggesting that
4GalT-IV is involved in the synthesis of
6-sulfosialyl-Lewis X.
Relative values of the enzymatic activities of 4GalT-IV for various
acceptor substrates
4GalT-IV for several glycans with or
without 6-O-sulfation at GlcNAc are summarized in Table VI. AgL2L4 and 6S-core2-O-pNP
had an inhibitory effect on
4GalT-IV activities at high
concentrations (Fig. 5), so that kinetic
constants were calculated using only the data obtained with lower
concentrations of these substrates. The Km value for
6SGN was lower than that for GlcNAc, and the
Vmax/Km value for 6SGN was
1900-fold higher than that for GlcNAc. Similarly, the
Vmax/Km values for 6S-biGP
and 6S-core2-O-pNP were much higher than those for biGP and
core2-O-pNP, respectively. These results indicate that
6-O-sulfation of GlcNAc residues is important for efficient catalytic activity of
4GalT-IV.
Kinetic analysis of 4GalT-IV for several oligosaccharides containing
6-O-sulfated or non-substituted GlcNAc
View larger version (17K):
[in a new window]
Fig. 5.
Enzymatic activities at various
concentrations of acceptor substrates. The activities were
measured as under "Experimental Procedures." Acceptor substrates
used were 6SGN ( ), core2-O-pNP (
),
6S-core2-O-pNP (
), and agL2L4 (
).
4GalT-IV in Various Human Tissues--
Lo
et al. (24) and Schwientek et al. (26) reported
the expression profile of
4GalT-IV in human adult and fetal tissues, but they had not examined its expression in colon or lymph node, where
6SGN-specific
4GalT should be expressed. To clarify the expression
pattern more extensively, we analyzed it using commercial Northern blot
membranes. As shown in Fig. 6, a 2.4-kb
transcript of
4GalT-IV was expressed ubiquitously. Relatively high
expression levels of the enzyme were observed in kidney, placenta,
lymph node, prostate, stomach, thyroid, tongue, and trachea. Recently, it has been shown that the 6-sulfosialyl-Lewis X determinant is present
in colonic mucosa (53); in colon,
4GalT-IV was also moderately
expressed (Fig. 7).
View larger version (29K):
[in a new window]
Fig. 6.
Northern blot analysis of
4GalT-IV transcript. The amount of
poly(A)+ RNA was normalized to the mRNA expression
levels of
-actin. The blots were hybridized with a
32P-labeled cDNA probe specific for human
4GalT-IV.
On the left, the migration position standard is
indicated.
View larger version (21K):
[in a new window]
Fig. 7.
Proposed schemes for biosynthesis of
(A) keratan sulfate and (B)
6-sulfosialyl-Lewis X structure.
4GalT was purified and identified as
4GalT-IV. (ii) The substrate specificities of the seven
4GalTs so
far cloned were investigated, and
4GalT-IV is the only 6SGN-specific
4GalT among them and is involved in the synthesis of several
6-O-sulfated N-acetyllactosamine glycans. (iii)
Porcine
4GalT-IV requires basic lipidous compounds for the catalytic
activity at least in a highly purified state. It has been reported that
sphingosine increases several times the enzymatic activities of
1
3-fucosyltransferase, GM2:
1
3GalT,
GM3:
1
4-N-acetylgalactosaminyltransferase (54), and
glycoprotein sulfotransferase (55) and decreases that of
1
3-glucuronyltransferase (56). However, to our knowledge, this is
the first demonstration that basic lipidous compounds are essential for
the catalytic activity of glycosyltransferases. Sphingosine is an
intermediate of the metabolism of sphingolipids and a precursor for
sphingosine 1-phosphate, a second messenger molecule (reviewed in Refs.
57-59); however, it has been believed that sphingosine is present at
quite low levels in living cells. The molecular aspect of the
requirement is unclear, but one explanation is that in the process of
purification, a certain basic compound, which binds to the enzyme in
the crude extract, is detached from the enzyme protein. We are in the
process of resolving this issue by analyzing biochemically the binding
of recombinant
4GalT-IV to various basic compounds.
1
4GlcNAc. Recently, Akama
et al. (60) showed that GlcNAc6ST-5 (CGn6ST) is involved in
KS synthesis. Fukuta et al. (61) identified a Gal6ST, which
prefers an internal Gal residue in the
di-N-acetyllactosamine chain (62), suggesting that
6-O-sulfation of Gal is a later step in KS synthesis. Among
3GnTs so far cloned,
3GnT-2 has strong enzymatic activity for
p-lacto-N-neohexaose (63), suggesting that the
enzyme may be a candidate involved in the elongation of the KS backbone
structure, although there is no evidence that
3GnT-2 has
3GnT
activity for KS elongation. In this study, we showed that among the
seven
4GalTs, only
4GalT-IV acts on KS-related glycans, agL2L2
and agL2L4. From these results, it is proposed that KS is synthesized
by the sequential actions of four enzymes, GlcNAc6ST-5,
4GalT-IV,
3GnT, and then Gal6ST (Fig. 7A).
4GalT,
2,3-sialyltransferase, and
1,3-fucosyltransferase. It has been shown that in lymph
nodes, this structure is attached on
1,6-branching GlcNAc in
O-linked core2 (14) or on elongated core1 (64) and that the
GlcNAc 6-O-sulfation is performed by GlcNAc6ST-2. However,
Uchimura et al. (10) showed that GlcNAc6ST-1 is responsible
for the synthesis of 6-sulfosialyl-Lewis X. Next, galactosylation may
be catalyzed by
4GalT-IV as indicated from our results that only
4GalT-IV efficiently acts on 6S-core2-O-pNP among the
seven
4GalTs. Ujita et al. (34) reported that among
4GalT-I to -V,
4GalT-IV efficiently acts on non-sulfated
core2-O-pNP. Here we showed that 6S-core2-O-pNP is a better substrate for
4GalT-IV than core2-O-pNP. From
these results, a biosynthetic scheme for 6-sulfosialyl-Lewis X is
proposed as shown in Fig. 7B.
4GalT-IV is extensively
expressed in human tissues as shown in Fig. 6, and there seems to be no
relationship between expression profiles of 6-sulfosialyl-Lewis X and
4GalT-IV. This indicates that
4GalT-IV is not a rate-limiting
enzyme for biosynthesis of 6-sulfosialyl-Lewis X and that the preceding
6-O-sulfation of GlcNAc may be a rate-limiting step. The next subject
of study should be whether 6-sulfosialyl-Lewis X is synthesized in
cultured cells defective in
4GalT-IV expression.
4GalT-IV is expressed in various human tissues including lymph node
as shown in Fig. 6. This result indicates that the
6-O-sulfated N-acetyllactosamine structure occurs
extensively. In fact, this sulfated glycan moiety has been shown to be
present in various glycoproteins including ovomucin (65), glycoproteins
derived from several culture cells (66), thyroglobulin (67), zona pellucida glycoproteins (68, 69), gp120 of human immunodeficiency virus
type 1 (70), respiratory mucins (71, 72), carcinoembryonic antigen
(73), hyosophorin (74), and oviducal mucins (75). 6SGN-specific
4GalT-IV identified in this study may be useful to elucidate the
biological roles of these sulfated moieties.
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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.
¶ Present address: High Throughput Factory, RIKEN Harima Institute, 1-1-1, Kouto, Mikazukichou, Sayo-gun, Hyogo 679-5148, Japan.
To whom correspondence should be addressed: Dept. of
Biochemistry, Sasaki Institute, Kanda-Surugadai 2-2, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: 81-3-3294-3286; Fax: 81-3-3294-2656; E-mail: yamashita@sasaki.or.jp.
Published, JBC Papers in Press, January 2, 2003, DOI 10/.1074/jbc.M211480200
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ABBREVIATIONS |
---|
The abbreviations used are:
GlcNAc6ST, GlcNAc
6-O-sulfotransferase;
KS, keratan sulfate;
agL2L2, SO6GlcNAc
1
3Gal
1
4(SO
6)GlcNAc;
agL2L4, SO
6GlcNAc
1
3(SO
6)Gal
1
4(SO
6)GlcNAc;
agLST-b, Neu5Ac
2
6GlcNAc
1
3Gal
1
4Glc;
4GalT,
1,4-galactosyltransferase;
biGP, GlcNAc
1
2Man
1
3(GlcNAc
1
2Man
1
6)Man
1
4GlcNAc
1
4GlcNAc;
core 2, Gal
1
3(GlcNAc
1
6)GalNAc
1
;
core 3, GlcNAc
1
3GalNAc
1
;
GL, GlcNAc
1
3Gal
1
4Glc;
GlcCer, glucosylceramide;
L2L2, Gal
1
4(SO
6)GlcNAc
1
3Gal
1
4(SO
6)GlcNAc;
L2L4, Gal
1
4(SO
6)GlcNAc
1
3(SO
6)Gal
1
4(SO
6)GlcNAc;
LST-b, Gal
1
3(Neu5Ac
2
6)GlcNAc
1
3Gal
1
4Glc;
Man, mannose;
Neu5Ac, N-acetylneuraminic acid;
pNP, p-nitrophenyl;
RCA-I, R. communis agglutinin-I;
6S-biGP, SO
6GlcNAc
1
2Man
1
3(Man
1
6)Man
1
4GlcNAc
1
4(Fuc
1
6)GlcNAc;
6S-core2, Gal
1
3(SO
6GlcNAc
1
6)GalNAc
1
;
6SGN, GlcNAc 6-O-sulfate;
PAPS, adenosine 3'-phosphate
5'-phosphosulfate;
C-MF, the membrane fraction derived from
pcDNA3-transfected COS-7 cells.
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