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J. Biol. Chem., Vol. 277, Issue 1, 169-177, January 4, 2002
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From the
Received for publication, September 19, 2001, and in revised form, October 22, 2001
The O-linked oligosaccharides
(O-glycans) in mammalian glycoproteins are classified
according to their core structures. Among the most common is the core 1 disaccharide structure consisting of
Gal The addition of O-glycans to proteins is a common
post-translational modification on many secreted and membrane-bound
glycoproteins (1-4). The mucin-type O-glycans, which
contain a core GalNAc in The core 1 disaccharide is synthesized by the action of an enzyme
activity designated the core 1 UDP-Gal:GalNAc- Core 1 As a step toward the cloning of the gene(s) encoding the core 1 Materials
GalNAc Core 1 The assay for core 1 UDP-GlcNAc:Gal Core 2 UDP-GlcNAc:GalNAc- Core 3 UDP-Gal:GlcNAc GDP-Fucose:GlcNAc CMP-NeuAc:GalNAc- Preparation of Asialo-BSM UltraLinkTM
Bovine submaxillary mucin (BSM) (100 mg) was dissolved in 20 ml
of 0.1 M MOPS and 0.6 M sodium citrate (pH
7.5), and coupled to 0.6 g of UltraLinkTM beads
overnight at 4 °C, followed by blocking with 3 M
ethanolamine (1 h, 20 °C). Following washing with 1 M
NaCl and equilibration with water, the coupled BSM (~3 mg/ml) was
desialylated by incubation with an equal volume of 2 N HCl
at 100 °C for 90 s. The beads were then chilled in ice,
neutralized with NaOH, and equilibrated with buffer 50 mM
Tris-HCl (pH 7.0), 150 mM NaCl, and 20 mM
MnCl2.
Purification of Core 1 Step 1: Homogenization, Subcellular Fractionation, Isolation of
Membranes, and Solubilization--
Rat liver (500 g) was freshly
obtained and washed with cold 150 mM NaCl in 25 mM Tris-HCl (pH 7.4) and homogenized with 2,000 ml of 25 mM Tris-HCl (pH 7.5) and 0.25 M sucrose
containing 1 mM dithiothreitol (DTT), 1 mM
PMSF, 2 µg/ml leupeptin, 1 mM benzamidine, and 0.7 µg/ml pepstatin A in a Waring commercial blender. In all steps of the
purification, protein was determined by absorbance at 280 nm or by the
BCA assay (Pierce) according to the manufacturer's protocol using
bovine serum albumin as a standard. The homogenate was centrifuged at
1,000 × g for 30 min, and the supernatant was decanted
and centrifuged at 100,000 × g for 60 min. The pellets were suspended in 5 volumes of 50 mM Tris-HCl (pH 9.0) and
0.25 M sucrose containing 2% Triton X-100, 1 mM PMSF, 2 µg/ml leupeptin, 1 mM benzamidine,
and 0.7 µg/ml pepstatin A and sonicated for 10 s four times
in an ice bath. These sonicated membranes were solubilized on
ice for 1 h and then centrifuged at 100,000 × g for 60 min. The supernatant was collected (~1,000 ml) and the pH was
adjusted to 6.5 using 1 M MES.
Step 2: SP-Sepharose Chromatography--
The extracted membrane
preparation was applied to a column of SP-Sepharose FF (6 × 20 cm; Amersham Biosciences), previously equilibrated with 25 mM MES (pH 6.5), 0.1% Triton, 5 mM
MnCl2, containing 1 mM PMSF, 2 µg/ml
leupeptin, 1 mM benzamidine, and 0.7 µg/ml pepstatin A. By using the same buffer, the column was washed, and the core 1 Step 3: Asialo-BSM-UltraLinkTM
Chromatography--
The eluate from the SP-Sepharose column was
dialyzed and concentrated down to 50 ml using an Omicon concentrator.
The concentrated sample was loaded onto a column of
asialo-BSM-UltralinkTM (1.2 × 5 cm) equilibrated with
25 mM Tris-HCl (pH 7.0), 0.01% Triton X-100, 20 mM MnCl2, 150 mM NaCl, containing 1 mM PMSF, 2 µg/ml leupeptin, 1 mM benzamidine,
and 0.7 µg/ml pepstatin A. After washing the column in the same
buffer, the core 1 Step 4: Superose 12 Chromatography--
The core 1 SDS-PAGE
SDS-PAGE was performed in Tris glycine buffer on
precast acrylamide gradient gels (NOVEX) as described by the
manufacturer. Proteins were visualized by silver staining (27). Gels
were scanned on a UMAX Astra 2100U Scanner. Stained protein bands were identified and quantified by scanning using the NIH Image program (rsb.info.nih.gov/nih-image/).
Generation of 3H-Galactosylated Asialo-BSM
To effect desialylation, bovine submaxillary mucin (BSM) (5 mg)
was dissolved in 0.80 ml of 1 N HCl and incubated at
100 °C for 90 s and then cooled in ice. The reaction mixture
was neutralized with NaOH and desalted on a G-25 Sephadex, PD-10 column
(Amersham Biosciences) equilibrated with water. The void volume was
collected and concentrated to 1 ml in a Centriprep-30 and designated
asialo-BSM. This acceptor was used directly for the core 1 Identification of the Enzymatic Reaction Product
The product using asialo-BSM as an acceptor was treated with
O-glycosidase in a total reaction volume of 100 µl by
adding 25 mM Tris-HCl (pH 7.4), 150 mM NaCl,
and O-glycosidase (~12.5 milliunits) and a drop of
toluene. This reaction mixture was incubated at 37 °C for 48 h.
The whole sample was then applied to a column of Sephadex G-25
(Superfine) and eluted with H2O. Fractions (100 µl) were
collected, and the radioactive peak was pooled and passed through 1-ml
mixed bed resin (Bio-Rad), and the column was washed with 5 ml of
H2O. The product was collected and dried in
vacuo. The dried core 1 N-terminal and Internal Amino Acid Sequence of Core 1 Purified core 1 Characterization of the Core 1 Rat liver was chosen as a tissue source for the purification of
core 1 Rat Liver Core 1 To investigate whether rat liver core 1 Purification of Core 1 The protocol developed for purification of the core 1
Purification, Characterization, and Subunit Structure of
Rat Core 1
1,3-Galactosyltransferase*
§,
, and§**
W. K. Warren Medical Research Institute,
the Departments of ** Medicine and ¶ Biochemistry and
Molecular Biology, and the § Oklahoma Center for Medical
Glycobiology, University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73104
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
3GalNAc
1Ser/Thr, which is also the precursor for
many extended O-glycan structures. The key enzyme for
biosynthesis of core 1 O-glycan from the precursor
GalNAc--Ser/Thr is UDP-Gal:GalNAc--Ser/Thr 3-galactosyltransferase (core1 3-Gal-T). Core 1 3-Gal-T
activity, which requires Mn2+, was solubilized from rat
liver membranes and purified 71,034-fold to apparent homogeneity
(>90% purity) in 5.7% yield by ion exchange chromatography on
SP-Sepharose, affinity chromatography on immobilized asialo-bovine
submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two
peaks of core 1 3-Gal-T activity were identified in the final step
on Superose 12. One peak of activity contained protein bands on
non-reducing SDS-PAGE of ~84- and ~86-kDa disulfide-linked dimers,
whereas the second peak of activity contained monomers of ~43 kDa.
Reducing SDS-PAGE of these proteins gave ~42- and ~43-kDa monomers.
Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same
novel N-terminal sequence. The purified enzyme, which is remarkably
stable, has an apparent Km for UDP-Gal of 630 µM and an apparent Vmax of 206 µmol/mg/h protein using GalNAc1-O-phenyl as the
acceptor. The reaction product was generated using asialo-bovine
submaxillary mucin as an acceptor; treatment with
O-glycosidase generated the expected disaccharide Gal13GalNAc. These studies demonstrate that activity of the core
1 1,3-Gal-T from rat liver is contained within a single, novel,
disulfide-bonded, dimeric enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycosidic linkage to Ser or Thr, are
synthesized in the Golgi apparatus by the sequential action of
glycosyltransferases that transfer individual sugars from nucleotide
sugar donors (5, 6). Whereas all mucin-type O-glycans
contain the common GalNAc1-Ser/Thr linkage, at least eight different
O-glycan core structures have been described that differ in
the types of sugars and their linkage. Two of the most common
mucin-type O-glycans are based on the core 1 and core 2 structures. Core 1 has the disaccharide structure Gal13GalNAc1R and core 2 has the trisaccharide structure
Gal13(GlcNAc16)GalNAc1R. In addition to serving as an
precursor to the core 2 structure, the core 1 can also be modified by
sialylation and fucosylation to generate a wide range of other
structures (3). Alternatively, the unmodified core 1 disaccharide is
recognized as a cancer-associated antigen (T- or Thomsen-Friedenreich
antigen) (1). In addition, the core 1 O-glycan may be
directly extended by addition of GlcNAc to form an extended core 1 structure (7-9).
-R
1,3-galactosyltransferase (core 1 3-Gal-T, EC 2.4.1.122), where R
is Ser/Thr through direct transfer to the acceptor -linked GalNAc.
Thus, the core 1 3-Gal-T may be considered to play a pivotal and
decisive role in mucin and glycoprotein biosynthesis. However, although
the core 1 3-Gal-T activity is present in most mammalian tissue, the
enzyme has not been purified to homogeneity, and the possible diversity
of different enzymes with this activity is unknown (10, 11). The core 1 O-glycan structure is required for the action of the core 1 1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-transferase), which allows synthesis of core 2-based
O-glycans of the sequence Gal14GlcNAc16
(Gal13)GalNAc1Ser/Thr (6, 12-15). The sialylated and
fucosylated derivative of a core 2-based O-glycan is
required for recognition of the leukocyte mucin-type ligand PSGL-1 by
P-selectin (9, 16). The inability to express the core 1 3-Gal-T
results in expression of truncated O-glycans, such as the
unsubstituted GalNAc1Ser/Thr (Tn antigen) or the sialylated
derivative NeuAc26GalNAc1Ser/Thr (sialyl Tn antigen). Expression of the Tn and sialyl-Tn antigens has been associated with
tumor progression (17, 18). Several tumor cell lines, including Jurkat,
a human T leukemic cell line (19), and LSC cells, a human colon
carcinoma cell line (20), express sialyl Tn antigen, which appears to
result from a lack of the core 1 3-Gal-T activity.
3-Gal-T may play an important role in other diseases, such as
Tn syndrome and IgA nephropathy. It has been proposed that exposure of
the Tn antigen in erythrocytes (Tn syndrome) (1) and in the hinge
region of IgA (IgA nephropathy) (21-23) results in immune recognition
and attack by a naturally circulating anti-Tn. These diseases may be
caused by deficiency of core 1 3-Gal-T (24-26), but the exact
pathogenesis is unknown because the core 1 3-Gal-T has not been
purified, and the gene(s) encoding core 1 3-Gal-T has not been cloned.
3-Gal-T, in this paper we describe the ~71,000-fold purification of core 1 3-Gal-T from rat liver to apparent homogeneity and the
characterization of the purified protein. N-terminal sequencing demonstrates that a single polypeptide was purified that exists as an
active monomer and as a disulfide-bonded dimer. This is the first
isolation of a core 1 3-Gal-T.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-O-phenyl, Gal1,3GalNAc--phenyl
UDP-Gal, DTT, ATP, UDP, PMSF, benzamidine, leupeptin, pepstatin A,
Triton X-100, bovine submaxillary mucin (type I-S), and the
disaccharides Gal1-3GalNAc, Gal1-4GlcNAc, Gal1-6GalNAc,
Gal1-6Gal, and Gal1-3GlcNAc were obtained from Sigma. MES,
MOPS, Tris, methanol, 1-butanol, and scintiverse-BD were obtained from
Fisher. UDP-[6-3H]Gal (40-60 Ci/mmol) was obtained from
American Radiolabeled Chemicals, Inc. The BCA protein assay kit and the
UltraLinkTM Bio-support Medium were obtained from Pierce.
Peptide N-glycosidase F (N-glycanase) and
O-glycosidase were obtained from Roche Molecular Biochemicals. Sep-Pak C18 cartridges was obtained from
Waters Associates. Precast SDS-PAGE gels were obtained from
Invitrogen/NOVEX. Mixed bed resin AG 501-X8 was obtained from Bio-Rad.
The synthetic glycopeptide 4-GP-1 with the sequence
Glu-Tyr-Glu-Tyr-Leu-Asp-Tyr-Asp-Phe-Leu-Pro-Glu-(GalNAc1-O-Thr)-Glu-Pro-Pro-Glu-Met and having the GalNAc-substituted Thr, as indicated, was
synthesized as described in Leppänen et al. (16).
Triose (GlcNAc1,3Gal1,4Glc) was prepared by treatment of the
tetrasaccharide LNneoT (Gal1,4GlcNAc1,3Gal1,4Glc) with jack
bean -galactosidase and subsequent purification of the trisaccharide
(triose) from the released galactose by gel filtration on a column
(3 × 200 cm) of Bio-Gel P-2 equilibrated in water.
3-Gal-T Assay
3-Gal-T contained 100 mM
MES, pH 6.8, 0.2% Triton X-100, 20 mM MnCl2, 1 mM GalNAc1-O-phenyl, 0.4 mM UDP-[3H]Gal (100,000-150,000 dpm/nmol), 2 mM
ATP, and 5-25 µl of enzyme in a total volume of 50 µl. Mixtures
were incubated at 37 °C for 30-60 min and stopped by adding 950 µl of cold H2O. The mixtures were loaded onto 500-mg
Sep-Pak C18 cartridges, previously activated with 2 ml of
ethanol and equilibrated with 10 ml of water. Following application of
the diluted reaction mixture, the columns were washed with 15 ml of
water, eluted with 2 ml of 1-butanol, and radioactivity determined by
liquid scintillation counting in 10 ml of Scintiverse-BD. A unit of
activity is defined as that amount of enzyme transferring 1 nmol of Gal
from UDP-Gal to the acceptor GalNAc1-O-phenyl per h at
37 °C. When the synthetic glycopeptide 4-GP-1 was used as an
acceptor, the assays were conducted as above substituting the 4-GP-1
for GalNAc1-O-phenyl, except that methanol, rather than
1-butanol, was used for elution of product glycopeptides from the
Sep-Pak C18 cartridges.
1,3GalNAc-R(GlcNAc to GalNAc) 6-GlcNAc
Transferase (Core 2 6-Gn-T) Assay
6-Gn-T activities were measured in a total volume of
50 µl containing 0.1 M MES (pH 6.5), 0.125% Triton
X-100, 10 mM ATP, 0.125 M GlcNAc, 2 mM Gal1,3GalNAc--phenyl, 1 mM
UDP-[3H]GlcNAc (10 cpm/pmol), and 20 µl of enzyme
sample. The reactions were incubated at 37 °C for 1.5 h, and
the product was estimated by Sep-Pak C18 column, as
described above for the core 1 3-Gal-T assay.
-R 3-GlcNAc Transferase (Core 3 3-Gn-T) Assay
3-Gn-T activities were measured in a total volume of
50 µl containing 0.1 M MES (pH 6.5), 15 mM
MnCl2, 0.2% Triton X-100, 10 mM ATP, 4 mM GalNAc--phenyl, 1 mM
UDP-[3H]GlcNAc (10 cpm/pmol), and 20 µl of enzyme
samples. The reactions were incubated at 37 °C for 1.5 h, and
the product was estimated by Sep-Pak C18 column, as
described above for the core 1 3-Gal-T assay.
1,4 Galactosyltransferase (4-Gal-T)
Assay
4-Gal-T activities were measured in a total volume of 50 µl
containing 0.1 M Tris-HCl (pH 7.2), 15 mM
MnCl2, 0.2% Triton X-100, 10 mM ATP, 5 mM GlcNAc1,3Gal1,4Glc (triose), 0.4 mM
UDP-[3H]Gal (20 cpm/pmol), and 20 µl of enzyme
samples. The reactions were incubated at 37 °C for 1.5 h, and
the product was estimated following chromatography on QAE-Sephadex A-25
(1 ml) column and measurement of the unbound radioactivity.
1,3-Fuc-transferases (1,3-Fuc-T)
Assay
1,3-Fuc-T activities were measured in a total volume of 50 µl containing 0.1 M MES (pH 6.5), 15 mM
MnCl2, 0.2% Triton X-100, 10 mM ATP, 10 mM Gal1,4GlcNAc1,3Gal1,4Glc (LNnT), 0.4 mM GDP-[3H]fucose (20 cpm/pmol), and 20 µl
of enzyme samples. The reactions were incubated at 37 °C for
1.5 h, and the product was estimated following chromatography on
QAE-Sephadex A-25 (1 ml) column and measurement of the unbound radioactivity.
-R 2,6-Sialyltransferase (2,6-ST)
Assay
2,6-ST activities were measured in a total volume of 100 µl
containing 0.1 M MES (pH 6.5), 5 mM EDTA, 0.2%
Triton X-100, 10 mM ATP, 150 µg of asialo-BSM, 1 mM CMP-[3H]NeuAc (10 cpm/pmol), and 20 µl
of enzyme samples. The reactions were incubated at 37 °C for
1.5 h. The reaction mixture was diluted with 400 µl of
H2O and 500 µl of ice-cold 10% trichloroacetic acid. The
mixtures were kept on ice for 30 min and centrifuged at 14,000 × g for 10 min. The pellets were washed with 1 ml of cold
0.01% HCl in acetone 3 times and dried on a speed-vac concentrator. The dried residues were dissolved in 0.5 ml of 0.2 N KOH
and mixed with 5 ml of ScintiVerse-BD and counted.
3-Gal-T
3-Gal-T was eluted in one step by application of 1 M
NaCl in equilibrating buffer.
3-Gal-T was eluted with 1 M NaCl in
the buffer lacking Mn2+. Fractions (1 ml) were collected,
and activity of core 1 3-Gal-T was assayed. Fractions containing the
core 1 3-Gal-T activity were pooled.
3-Gal-T
from Asialo-BSM UltraLinkTM was concentrated down to 200 µl, using Centriprep 30 and Centricon 30 (Omicon), and loaded on
Superose 12 (1.0 × 35 cm; Amersham Biosciences), which was
pre-equilibrated with 25 mM Tris-HCl (pH 7.2), 0.005% Triton X-100, and 150 mM NaCl. Core 1 3-Gal-T was eluted
with the same buffer, and fractions (0.25 ml) were collected and
assayed for activity. Two peaks of activity were recovered from the
Superose 12 step, and those fractions containing peak levels of
activity were pooled. This pooled material represented the purified
core 1 3-Gal-T.
3-Gal-T
reaction. The reaction mixture in 100 µl total volume contained
asialo-BSM (160 µg), UDP-[3H]Gal (0.4 mM)
(100,000 dpm total), Triton X-100 (0.2%), ATP (2 mM),
MnCl2 (20 mM), and purified core 1 3-Gal-T
(20 units of activity). The reaction mixture was incubated at 37 °C
for 2 h and then applied to a column of Sephadex G-25 (0.5 × 6 cm; Superfine; Amersham Biosciences) and eluted with water. Fractions
(~100 µl) were collected and pooled, and the first radioactive peak
was concentrated to 50 µl.
3-Gal-T product was dissolved in 25 µl of H2O and mixed with 15 µl containing 2.5 µl of 1 mg/ml authentic Gal1-3GalNAc standard. The mixed sample was
analyzed on a Dionex HPAEC (Dionex Corp.) equipped with a PA-1 column
(4 × 250 mm) and pulsed amperometric detector. Fractions (0.33 ml) were collected, and the elution of the radioactive product was
monitored by liquid scintillation counting. The elution positions of
the unlabeled standards Gal1-3GalNAc, Gal1-4GlcNAc,
Gal1-6GalNAc, Gal1-6Gal, and Gal1-3GlcNAc was directly
determined for each individual glycan using pulsed amperometric detection.
3-Gal-T
3-Gal-T (~10 µg) was analyzed on a
4-12% SDS-PAGE (NOVEX) under non-reducing conditions and then
transferred to a polyvinylidene difluoride membrane (Applied
Biosystems, Inc.). After staining with Coomassie Brilliant Blue, the
top two bands of 84 and 86 kDa (Fig. 3), respectively, were excised and
the N-terminal sequence determined by Edman degradation (Applied
Biosystems, Inc. protein sequencer). Similarly, the protein band at
42/43 kDa in non-reducing SDS-PAGE (Fig. 3) was excised and the
N-terminal sequence determined by Edman degradation. To obtain internal
peptide sequence, purified enzyme (~5 µg) was resolved by SDS-PAGE
(4-12%) under non-reducing conditions, and the top two bands of 84/86 kDa were excised from the gel. These proteins were digested in situ with trypsin, and tryptic peptides were isolated by
reverse-phase high pressure liquid chromatography. The size of one
purified peptide was determined by matrix-assisted
laser-desorption/ionization-mass spectrometry (MALDI-MS) and confirmed
by direct sequencing by Edman degradation at the Yale University
Protein Sequencing Facility.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-Gal-T Assay
3-Gal-T, because our preliminary experiments demonstrated that rat liver homogenates contain relatively high core 1 3-Gal-T activity compared with other organs. Core 1 3-Gal-T was assayed by
monitoring the transfer of [3H]Gal from
UDP-[3H]Gal to the synthetic acceptor
GalNAc1-O-phenyl. The product was isolated by Sep-Pac
chromatography by exploiting the hydrophobicity of the acceptor and
product. Core 1 3-Gal-T activity in the solubilized rat liver
membrane fraction was linear with respect to added protein from 50 to
400 µg (Fig. 1A). Activity
was also linear with respect to time between 20 and 90 min (Fig.
1B). The core 1 3-Gal-T activity demonstrated an absolute
requirement for divalent cation, with optimal activity being observed
with 10 mM Mn2+ (Fig. 1C).
View larger version (13K):
[in a new window]
Fig. 1.
Enzymatic characterization of Core 1 3-Gal-T in rat liver extract. Core 1 3-Gal-T activity was determined as described under "Experimental
Procedures" in extracts of rat liver, as a function of protein
concentration (A), time of incubation (B), and
Mn2+ concentration (C).
3-Gal-T Is a Membrane-associated Protein
3-Gal-T was
membrane-associated, the post-nuclear supernatant fraction of rat liver homogenates was centrifuged at 106,000 × g for 60 min.
Greater than 97% of the enzyme activity was pelleted under these
conditions (data not shown). Detergent (2% Triton X-100), but not 1 M NaCl, caused the quantitative release of core 1 3-Gal-T activity from the membrane into the 106,000 × g supernatant, indicating the enzyme is membrane-associated.
3-Gal-T
3-Gal-T
from rat liver resulted in a 71,034-fold purification, as summarized in
Table I and as discussed below.
Rat liver core 1 3-galactosyltransferase purification
Steps 1 and 2: Homogenization, Subcellular Fractionation, and
Isolation of Membranes and Solubilization--
Rat livers were
homogenized, and the homogenate was centrifuged to obtain a
post-nuclear supernatant. The membranes in the post-nuclear supernatant
were collected by centrifugation at 100,000 × g for 60 min, and the supernatant was decanted and discarded. This membrane
preparation was solubilized in buffer containing 2.0% Triton X-100.
Enzyme assays demonstrated that this solubilized membrane preparation
contained core 1 3-Gal-T activity at 9.8 nmol/h/mg protein, and
<10% of the activity remained in the unsolubilized material.
Step 3: SP-Sepharose FF Chromatography--
The solubilized
membrane proteins from step 2 were chromatographed on a column of the
anion exchanger SP-Sepharose. Elution of bound proteins was carried out
in a single step by application of 1 M NaCl in buffer (data
not shown). With this step the core 1 3-Gal-T was purified
~3-4-fold with 31.3% recovery. Although the degree of purification
and recovery was not high, this procedure was necessary to separate
contaminating proteins, including some glycosidases, from the core 1 3-Gal-T activity.
Step 4: Asialo-BSM-UltraLinkTM
Chromatography--
Bovine submaxillary mucin contains a variety of
short core 1 type O-glycans including sialyl Tn (28).
Desialylation exposes the Tn antigen, thus making asialo-BSM an
excellent acceptor substrate for the core 1 3-Gal-T. During the
course of trying different affinity approaches, we discovered that core
1 3-Gal-T bound tightly to a column of
asialo-BSM-UltralinkTM in the presence of Mn2+.
The enzyme was eluted as a broad peak from this column with using
buffer containing 1 M NaCl and lacking Mn2+
(Fig. 2A). This affinity step
was the most important step for purification, resulting in an increase
of specific activity to 162,333 from 33.8 nmol/hr/mg with a 4,802-fold
purification.
|
Step 5: Superose 12 Chromatography--
The core 1 3-Gal-T from
the asialo-BSM affinity column was then further purified by gel
filtration chromatography on Superose 12 (Fig. 2B). Core 1 3-Gal-T was recovered in a broad peak with ~65% recovery from
this column. This 5-step protocol resulted in a 71,034-fold
purification of the core 1 3-Gal-T from rat liver homogenates, with
a 5.7% yield and a total of 41 µg of total protein at ~15 µg/ml
(Table I).
SDS-PAGE
The core 1 3-Gal-T activity from Superose 12 was analyzed by
SDS-PAGE under non-reducing conditions, and the protein was detected by
silver staining (Fig. 2C). The activity of the core 1 3-Gal-T from the Superose 12 column was associated with three major
protein bands of ~84 and 86 kDa (fractions 32-40) and ~43 kDa
(e.g. fraction 50), which were also apparent in the starting material (Fig. 2C). In multiple analyses we found that
activity of core 1 3-Gal-T consistently correlated with elution of
these major protein peaks from Superose 12 (Fig. 2, B
and C). The fractions 32-40 and 49-51 from the Superose 12 column were pooled, and these represent the purified rat liver core 1 3-Gal-T. The analysis of the purity of this pooled material by
SDS-PAGE is below.
These data suggested that the core 1 3-Gal-T occurs as an ~43-kDa
monomer and an 84/86-kDa dimer. To define whether the 84/86 kDa was a
disulfide-bonded dimer of the ~43-kDa monomers, the purified core 1 3-Gal-T was analyzed in reducing versus non-reducing SDS-PAGE. The results in Fig. 3
(top) show that in non-reducing SDS-PAGE the major material
is a doublet of 84/86 kDa with minor bands at 42/43 kDa. A scan of the
material in the non-reduced purified core 1 3-Gal-T is shown in Fig.
3 (bottom) and reveals that >90% of detectable protein is
contained within the doublet of 84/86 kDa and the minor bands at 42/43
kDa. However, in reducing SDS-PAGE the major material occurs as a close
doublet of the 42/43-kDa proteins (Fig. 3, top). These
results indicate that the 84/86-kDa material represents a
disulfide-bonded dimeric form of the core 1 3-Gal-T, which upon
reduction behaves as a doublet of 42/43-kDa proteins. This
interpretation is confirmed by direct protein sequencing, as described
below in more detail.
|
To assess further purity of the core 1 3-Gal-T, we assayed the
purified enzyme for the presence of contaminating glycosyltransferases that might co-purify with the enzyme. We also tested the initial rat
liver homogenate as a control. The enzymes tested included the
UDP-GlcNAc:Gal1,3GalNAc-R(GlcNAc to GalNAc) 6-GlcNAc-transferase (core 2 6-Gn-T), UDP-GlcNAc:GalNAc--R 3-GlcNAc-transferase (core 3 3-Gn-T), UDP-Gal:GlcNAc 1,4-galactosyltransferase
(4-Gal-T), GDP-fucose:GlcNAc 1,3-Fuc-transferases (1,3-Fuc-T),
and CMP-NeuAc:GalNAc--R 2,6-sialyltransferase (2,6-ST). Each
enzyme was assayed with specific acceptors and radiolabeled sugar
nucleotide donors (Table II), and the
products were isolated and identified as described under
"Experimental Procedures". All enzyme activities, except for the
core 3 3-Gn-T, were detected in the rat liver homogenate. However,
the purified core 1 3-Gal-T did not contain detectable levels of any
other enzyme assayed (Table II). These results are consistent with
SDS-PAGE analyses and confirm that the core 1 3-Gal-T from rat liver
is highly purified.
|
We considered the possibility that the different sizes observed for the
purified core 1 3-Gal-T could result of differential glycosylation.
However, the apparent molecular weight of the purified core 1 3-Gal-T was not directly altered by treatment with peptide N-glycosidase F (N-glycanase) to remove potential
N-glycans (data not shown). Subsequent cloning of the
cDNA encoding this human enzyme and expression of the recombinant
purified core 1 3-Gal-T, as reported in the accompanying manuscript
(61), confirms that the human purified core 1 3-Gal-T lacks
N-glycans, because it lacks any potential
N-glycosylation sequons. That study also suggests that the
core 1 3-Gal-T has few if any other types of post-translational glycosylation, such as O-glycans.
N-terminal and Internal Amino Acid Sequence
To define directly the relationship between the 84/86-kDa dimers
and the 42/43-kDa monomers, we sequenced the N terminus of the 84- and
86-kDa proteins separately and the combined 42/43-kDa monomers. The
results shown in Fig. 4 demonstrate that
the 84- and 86-kDa material have identical N-terminal sequences to the combined 42/43-kDa material. These results and the behavior of the
84/86-kDa material in reducing SDS-PAGE demonstrate that the 84/86-kDa
material represents dimeric forms of the 42/43-kDa monomeric material.
These results also indicate that all detectable protein by SDS-PAGE
analysis of the non-reduced core 1 3-Gal-T (Fig. 3, top
and bottom) are contained within the 84/86- and 42/43-kDa bands. Furthermore, these results are consistent with the scan of the
purified material (Fig. 3, bottom) indicating that the enzyme is >90% pure. SDS-PAGE analysis of the reduced core 1 3-Gal-T (Fig. 3, top) showed the presence of some other
minor protein bands primarily of lower size than the 42/43-kDa bands.
Because direct protein sequencing of all detectable protein in the
non-reduced material (Fig. 3, top and bottom,
Fig. 4) showed that all proteins have the same N-terminal sequence, the
other protein bands smaller than the 42/43-kDa material may represent
proteolytic fragments of the enzyme processed in the C terminus. Due to
their minor nature, they were not analyzed further.
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The monomeric form of the enzyme may arise during the brief treatment
of the liver extract with DTT in early steps of the purification. The
results from Superose 12 chromatography demonstrate that both the
dimeric and monomeric forms of the enzyme are active. Interestingly,
the core 1 3-Gal-T must be active in reducing conditions, because
the initial post-nuclear supernatant, which contained DTT, had fully
active enzyme (Table I). To obtain more sequence information the mass
of one major tryptic peptide was identified and found to be 1385.1 Da
by MALDI-MS and direct sequencing. The deduced primary structure of the
internal peptide is also shown in Fig. 4. It consists of 11 amino acids
with a calculated molecular mass of 1384.53 Da, consistent with the
mass found by MALDI-MS.
Identification of the Product of Core 1 3-Gal-T
Asialo-BSM is an excellent acceptor of core 1 3-Gal-T, and the
reaction product from this acceptor was used to characterize the
structure of the oligosaccharide generated by the activity. Following
incorporation of [3H]Gal into asialo-BSM from
UDP-[3H]Gal by the purified core 1 3-Gal-T, the
[3H]asialo-BSM was separated from
UDP-[3H]Gal and other small molecules by gel filtration
on Sephadex G-25. The [3H]asialo-BSM appearing in the
void volume was recovered and treated with O-glycosidase,
which specifically cleaves core 1 O-glycan disaccharide from
Ser/Thr and requires the unmodified disaccharide for recognition and
cleavage (29, 30). The 3H-labeled material released by
O-glycosidase was recovered in included fractions following
chromatography on Sephadex G-25 and further purified by passage over a
mixed bed ion exchange column. The 3H-labeled material was
analyzed by high performance high pH anion exchange chromatography
(HPAEC) on a Dionex PA-1 column and compared in its elution with the
defined disaccharide standards. The radioactive product of the core 1 3-Gal-T reaction released from asialo-BSM product by
O-glycosidase eluted identically to the standard
Gal13GalNAc (Fig. 5). (The leading
edge of the peak of radioactive product in Fig. 5 was also seen in the
elution of the standard Gal13GalNAc.) The combined evidence that
the product of the reaction with the purified core 1 3-Gal-T is
released by the substrate-specific O-glycosidase and the
released product has identical elution with authentic Gal13GalNAc
upon Dionex HPAEC confirm that the purified core 1 3-Gal-T
synthesizes the expected core 1 product.
|
Characterization of the Enzyme Activity of the Purified Core 1 3-Gal-T
The kinetics of the purified core 1 3-Gal-T toward the simple
acceptor GalNAc1-O-phenyl and toward a complex
glycopeptide with O-linked GalNAc were determined. For the
acceptor GalNAc1-O-phenyl the apparent
Km was 760 µM (Fig.
6, A and B), and
the apparent Km for UDP-Gal was 630 µM
(Fig. 7, A and B, respectively). The purified enzyme had an apparent
Vmax of 206 µmol/mg/h protein using
GalNAc1-O-phenyl as the acceptor. An unusual feature of
the purified core 1 3-Gal-T was its relatively poor recognition of
UDP, because unlike many other galactosyltransferases the core 1 3-Gal-T was unable to bind efficiently to UDP-hexanolamine. As shown
in Fig. 8, the IC50 for
inhibition of the enzyme in rat liver detergent extracts by UDP was
estimated to be ~1.4 mM. We also tested the ability of
the purified core 1 3-Gal-T to utilize as an acceptor the synthetic
glycopeptide 4-GP-1, which was generated based on the proposed
N-terminal sequence of the human P-selectin glycoprotein ligand-1
(PSGL-1) (9, 16). This glycopeptide contains GalNAc1-Thr at a single
position corresponding to Thr-57 in the native sequence. As shown in
Fig. 6A, 4-GP-1 was an excellent acceptor and perhaps
slightly better than GalNAc1-O-phenyl, although sufficient quantities of the synthetic glycopeptide were not available for use to determine precisely its Km value.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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These results demonstrate that the rat liver core 1 UDP-Gal:GalNAc--R 3-galactosyltransferase (core 1 3-Gal-T) has
been purified 71,034-fold. The protein occurs as a disulfide-bonded dimer of 84/86 kDa and a monomeric species of 42/43 kDa. Both the
dimeric and monomeric forms are enzymatically active. Like most of
other glycosyltransferases, core 1 3-Gal-T requires Mn2+
for its activity.
An unusual feature of the core 1 3-Gal-T is its relatively high
apparent Km for UDP-Gal of ~630 µM
and a remarkably high IC50 for UDP of ~1.4
mM. Many other galactosyltransferases purified to date have
relatively low Km values in the range of 10-80
µM, and the IC50 and Ki
values of the enzymes for UDP are in the same range (31-34). Such a
high Km value for UDP-Gal for the core 1 3-Gal-T
suggests a tight regulation of its activity by availability and
concentration of sugar nucleotides and a strong dependence of its
catalytic efficiency and rate on levels of enzyme. Whether the
relatively high Km value of the core 1 3-Gal-T
for UDP-Gal plays a role in regulating its catalytic rate and
efficiency is presently unknown. Although the subcellular localization
of the core 1 3-Gal-T is not known, it is likely that the enzyme
acts after the polypeptide
N-acetylgalactosaminyltransferases that initiates mucin-type
O-glycan biosynthesis. These polypeptide GalNAc-transferases
have been found in some cells to be localized diffusely throughout the
Golgi apparatus (35). Thus, the core 1 3-Gal-T is likely to function
within these same Golgi compartments, where other
galactosyltransferases with lower Km requirements for UDP-Gal are also functioning. Direct enzyme assays demonstrated that the activity of the core 1 3-Gal-T is co-localized with the
1,4-galactosyltransferase in the trans-Golgi (36). This is
consistent with findings by Hendricks et al. (37) that a 58-kDa resident protein of the cis-Golgi had terminal GalNAc residues in O-linkage. Thus, it is likely that the core 1 3-Gal-T
is localized in the distal Golgi compartments. However, more precise
subcellular localization of the core 1 3-Gal-T in many types of
cells may require studies using specific antibodies to the enzyme. We
are in the process of generating such antibodies for these studies.
The core 1 3-Gal-T is like other so-called inverting
glycosyltransferases in that it generates a -linked product from an -linked sugar nucleotide donor UDP-Gal. Recent crystal structures of
inverting glycosyltransferases suggest that acceptor binding may depend
on sugar nucleotide binding (38, 39), such as seen for the ordered Bi
Bi kinetics of N-acetylglucosaminyltransferase I. It would
appear, however, that the core 1 3-Gal-T might be an interesting
exception to this observation, because we found that sugar nucleotide
is not required for acceptor binding, because in the presence of
Mn2+ alone the enzyme binds tightly to its acceptor
substrate asialo-BSM. However, it is possible that the core 1 3-Gal-T could first bind either sugar nucleotide or acceptor and
then bind the second reactant before proceeding with the reaction. It
will be interesting in future studies to explore the crystal structure
of the core 1 3-Gal-T in complex with donor, acceptor, and metal.
The lack of N-glycosylation and probable lack of other
significant post-translational modifications may make the core 1 3-Gal-T an excellent candidate for crystallization.
A critical and novel step in the purification of the rat liver core 1 3-Gal-T is affinity chromatography on immobilized asialo-BSM. Asialo-BSM represents a type of natural mucin acceptor substrate of
core 1 3-Gal-T, and the enzyme binds so tightly to the support in
the presence of Mn2+ that it can only be eluted in a broad
peak with high salt (e.g. 1 M NaCl) and only
upon depletion of Mn2+. This is the most important step in
the purification resulting in ~4800-fold purification. On SDS-PAGE
under non-reducing conditions, the core 1 3-Gal-T migrates as a
doublet of 84 and 86 kDa with similar intensity, and both have the same
N-terminal amino acid sequence. The size difference may result from
either differential glycosylation or C-terminal cleavage. Under
reducing SDS-PAGE the enzyme migrates as a close 42/43-kDa doublet, and
these proteins share the same unique N terminus as the 84- and 86-kDa
dimeric forms of the enzyme.
Despite the obvious importance of the core 1 3-Gal-T to our overall
understanding of mucin and glycoprotein biosynthesis, several previous
studies (11, 40) have not been successful in purifying the enzyme to
apparent homogeneity. It was reported earlier (41) that the core 1 3-Gal-T was purified from swine trachea. However, the fold
purification of the enzyme was <2,000-fold, and the purified protein
was reported to have an apparent mass of 84 kDa in reducing
SDS-PAGE. However, as we have shown the size of the purified enzyme
from rat liver in reducing SDS-PAGE is 42/43 kDa. Furthermore, we have
now cloned the cDNA encoding the core 1 3-Gal-T from many
different organisms, including humans, mice, rats, Caenorhabditis
elegans, and Drosophila melanogaster, as described in
the accompanying paper (61). The sizes of the enzyme predicted from
these sequences range from ~40 to 50 kDa. No sequence information was
provided for the enzyme purified from swine trachea (41), so the
protein of 84 kDa that was "purified" remains unknown. It was also
reported that core 1 3-Gal-T purified from swine trachea has a
Km for UDP-Gal of 20 µM (41). By
contrast, we find that the purified enzyme from rat liver has a
Km for UDP-Gal of 630 µM. Thus, it is
clear that the enzyme purified from swine trachea is not likely to be
similar to the core 1 3-Gal-T we have purified from rat liver. In
addition, some kinetic studies were carried out on the core 1 3-Gal-T in a preparation from rat liver (11). The activity in this
preparation was enriched 170-fold and the preparation had other enzyme
containing activities. By contrast, we have achieved ~71,000-fold
purification of the core 1 3-Gal-T from rat liver, and the purified
enzyme was devoid of other potentially contaminating
glycosyltransferase activities.
During the course of our many attempts to devise a strategy for
purification of this enzyme, we have developed some insights into
perhaps why this enzyme has presented unusual difficulties in
purification. First, attempts to extract the enzyme in rat liver
membranes with 2% Triton X-100 at <pH 8.5 resulted in extraction of
<50% of the activity, whereas extraction with 2% Triton X-100 at pH
9.0 resulted in extraction of >90% of the activity. Second, many
different galactosyltransferases that utilize UDP-Gal as the donor have
been successfully purified by affinity chromatography on
UDP-hexanolamine-Sepharose or a similar affinity support. However, we
found that the rat liver core 1 3-Gal-T does not bind immobilized UDP-hexanolamine even in the presence of Mn2+. Consistent
with this finding is the unusually high IC50 value for UDP
(~1.4 mM). Third, the enzyme binds tightly to immobilized asialo-BSM coupled to UltraLink, but binding requires the presence of
Mn2+ and omission of Mn2+ in high salt for
elution. Fourth, we also found that a significant difference between
UltraLink and other affinity supports in that UltraLink demonstrates
little if any nonspecific adsorption of protein. Although we cannot be
certain, a combination of these factors may have contributed to
previous difficulties in purifying this enzyme.
Several other glycosyltransferases have been shown to occur as
disulfide-bonded homodimers, including 1,4
N-acetylgalactosaminyltransferase (GM2/GD2/GA2 synthase)
(42, 43), 1,4 galactosyltransferase (44, 45), human milk 1,3/4
fucosyltransferase (46), the spinach galactosyltransferase
monogalactosyldiacylglycerol synthase (47), and -galactoside
2,6-sialyltransferase (48). Interestingly, the dimeric form of the
-galactoside 2,6-sialyltransferase lacks catalytic activity but
retains the ability to bind galactose (48). Our studies demonstrate
that both the dimeric and monomeric forms of the rat liver core 1 3-Gal-T are enzymatically active. Although both forms may exist in
the native state, the monomeric form may arise from the inclusion of
DTT in the initial step of homogenization. More detailed studies will
be needed to define the relative occurrence of the dimeric and
monomeric forms of the core 1 3-Gal-T in liver and other tissues.
Nevertheless, our studies and those cited above raise many questions
about the possible relationship between enzymatic properties and
oligomerization for glycosyltransferases, which be important for
regulating enzyme activity and targeting enzymes to correct subcellular compartments.
Many glycosyltransferases in vertebrates occur in relatively large gene
families. These include the families of galactosyltransferases (49),
N-acetylgalactosaminyltransferases (50, 51),
fucosyltransferases (52, 53), and sialyltransferases (54). By contrast,
several other glycosyltransferases in vertebrates are encoded by unique and single copy genes, such as
N-acetylglucosaminyltransferases-I (55-58), -III (59), and
1-6-fucosyltransferase (60). Although we obtained a doublet of
protein bands of 84/86 and 42/43 kDa in SDS-PAGE, N-terminal sequencing
indicated that all protein species have the same unique N terminus.
This indicates that all rat liver core 1 3-Gal-T activity is
contained within a single polypeptide. To address this possibility, we
describe in the accompanying manuscript (61) the cloning of the
vertebrate gene encoding the core 1 3-Gal-T. Those data demonstrate
that the core 1 3-Gal-T is encoded by a single gene, consistent with
the finding of a single N terminus for the purified protein.
The availability of highly purified core 1 3-Gal-T should have
several important ramifications in the field of glycobiology. Already
this enzyme has been used successfully to construct synthetic glycopeptides containing specifically placed O-glycans to
allow exploration of the ligand binding specificity of human P-selectin (9, 16). In addition, the availability of the core 1 3-Gal-T and, as
described in our accompanying manuscript (61), the gene encoding the
enzyme will allow assessment of the postulated role of the core 1 3-Gal-T in Tn syndrome and IgA nephropathy (1, 21, 23-26).
| |
ACKNOWLEDGEMENTS |
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We thank Dr. Anne Leppänen for providing the synthetic glycopeptide GSP-1, Dr. Kevin Moore for providing rat liver membrane fractions, and Michele Arcade for manuscript preparation.
| |
FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO1 AI48075-01 (to R. D. C.).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.
To whom correspondence may be addressed: Dept. of Biochemistry
and Molecular Biology, University of Oklahoma Health Sciences Center,
975 N.E. 10th St., BRC Rm. 417, Oklahoma City, OK 73104. Tel.:
405-271-2481; Fax: 405-271-3910; E-mail: richard-cummings@ouhsc.edu.
To whom correspondence may be addressed: Novazyme
Pharmaceuticals, Inc., 800 Research Pkwy., Ste. 200, Oklahoma City, OK
73104. Tel.: 405-271-8144; Fax: 405-271-1030; E-mail: wcanfield@novazyme.com.
Published, JBC Papers in Press, October 22, 2001, DOI 10.1074/jbc.M109056200
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 H. Yoshida, T. J Fuwa, M. Arima, H. Hamamoto, N. Sasaki, T. Ichimiya, K.-i. Osawa, R. Ueda, and S. Nishihara Identification of the Drosophila core 1 {beta}1,3-galactosyltransferase gene that synthesizes T antigen in the embryonic central nervous system and hemocytes Glycobiology, December 1, 2008; 18(12): 1094 - 1104. [Abstract] [Full Text] [PDF]
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T. Ju, R. P. Aryal, C. J. Stowell, and R. D. Cummings Regulation of protein O-glycosylation by the endoplasmic reticulum-localized molecular chaperone Cosmc J. Cell Biol., August 12, 2008; 182(3): 531 - 542. [Abstract] [Full Text] [PDF]
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Q. Zheng, I. Van Die, and R. D Cummings A novel {alpha}1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures Glycobiology, April 1, 2008; 18(4): 290 - 302. [Abstract] [Full Text] [PDF]
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 S. Tan and P.-W. Cheng Mucin Biosynthesis: Identification of the cis-Regulatory Elements of Human C2GnT-M Gene Am. J. Respir. Cell Mol. Biol., June 1, 2007; 36(6): 737 - 745. [Abstract] [Full Text] [PDF]
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 S. A. Williams, L. Xia, R. D. Cummings, R. P. McEver, and P. Stanley Fertilization in mouse does not require terminal galactose or N-acetylglucosamine on the zona pellucida glycans J. Cell Sci., April 15, 2007; 120(8): 1341 - 1349. [Abstract] [Full Text] [PDF]
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 W. S. Alexander, E. M. Viney, J.-G. Zhang, D. Metcalf, M. Kauppi, C. D. Hyland, M. R. Carpinelli, W. Stevenson, B. A. Croker, A. A. Hilton, et al. Thrombocytopenia and kidney disease in mice with a mutation in the C1galt1 gene PNAS, October 31, 2006; 103(44): 16442 - 16447. [Abstract] [Full Text] [PDF]
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T. Ju, Q. Zheng, and R. D. Cummings Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans Glycobiology, October 1, 2006; 16(10): 947 - 958. [Abstract] [Full Text] [PDF]
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 T. Kudo, T. Iwai, T. Kubota, H. Iwasaki, Y. Takayama, T. Hiruma, N. Inaba, Y. Zhang, M. Gotoh, A. Togayachi, et al. Molecular cloning and characterization of a novel UDP-Gal:GalNAc{alpha} peptide beta1,3-galactosyltransferase (C1Gal-T2), an enzyme synthesizing a core 1 structure of O-glycan. VOLUME 277 (2002) PAGES 47724-47731 J. Biol. Chem., August 25, 2006; 281(34): 24999 - 24999. [Full Text] [PDF]
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E. M. Comelli, S. R. Head, T. Gilmartin, T. Whisenant, S. M. Haslam, S. J. North, N.-K. Wong, T. Kudo, H. Narimatsu, J. D. Esko, et al. A focused microarray approach to functional glycomics: transcriptional regulation of the glycome Glycobiology, February 1, 2006; 16(2): 117 - 131. [Abstract] [Full Text] [PDF]
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 H. Wang, A. Spang, M. A. Sullivan, J. Hryhorenko, and F. K. Hagen The Terminal Phase of Cytokinesis in the Caenorhabditis elegans Early Embryo Requires Protein Glycosylation Mol. Biol. Cell, September 1, 2005; 16(9): 4202 - 4213. [Abstract] [Full Text] [PDF]
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S. Legardinier, D. Klett, J.-C. Poirier, Y. Combarnous, and C. Cahoreau Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in MimicTM insect cells Glycobiology, August 1, 2005; 15(8): 776 - 790. [Abstract] [Full Text] [PDF]
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 M. N. Arbeitman, A. A. Fleming, M. L. Siegal, B. H. Null, and B. S. Baker A genomic analysis of Drosophila somatic sexual differentiation and its regulation Development, May 1, 2004; 131(9): 2007 - 2021. [Abstract] [Full Text] [PDF]
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L. Xia, T. Ju, A. Westmuckett, G. An, L. Ivanciu, J. M. McDaniel, F. Lupu, R. D. Cummings, and R. P. McEver Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans J. Cell Biol., February 2, 2004; 164(3): 451 - 459. [Abstract] [Full Text] [PDF]
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T. Correia, V. Papayannopoulos, V. Panin, P. Woronoff, J. Jiang, T. F. Vogt, and K. D. Irvine Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila PNAS, May 27, 2003; 100(11): 6404 - 6409. [Abstract] [Full Text] [PDF]
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T. Ju and R. D. Cummings A unique molecular chaperone Cosmc required for activity of the mammalian core 1 beta 3-galactosyltransferase PNAS, December 24, 2002; 99(26): 16613 - 16618. [Abstract] [Full Text] [PDF]
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T. Kudo, T. Iwai, T. Kubota, H. Iwasaki, Y. Takayma, T. Hiruma, N. Inaba, Y. Zhang, M. Gotoh, A. Togayachi, et al. Molecular Cloning and Characterization of a Novel UDP-Gal:GalNAcalpha Peptide beta 1,3-Galactosyltransferase (C1Gal-T2), an Enzyme Synthesizing a Core 1 Structure of O-Glycan J. Biol. Chem., November 27, 2002; 277(49): 47724 - 47731. [Abstract] [Full Text] [PDF]
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A. Leppanen, L. Penttila, O. Renkonen, R. P. McEver, and R. D. Cummings Glycosulfopeptides with O-Glycans Containing Sialylated and Polyfucosylated Polylactosamine Bind with Low Affinity to P-selectin J. Biol. Chem., October 11, 2002; 277(42): 39749 - 39759. [Abstract] [Full Text] [PDF]
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Z. S. Kawar, I. Van Die, and R. D. Cummings Molecular Cloning and Enzymatic Characterization of a UDP-GalNAc:GlcNAcbeta -R beta 1,4-N-Acetylgalactosaminyltransferase from Caenorhabditis elegans J. Biol. Chem., September 13, 2002; 277(38): 34924 - 34932. [Abstract] [Full Text] [PDF]
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T. Iwai, N. Inaba, A. Naundorf, Y. Zhang, M. Gotoh, H. Iwasaki, T. Kudo, A. Togayachi, Y. Ishizuka, H. Nakanishi, et al. Molecular Cloning and Characterization of a Novel UDP-GlcNAc:GalNAc-peptide beta 1,3-N-Acetylglucosaminyltransferase (beta 3Gn-T6), an Enzyme Synthesizing the Core 3 Structure of O-Glycans J. Biol. Chem., April 5, 2002; 277(15): 12802 - 12809. [Abstract] [Full Text] [PDF]
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T. Ju, K. Brewer, A. D'Souza, R. D. Cummings, and W. M. Canfield Cloning and Expression of Human Core 1 beta 1,3-Galactosyltransferase J. Biol. Chem., January 4, 2002; 277(1): 178 - 186. [Abstract] [Full Text] [PDF]
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