 |
INTRODUCTION |
Many of the functional moieties of complex glycoconjugates are in
the terminal sequences of N- and O-glycans of
glycoproteins and in glycolipids, which are recognized by a growing
number of carbohydrate binding proteins (1-4). A common terminal motif that is modified in a variety of ways by the additions of other sugars
and sulfate groups is the lactosamine sequence Gal
4GlcNAc-R (LacNAc
or LN),1 which is generated
by a large family of UDP-Gal:GlcNAc-R
1,4-galactosyltransferases (4GalTs) acting on terminal
GlcNAc residues (5). However, another common terminal motif found in
vertebrate and invertebrate glycoconjugates is the GalNAc4GlcNAc-R
(LacdiNAc or LDN) sequence. The LDN motif occurs in mammalian pituitary
glycoprotein hormones, where the terminal GalNAc residues are
4-O-sulfated (6) and function as a recognition markers for
clearance by the endothelial cell Man/S4GGnM receptor (7).
However, nonpituitary mammalian glycoproteins also contain LDN
determinants (8-11), indicating that expression of LDN determinants in
vertebrate glycoconjugates is more widespread than once thought. In
addition, LDN and modifications of LDN sequences are common antigenic
determinants in many parasitic nematodes and trematodes (12-17).
The LDN structure can be considered a variant of the more typical LN
structure generated by a family of 4GalTs that includes the best
characterized of all glycosyltransferases, the 4GalT I or lactose
synthase (18-26). As more members of this family have been studied and
the cDNAs encoding them have been cloned, it is evident that they
share highly homologous regions within their amino acid sequences
(27-36). Interestingly, these regions of homology are also found
within the amino acid sequence of a snail UDP-GlcNAc:GlcNAc-R 1,4-N-acetylglucosaminyltransferase (37-39). This latter
finding raised the possibility that the 4GalNAcT enzyme(s) might
also have amino acid sequence homology to members of the 4GalT
family. However, despite many studies reporting on the activity of a
putative 4GalNAcT capable of generating LDN sequences (11, 40-46),
the gene(s) encoding the putative 4GalNAcT responsible for LDN
synthesis has not been identified.
In searching for the putative 4GalNAcT required for LDN synthesis,
we examined genes in Caenorhabditis elegans. The C. elegans genome contains three open reading frames that encode
proteins with sequence homology to the 4GalT family. One of these
open reading frames (ORF R10E11.4; sqv-3) is predicted to encode a protein involved in vulval invagination (47) and is likely to be a
UDP-Gal:xylose -R 1,4-galactosyltransferase (33, 48). Another of
these open reading frames (ORF W02B12.11) encodes a protein for which
no enzymatic activity has yet been reported. The third open reading
frame (ORF Y73E7A.7) was identified more recently than the two
mentioned above and therefore had not been reported in previous studies
(27, 31). In this study, we have cloned a cDNA corresponding to the
latter open reading frame and demonstrate that it encodes a
4GalNAcT, which we have termed Ce4GalNAcT. Ce4GalNAcT is
active when expressed in mammalian cells in generating LDN determinants
on N-glycans of glycoproteins.
| |
EXPERIMENTAL PROCEDURES |
Materials--
All chemicals and reagents used in this study,
unless otherwise indicated, were from Sigma. The C. elegans
cDNA library was a gift from Dr. Robert Barstead (Oklahoma Medical
Research Foundation, Oklahoma City, OK). The QIA Quick gel extraction
kit was from Qiagen (Valencia, CA). Restriction enzymes were from New
England Biolabs (Beverly, MA). The pCR 2.1 vector was from Invitrogen. The pcDNA3.1(+)-TH was a gift from Dr. Alireza R. Rezaie
(Department of Biochemistry and Molecular Biology, St. Louis University
School of Medicine, St. Louis, MO). FuGENE 6 and Complete protease
inhibitor mixture were from Roche Molecular Biochemicals.
N-Glycanase was from Glyko (Novato, CA). HighSignal West
Pico Chemiluminescent Substrate was from Pierce.
GlcNAc1-3GalNAc
1-O-pNP (core 3-O-pNP) and
GlcNAc1-6GalNAc1-O-pNP (core 6-O-pNP) were
obtained from Toronto Research Chemicals (Toronto, Canada). Acceptor
compounds (see Table II) 1-3, 5, 9,
and 12 were purchased from Sigma, 4 was from
Koch-Light Laboratories, and 6-8 were from Toronto Research
Chemicals. Compounds 10 and 11 were a kind gift
from Dr. L. Anderson (University of Wisconsin, Madison, WI), and
14-17 were from Dr. J. Lönngren (University of
Stockholm). Compounds 13 (39) and 18-21 (32)
were synthesized as described previously. Radiolabeled nucleotide
sugars were obtained from PerkinElmer Life Sciences and were diluted
with unlabeled nucleotide sugars (Sigma) to give the desired specific radioactivity.
Cloning and Sequencing of the Ce4GalNAcT cDNA--
A
BlastP search of the NCBI nonredundant protein data base for homologues
of the human 4GalT I (accession number CAA39074) identified a
hypothetical protein encoded by an open reading frame in the C. elegans genome designated Y73E7A.7. A cDNA was amplified by
PCR from a mixed stage C. elegans cDNA library using
primers corresponding to the 5'- and 3'-ends of this open reading frame (5'-GCCACCATGGCTTTTCGTCATTTGGC-3'; 5'-CTAAAAACACGTTGGAAAGTCC-3'). Amplification was carried out at 95 °C for 2:30 min followed by 35 cycles at 95 °C for 50 s, 53 °C for 50 s, and 72 °C
for 1:50 min and then at 72 °C for 10 min. The PCR product was
purified from an agarose gel slice using a QIA Quick gel extraction
kit, cloned into the pCR 2.1 vector, and sequenced on both strands at
the Sequencing Facility of the Oklahoma Medical Research Foundation (Oklahoma City, OK).
Construction of an Expression Vector Encoding a Soluble,
Epitope-tagged Form of Ce4GalNAcT--
A PsiI
(partial)/PvuII DNA fragment starting at bp 87 of the
Ce4GalNAcT open reading frame and extending beyond the stop codon
was subcloned into the EcoRV site of the pcDNA
3.1(+)-TH vector. The resulting vector (pCMV-SH-Ce4GalNAcT) encodes
a fusion protein, designated SH-Ce4GalNAcT, which consists of a
signal peptide at the N terminus followed by an HPC4 epitope and then the catalytic domain of the Ce4GalNAcT (beginning at
Lys30, the first amino acid after the transmembrane
domain). The HPC4 epitope is recognized by the
Ca2+-dependent monoclonal antibody HPC4 (49,
50). SH-Ce4GalNAcT is under the transcriptional control of the
cytomegalovirus promoter, which is present in the vector.
Expression of SH-Ce4GalNAcT--
CHO-Lec8 and CHO-Lec2 cells
were transfected with pCMV-SH-Ce4GalNAcT using FuGENE 6, according
to the manufacturer's instructions, and cultured in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum and 600 µg/ml Geneticin to select for stably transformed cells. After 4 weeks
of culturing in medium containing Geneticin, the cells were cultured in
the same medium without Geneticin, and the culture medium was harvested
every 3 days and used to purify SH-Ce4GalNAcT. To assay
intracellular 4GalNAcT activity and for Western blots, cells were
washed with 75 mM sodium cacodylate, pH 7.0, and lysed in a
buffer of 50 mM sodium cacodylate, pH 7.0, 20 mM MnCl2, 1% Triton X-100, 1× Complete
protease inhibitor mixture (EDTA-free). The lysates were centrifuged at
12,000 × g for 3 min, and the supernatants were used
for further analyses.
Purification of SH-Ce4GalNAcT--
Medium
containing SH-Ce4GalNAcT was centrifuged at 1,500 × g for 5 min to remove cellular debris and then incubated
with HPC4-UltraLink beads (5 mg of HPC4 antibody/ml of beads; 0.1 µl of beads/ml of medium) for 1 h at room temperature on a rotating platform. The beads were collected by centrifugation at 600 × g for 3 min and washed three times with 10 ml of 100 mM sodium cacodylate, pH 7.0, 2 mM
CaCl2. The beads were then resuspended in the same buffer
with the addition of 20 mM MnCl2 and used as the enzyme source. For Western blot analysis, the bound material was
released by incubating the beads in a buffer of 50 mM
sodium cacodylate, pH 7.0, 20 mM EDTA for 10 min at room
temperature and then collecting the supernatant.
SDS-PAGE and Western Blot Analyses--
Cell lysates were
treated with N-glycanase in a buffer of 20 mM
sodium phosphate, pH 7.5, 50 mM -mercaptoethanol, 0.1%
SDS, 0.75% Nonidet P-40 for 3 h at 37 °C. Control treatments
were carried out in the same way but without adding
N-glycanase. The lysates were then mixed with loading
buffer, resolved by SDS-PAGE (4-20% gradient), and transferred to a
nitrocellulose membrane. The membrane was blocked with 5% bovine serum
albumin in a buffer of 20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 2 mM CaCl2, 0.05% Tween
20 for 5 h at 4 °C. It was then incubated with the primary
antibody (mouse monoclonal anti-LDN IgM SMLDN1.1 (16) or HPC4 IgG) in
the same buffer (without bovine serum albumin) for 1 h at room
temperature, washed in the same buffer, and incubated with the
secondary antibody (horseradish peroxidase-conjugated, goat anti-mouse
IgM or IgG) as before. The membrane was then washed again, incubated in
HighSignal West Pico Chemiluminescent Substrate for 2 min at room
temperature, and exposed to a BioMax film (Eastman Kodak Co.) for 1 min. The film was then developed using a processing machine (Konica
SRX-101).
4GalNAcT Assays--
Standard assays were performed
essentially as described previously (45) in a 25-µl reaction mixture
containing 2.5 µmol of sodium cacodylate, pH 7.2, 12.5 nmol of
UDP-[3H]GalNAc (2.5 Ci/mol), 1 µmol of
MnCl2, 0.1 µmol of ATP, 0.1 µl of Triton X-100, 2 µl
of beads, and acceptor substrate, containing 25 nmol of terminal GlcNAc
at the nonreducing end unless otherwise indicated. Control assays
lacking the acceptor substrate were carried out to correct for
incorporation into endogenous acceptors, and all assays were carried
out in duplicate. All assays were linear with time for up to 180 min.
After incubation at 37 °C for 180 min, the reaction was stopped.
When oligosaccharides or glycopeptides were the acceptor, the labeled
product was separated from unincorporated label by chromatography on a
1-ml column of Dowex 1-X8 (Cl
form) according to Easton
et al. (51). When oligosaccharide acceptors with hydrophobic
aglycon (pNP) were used as the acceptor, the product was isolated using
Sep-Pak C-18 cartridges (Waters) as described (52). The isolated
products were assayed for incorporation of radioactivity by liquid scintillation.
High pH Anion Exchange Chromatography with Pulsed
Amperometric Detection (HPAEC-PAD)--
The product catalyzed by
SH-Ce4GalNAcT using GlcNAc1-O-pNP as acceptor was
isolated using a Sep-Pak C-18 cartridge (1 cm3) and
lyophilized. Three nmol of the product (dissolved in water) were
analyzed by a Dionex HPAEC-PAD system, using a PA-1 column with a 100 mM NaOH solution at a flow rate of 1 ml/min. The standard containing the authentic LDN structure
GalNAc1-4GlcNAc1-O-pNP was synthesized using bovine
4GalT I and GlcNAc1-O-pNP as the acceptor for
UDP-GalNAc in the standard assay described above. Commercially acquired
GlcNAc1-3GalNAc1-O-pNP (core 3-O-pNP) and
GlcNAc1-6GalNAc1-O-pNP (core 6-O-pNP) were
also used as standards.
Large Scale Synthesis of Product for 1H NMR
Analysis--
Synthesis was carried out overnight at 37 °C in a
1-ml reaction mixture containing 50 µmol of sodium cacodylate, pH
7.0, 300 nmol of GlcNAc1-S-pNP, 1 µmol of UDP-GalNAc,
20 µmol of MnCl2, 5 µmol of ATP, 3 µmol of
NaN3, and 100 µl of beads. The product was then isolated
using a Sep-Pak C-18 cartridge (1 cm3) and lyophilized.
400-Mz 1H NMR--
150 nmol of the product catalyzed
by SH-Ce4GalNAcT using GlcNAc1-S-pNP as acceptor were
treated with D2O (99.75 atom %; Merck) three times with
intermediate lyophilization. Finally, the sample was redissolved in 400 µl of D2O (99.96 atom %; Sigma-Aldrich). 1H
NMR spectroscopy was performed on a Bruker MSL 400 spectrometer operating at 400 MHz at a probe temperature of 300 K. Resolution enhancement was achieved by Lorentzian to Gaussian transformation. Chemical shifts are expressed in ppm downfield from internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured by
reference to internal acetone (
= 2.225 ppm in
D2O).
| |
RESULTS |
Isolation of the cDNA Encoded by Y73E7A.7
(Ce4GalNAcT)--
A potential C. elegans open reading
frame designated Y73E7A.7 was identified by a BlastP search as encoding
a homologue of the human 4GalT I. An identical cDNA
(GenBankTM accession number AY130767) was amplified by PCR
from a mixed stage C. elegans cDNA library using primers
corresponding to the 5'- and 3'-ends of this open reading frame,
establishing that the gene is expressed in vivo. The
cDNA of Y73E7A.7 encodes a predicted 383-amino acid protein with a
single transmembrane domain in a type 2 topology, which is a common
topological motif in glycosyltransferases. The protein encoded by
Y73E7A.7 is predicted to contain six potential N-glycosylation sites and two DVD motifs, which are
thought to participate in metal ion binding (53) (Fig.
1). Curiously, the last four potential
N-glycosylation sites share an identical sequon (NQT), the
significance of which is not clear at this time. The protein sequence
encoded by Y73E7A.7 is 35.5% identical to human 4GalT I (Fig.
2A) and is more closely
related to the first four members of the 4GalT family (I, II, III,
and IV) than to the other three (Fig. 2B).
View larger version (49K):
[in this window]
[in a new window]
|
Fig. 1.
cDNA and deduced protein sequence of
Y73E7A.7 (Ce4GalNAcT). The putative
transmembrane domain of the predicted protein encoded by Y73E7A.7 is
double underlined; the potentially
N-glycosylated Asp residues are in boldface
type within boxed sequons, and the DVD
motifs are single underlined. This cDNA
sequence has been deposited in GenBankTM under accession
number AY130767.
|
|
View larger version (51K):
[in this window]
[in a new window]
|
Fig. 2.
Protein sequence comparisons between the
protein encoded by Y73E7A.7 (Ce4GalNAcT) and
members of the 4GalT family. A,
alignment of Y73E7A.7 (Ce4GalNAcT) with human 4GalT I using the
Align and Boxshade programs. Black boxes,
identical residues; gray boxes, similar residues.
B, phylogenic analysis of Ce4GalNAcT and other 4GalT
family members using the ClustalW and Drawgram programs.
|
|
Expression and Purification of a Soluble, Recombinant Form of the
Protein Encoded by Y73E7A.7 (SH-Ce4GalNAcT)--
To assess whether
Y73E7A.7 encodes an active 4-galactosyltransferase or possibly a
4-N-acetylgalactosaminyltransferase, a soluble,
recombinant form of the protein was generated lacking the cytoplasmic N
terminus and transmembrane domain and containing the HPC4 peptide
epitope at the new N terminus. This construct was stably expressed in
Chinese hamster ovary CHO-Lec8 cells. These cells are impaired in the
transport of UDP-Gal into the Golgi (54) and consequently generate
hybrid- and complex-type N-glycans containing terminal
GlcNAc and O-glycans containing the simple Tn antigen
GalNAc1-Ser/Thr (55-57). The transfected cells expressing Y73E7A.7,
but not the control mock-transfected cells, acquired a novel
intracellular GalNAcT activity in the cell extracts capable of
utilizing UDP-GalNAc as the donor and GlcNAc1-S-pNP as
the acceptor (Fig. 3A). The
recombinant protein containing the HPC4 epitope from extracellular
medium was bound by HPC4-conjugated beads, confirming the 4GalNAcT
activity of the enzyme encoded by the Y73E7A.7 (Fig. 3A). A
Western blot of the material bound to the HPC4-conjugated beads
(Fig. 3B) confirmed that it corresponded to the
predicted size of the HPC4 epitope-tagged protein (43.1-kDa
peptide plus N-glycans) as discussed below. These data
demonstrate that Y73E7A.7 encodes an active 4GalNAcT and the
enzyme was designated the C. elegans
UDP-GalNAc:GlcNAc-R 1,4-N-acetylgalactosaminyltransferase
(Ce4GalNAcT), and the soluble, HPC4 epitope-tagged version was
designated SH-Ce4GalNAcT.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Expression and purification of the protein
encoded by Y73E7A.7 (SH-Ce4GalNAcT).
A, intracellular extracts of wild-type CHO-Lec8 cells (Lec8)
and CHO-Lec8 cells expressing a soluble, HPC4 epitope-tagged form of
the protein encoded by Y73E7A.7 (SH-Ce4GalNAcT; Lec8/GT) were tested
for GalNAcT (striped bars) and GalT
(black bars) activities using
GlcNAc1-S-pNP as acceptor. The material captured by HPC4
beads from the extracellular medium from both cell types was also
tested for these activities. The activity is indicated in pmol of donor
sugar transferred/h for 105 cells (extracts) or 10 ml of medium (beads). B, Western blot using the HPC4
monoclonal antibody of the material captured on HPC4 beads from 10 ml
of medium from Lec8/GT cells. The positions of molecular mass markers
are indicated on the left in kDa.
|
|
Donor and Substrate Specificity of SH-Ce4GalNAcT--
The
enzyme purified from the medium using HPC4-conjugated beads was used in
assays to further characterize its activity. In assays to determine its
specificity for nucleotide-sugar donors (Table
I), SH-Ce4GalNAcT efficiently utilized
UDP-GalNAc but did not significantly utilize UDP-Gal, UDP-GlcNAc, or
UDP-Glc. To define the acceptor specificity of Ce4GalNAcT, the
enzyme was tested with a wide variety of acceptors (Table
II). SH-Ce4GalNAcT efficiently
utilizes free GlcNAc and all substrates containing terminal -linked
GlcNAc in both N- and O-glycan type structures. SH-Ce4GalNAcT less effectively utilizes -linked GlcNAc or
6-sulfated GlcNAc and does not utilize acceptors with terminal
-linked Gal, Glc, or GalNAc. The acceptor substrate specificity of
SH-Ce4GalNAcT is therefore similar to the broad specificity reported
for human 4GalT I (32). In contrast, the snail 4-GlcNAcT has a
marked preference for acceptors with 1,6-linked terminal GlcNAc (39) (see Table II for a side-by-side comparison).
In view of the sequence homology between Ce4GalNAcT and the 4GalT
family, we examined whether the modifier protein -lactalbumin would
affect the acceptor specificity of SH-Ce4GalNAcT. -Lactalbumin, which is expressed in lactating mammary glands, associates with 4GalT I and switches its acceptor specificity from GlcNAc-R to free
Glc, thus forming lactose synthase (58). However, unlike its effect on
4GalT I, -lactalbumin does not induce SH-Ce4GalNAcT to
utilize Glc as an acceptor instead of GlcNAc (Table
III). -Lactalbumin does
appear to slightly depress activity of Ce4GalNAcT toward the free
GlcNAc acceptor, suggesting a possible weak interaction between the enzyme and -lactalbumin.
Product Characterization by HPAEC-PAD and 1H
NMR--
The product generated by SH-Ce4GalNAcT using
GlcNAc1-O-pNP as acceptor was analyzed by HPAEC-PAD (Fig.
4). The product co-eluted with the
authentic GalNAc1-4GlcNAc1-O-pNP standard but not
with two other disaccharide-O-pNP standards
(GlcNAc1-3GalNAc1-O-pNP and
GlcNAc1-6GalNAc1-O-pNP).
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
HPAEC-PAD analysis of the reaction
product catalyzed by SH-Ce4GalNAcT using
GlcNAc1-O-pNP as
acceptor. A, analysis of the reaction products obtained
in the absence of SH-Ce4GalNAcT. B, analysis of the
reaction products obtained in the presence of SH-Ce4GalNAcT. The
arrows indicate the elution positions of reference
compounds. a, GalNAc1-4GlcNAc1-O-pNP;
b, GlcNAc1-6GalNAc1-O-pNP; c,
GlcNAc1-3GalNAc1-O-pNP; d,
GlcNAc1-O-pNP. The peak corresponding to
GalNAc1-4GlcNAc1-O-pNP is shaded.
|
|
To further establish the structure of the product generated by
SH-Ce4GalNAcT using GlcNAc1-S-pNP as acceptor, the
product was analyzed by 1H NMR spectroscopy (Fig.
5). The spectrum shows two H-1 doublets at = 5.146 ppm and 4.540 ppm. The coupling constants of the H-1 doublets (10.5 and 8.5 Hz, respectively) indicate that both C-1
atoms are in -anomeric conformation (59). The doublet at 5.146 ppm
and the signal at = 2.013 ppm can be assigned to the H-1 and
the CH3-NAc of GlcNAc1-S-pNP by analogy to
the resonance positions in GlcNAc1-4GlcNAc1-S-pNP
(38). The doublet at = 4.540 ppm and the signal at = 2.077 ppm have shifts that are close to those reported for a
4-linked GalNAc residue (44, 45). The NMR spectrum therefore
confirms that the analyzed product is
GalNAc1-4GlcNAc1-S-pNP.
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
400-MHz 1H NMR analysis of the
reaction product catalyzed by SH-Ce4GalNAcT
using GlcNAc1-S-pNP as
acceptor. A, NMR spectrum with close-ups of diagnostic
areas. B, NMR data and comparison with data of two related
compounds. Data for compound a are from Ref. 44; data for
compound b are from Ref. 38.
|
|
In Vivo Synthesis of LDN Structures on N-Glycans by
SH-Ce4GalNAcT--
Since SH-Ce4GalNAcT was active in cell
extracts when expressed in CHO-Lec8 cells (Fig. 3A), we
examined whether it can generate LDN structures on endogenous glycan
acceptors in animal cells. Cell lysates from nontransfected CHO-Lec8
and CHO-Lec2 cells and transfected CHO-Lec8 and CHO-Lec2 cells
expressing SH-Ce4GalNAcT were examined for the presence of LDN
determinants by a Western blot analysis using a monoclonal antibody
SMLDN1.1 against LDN (16) (Fig.
6A). As indicated above the
CHO-Lec8 cells are deficient in UDP-Gal transport into the Golgi (54),
whereas the CHO-Lec2 cells are deficient in CMP-sialic acid transport
into the Golgi and hence generate nonsialylated glycans terminating in
Gal residues (60). Nontransfected CHO-Lec8 and CHO-Lec2 cells did not
express detectable levels of LDN determinants as detected by SMLDN1.1 (Fig. 6A). By contrast, both cell lines expressing
SH-Ce4GalNAcT expressed the LDN epitope on several glycoproteins. It
would be predicted that the Ce4GalNAcT might only add GalNAc to
N-glycans in CHO cells, since CHO cells produce
O-glycans of the core 1 structure (Gal3GalNAc1
Ser/Thr) lacking in GlcNAc residues (61, 62). Cell extracts derived
from CHO cell lines expressing SH-Ce4GalNAcT were treated with
N-glycanase to determine whether LDN determinants were
present in N-glycans. N-Glycanase treatment
quantitatively removed the LDN-reactive epitopes from glycoproteins,
demonstrating that LDN was expressed exclusively on
N-glycans by the SH-Ce4GalNAcT. Transfected CHO-Lec2
cells expressed lower levels of LDN determinants than transfected
CHO-Lec8, possibly due to competition from endogenous 4GalTs, since
the cells expressed equivalent amounts of SH-Ce4GalNAcT as detected
by a Western blot using the HPC4 antibody (Fig. 6B). The
latter experiment also confirmed the molecular weight of
SH-Ce4GalNAcT, demonstrating that N-glycanase treatment
shifted the 59.4-kDa protein to 43.1 kDa, the predicted peptide size of
SH-Ce4GalNAcT.
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
In vivo synthesis of
LDN-containing glycans. Western blots of cellular extracts of
wild-type CHO-Lec8 cells (lane 1), CHO-Lec8 cells
expressing SH-Ce4GalNAcT (lanes 2 and
3), wild-type CHO-Lec2 cells (lane 4),
and CHO-Lec2 cells expressing SH-Ce4GalNAcT (lanes
5 and 6). The extracts in lanes
3 and 6 have been treated with
N-glycanase. The membranes were probed with monoclonal
antibodies against LDN (A) or the HPC4 tag (B).
The positions of molecular mass markers are indicated on the
left in kDa.
|
|
| |
DISCUSSION |
The results presented here provide several new insights into
the biosynthesis of animal cell glycoproteins. We have identified a
specific N-acetylgalactosaminyltransferase Ce4GalNAcT
from C. elegans capable of utilizing UDP-GalNAc as
the donor for the transfer of GalNAc residues to terminal GlcNAc
residues in a wide variety of acceptors to generate the LacdiNAc (LDN)
sequence GalNAc4GlcNAc-R. The enzyme is a member of the
4-galactosyltransferase family, although Ce4GalNAcT is unable to
utilize UDP-Gal as the donor. In vertebrate cells, the recombinant form
of Ce4GalNAcT is fully functional and capable of generating the LDN
structure in complex-type N-glycans of glycoproteins. This
represents the first identification of a 4GalNAcT capable of
generating the LDN sequence in animal glycoconjugates.
Although the LacNAc (LN) sequence Gal4GlcNAc-R is a more general
terminal modification in vertebrate glycoconjugates, the LDN sequence
also occurs in several vertebrate glycoproteins and glycolipids,
including pituitary glycoprotein hormones (63) and other
glycoconjugates (8, 11, 64-66). A hormone-specific 4GalNAcT enzyme,
active in the pituitary gland and other tissues, acts preferentially on
glycoproteins containing a specific peptide motif (46, 63, 67-70). The
GalNAc residue added to these hormones is subsequently
4-O-sulfated (71-73), and the resulting terminal GalNAc-4-SO4 acts as a clearance signal that regulates
their circulatory half-lives (6, 74-76). The addition of the LDN motif
to other glycoproteins, such as glycodelin (9, 66) and protein C (8), may also be cell- and protein-specific and may be important to the
functional activities of these glycoproteins. In addition to the
hormone-specific 4GalNAcT, a motif-independent 4GalNAcT activity
has been detected in extracts from many cells (69), including human 293 cells (11), bovine mammary gland (43), snails (40, 41), insect cells
(45), and schistosomes (42, 44). The LDN motif is also a more common
structural feature in invertebrate glycoconjugates compared with the LN
motif, especially as seen in many parasitic nematodes and trematodes
(12-17, 77). However, neither the enzyme(s) nor gene(s) encoding the
enzyme responsible for LDN synthesis in invertebrates have previously been defined.
Ce4GalNAcT is clearly a member of the 4GalT family of enzymes
with homology to the other members found in various species ranging
from C. elegans to mammals. Curiously, the GalT I or lactose synthase is capable of utilizing both UDP-Gal and UDP-GalNAc, and in
the presence of -lactalbumin, this enzyme is stimulated to utilize
UDP-GalNAc as the donor to generate LDN with free GlcNAc as the
acceptor (78). Thus, we considered the possibility that the LDN
structure might not be generated by a separate enzyme specific for
UDP-GalNAc. Therefore, it is especially interesting that the
Ce4GalNAcT, although a member of the 4GalT family, does not
utilize UDP-Gal. Two recent crystallographic studies on 4GalT I have
shed light on the amino acid residues that are important in donor and
acceptor recognition by the enzymes of the 4GalT family. The first
study demonstrated that changing a tyrosine residue
(Tyr289) in the bovine 4GalT I to isoleucine altered its
donor specificity from UDP-Gal to UDP-GalNAc (21). It is noteworthy
that the Ce4GalNAcT contains an isoleucine residue
(Ile257) at the corresponding position. The second study
identified 12 amino acids in the bovine 4GalT I that constitute its
acceptor binding site (79). These amino acids vary considerably among members of the 4GalT family, and Ce4GalNAcT has between 2 and 4 of these residues in common with each of the other members of this
family. The specific amino acids in Ce4GalNAcT responsible for its
sugar nucleotide and acceptor specificity await identification.
It is noteworthy that the soluble form of the Ce4GalNAcT, when
expressed in CHO-Lec8 or CHO-Lec2 cells, is capable of generating LDN
epitopes on cellular glycoproteins. Interestingly, a significant amount
of the total Ce4GalNAcT was present in cell extracts compared with
extracellular media (Fig. 3A). This implies that the soluble enzyme is sufficiently retained in the cell to allow productive interactions with intracellular acceptor glycoproteins. The mode of
retention of the soluble Ce4GalNAcT in CHO cells is not known. Targeting and retention in the Golgi apparatus for many
glycosyltransferases requires membrane anchoring, although other
domains of the enzymes are also important (80, 81). Similarly, we
previously observed that the soluble form of the
1,3-galactosyltransferase is also functional within cells (82).
However, soluble forms of some other glycosyltransferases inefficiently
glycosylate intracellular acceptors (83, 84). It is conceivable that
the high concentration of potential terminal GlcNAc-R acceptors in
CHO-Lec8 and CHO-Lec2 cells could cause the retention of Ce4GalNAcT
in appropriate Golgi compartments, based on the observation that many
glycosyltransferases show affinity for their acceptor substrates and
can be purified by affinity chromatography on immobilized acceptors
(85). The Ce4GalNAcT could also interact with some other
Golgi-resident protein, such as another glycosyltransferase, as
proposed in the kin recognition hypothesis (86). Overall, our results
support the possibility that Golgi retention of glycosyltransferases is likely to be a complex event mediated in part by multiple domains of
the enzymes and not necessarily by the transmembrane domains.
Although Ce4GalNAcT is able to act on most of the common types of
mammalian N- and O-glycans, there is only a
limited knowledge of the glycan structures produced in C. elegans. It has been reported that the LDN motif appears at the
reducing end of unusual O-glycans of C. elegans
with the predicted sequence R-GalNAc4GlcNAc-Ser/Thr (87). Whether
Ce4GalNAcT is responsible for synthesis of this type of
structure is currently unknown, as are the enzymes that can potentially
act to extend a glycan from the LDN motif.
The availability of a recombinant, well characterized 4GalNAcT
active in mammalian cells should help advance our understanding of this
type of glycosyltransferase and the structures and functions of
LDN-containing glycans. The enzyme can be a valuable tool for both the
in vitro and in vivo synthesis of LDN-based
glycan structures, which may be used for further studies on their
function in both vertebrates and invertebrates, as well as for studying
LDN-containing antigenic glycans and pharmaceutical or commercial products.
-R
,
TOP
ABSTRACT
INTRODUCTION
