* Epithelial Cell Biology Laboratory, Electron Microscopy Unit, Imperial Cancer Research Fund, Lincoln's Inn Fields, London
WC2A 3PX, UK; § Department of Biochemistry, Hospital for Sick Children and University of Toronto, Toronto M5G 1X8,
Canada; and
Sloan-Kettering Institute, New York 10021
The 2,3 sialyltransferase,
2,3 SAT (O),
catalyzes the transfer of sialic acid to Gal
1,3 N-acetyld-galactosamine (GalNAc) (core-1) in mucin type
O-glycosylation, and thus terminates chain extension. A
Core-2 branch can also be formed from core-1 by the core-2
1,6 N-acetyl-d-glucosamine transferase (
1,6
GlcNAc T) that leads to chain extension. Increased levels of the
2,3 SAT (O) and decreased levels of the
core-2
1,6 GlcNAc T are seen in breast cancer cells
and correlate with differences in the structure of the
O-glycans synthesized (Brockhausen et al., 1995
; Lloyd et al., 1996
). Since in mucin type O-glycosylation sugars
are added individually and sequentially in the Golgi apparatus, the position of the transferases, as well as their
activity, can determine the final structure of the O-glycans synthesized. A cDNA coding for the human
2,3
SAT (O) tagged with an immunoreactive epitope from
the myc gene has been used to map the position of the
glycosyltransferase in nontumorigenic (MTSV1-7) and
malignant (T47D) breast epithelial cell lines. Transfectants were analyzed for expression of the enzyme at the
level of message and protein, as well as for enzymic activity. In T47D cells, which do not express core-2
1,6
GlcNAc T, the increased activity of the sialyltransferase correlated with increased sialylation of core-1
O-glycans on the epithelial mucin MUC1. Furthermore, in MTSV1-7 cells, which do express core-2
1,6
GlcNAc T, an increase in sialylated core-1 structures is accompanied by a reduction in the ratio of GlcNAc:
GalNAc in the O-glycans attached to MUC1, implying
a decrease in branching. Using quantitative immunoelectron microscopy, the sialyltransferase was mapped
to the medial- and trans-Golgi cisternae, with some being present in the TGN. The data represent the first
fine mapping of a sialyltransferase specifically active in
O-glycosylation and demonstrate that the structure of
O-glycans synthesized by a cell can be manipulated by
transfecting with recombinant glycosyltransferases.
In eukaryotic cells, proteins are synthesized in the ER
from where they are transported to different locations, either within the cell or to the plasma membrane. Along the exocytic pathway, proteins undergo
various modifications including proteolytic cleavage, glycosylation, and sulfation. Within this pathway there are two main types of glycosylation, N-linked and mucin-type
O-linked. The addition of N-linked glycans is initiated in
the ER by the addition to asparagine of an oligosaccharide
chain via an intermediate lipid carrier. This chain is then
modified by trimming and the addition of sugars. In contrast, mucin-type O-linked glycosylation is thought to be
initiated in the cis-Golgi cisternae where the first sugar,
N-acetyl-d-galactosamine (GalNAc),1 is added to the hydroxyl groups of serine and threonine (Roth et al., 1994 After the addition of the first GalNAc to threonine or
serine, chains are extended via various core structures,
generally with polylactosamine units, and the final structures (added to the same core protein) can be different in
different tissues, depending on the profile of glycosyltransferases expressed (Brockhausen, 1996 The key glycosyltransferases involved in the changes
seen in breast malignancies are the
The position of the Cell Culture
The cell line MTSV1-7 was grown in DME supplemented with 10% FCS
(GIBCO BRL, Gaithersburg, MD), 10 µg/ml insulin (Sigma Chemical Co., St. Louis, MO), 5 µg/ml hydrocortisone (Sigma Chemical Co.), and
0.3 µg/ml glutamine. The retroviral infectants of MTSV1-7 were maintained in the same media with the addition of 2 µg/ml of puromycin
(Sigma Chemical Co.). T47D and AM12 cells were grown in DME supplemented with 10% FCS (GIBCO BRL) and 0.3 µg/ml glutamine. The
T47D transfectants were grown in the same medium as the parental line
but with the addition of 500 µg/ml of G418 (GIBCO BRL).
Development of the The cDNA encoding human The product was digested with HindIII/NotI and cloned into the
pcDNAIneo expression vector (Invitrogen, San Diego, CA). This neo3STMYC construct was sequenced, and expression of the myc epitope tag was checked by transient expression in COS cells before being used
for stable transfection of T47D cells.
For production of an amphotrophic Transfection and Transduction of Cell Lines
T47D cell line was transfected directly with the neo3STMYC construct using the method of calcium phosphate transfection described above with
the following alterations. The DNA precipitate was left on the cells overnight before glycerol shocking the cells for 3 min. The cells were then
washed three times with PBS before refeeding with fresh medium. 2 d
later, the dishes were split 1:10 into medium containing 500 µg/ml G418,
and selection was carried out until individual clones were isolated and expanded. Selected clones were referred to as T47D 3STMYC. Cells were
also transfected with the vector pcDNA1neo and a clone, T47D neo, was isolated.
For transduction of MTSV1-7 cells with the Northern Analysis of mRNA from Cell Lines
Total cellular RNA from the cell lines was isolated according to the
method of Chomczynski and Sacchi (1987) Detection of Sialyltransferase Expression by Western
Blot Analysis
Confluent cell cultures were washed with cold PBS and lysed in RIPA
buffer (20 mM sodium phosphate, pH 7.2, 50 mM sodium fluoride, 5 mM
EDTA, 1% Triton, 1% deoxycholate). After clarification of the lysates by
centrifugation at 15,000 g for 10 min at 4°C, the protein concentration of
the lysates was estimated using the Bio-Rad protein assay kit (Bio Rad
Laboratories, Hercules, CA). Samples equivalent to 50 µg were electrophoretically separated on a 5-15% gradient/3% stacking SDS-PAGE gel
and transferred onto Hybond-C membrane (Amersham Intl.). Immunoblots were blocked with 5% skimmed milk/0.1% Tween-20 in PBS for 2 h,
incubated with 0.7 µg/ml anti-myc mAb, 9E10, for 1 h, and rinsed in 1%
skimmed milk/0.1% Tween followed by peroxidase-conjugated rabbit anti-
mouse secondary antibody (Dako Ltd., High Wycombe, UK) for 1 h. The
bands were visualized using the enhanced chemiluminescence detection
kit (Amersham Intl.).
Measurement of The Carbohydrate Structural Analysis
Changes in the carbohydrate side chains of MUC1 expressed in the transfected MTSV1-7 and T47D cell lines were analyzed directly by high performance anion exchange chromatography (HPAEC) as previously described (Lloyd et al., 1996 FACS® Analysis
Reactivity of Peanut Lectin with Live Cells.
Cells were incubated with or
without neuraminidase and analyzed by FACS®can for Arachis hypogaea
peanut agglutinin (PNA) (Sigma Chemical Co.) lectin binding as described
previously (Burchell and Taylor-Papadimitriou, 1993 Detection of the Tagged Sialyltransferase in Permeabilized Cells.
Cells were
stained with 9E10 mAb after incubating the cells with 0.3% saponin for 20 min. Subsequent steps were as previously described (Burchell and TaylorPapadimitriou, 1993) with the exception that 0.1% saponin was included
in all incubations and washes, which were carried out at room temperature. The reactivity of an mAb (LE61) to keratin 18 (Lane, 1982 Immunofluorescence Staining
Cells were grown on glass coverslips, washed with PBS, and fixed with 4%
paraformaldehyde for 15 min. Cells were permeabilized with 0.1% Triton
for 5 min, and then nonspecific binding was blocked with 10% FCS/PBS
for 30 min. The cells were then incubated with the 9E10 mAb to the myc
epitope (10 µg/ml) or TEX-1, a rabbit antiserum which recognizes mannosidase II (diluted 1:50) (Slusarewicz, 1994 Immunoelectron Microscopy
Cells were fixed for 1 h at room temperature in 0.1% glutaraldehyde/4%
paraformaldehyde before being scraped, spun, and stored overnight in 2%
paraformaldehyde at 4°C. Double labeling was performed as described
previously (Slot et al., 1991 Immunogold Quantitation
Golgi apparatus profiles were selected at random, photographed, and
printed at a final magnification of 75,000. The compartments of the Golgi
apparatus were defined as described previously (Nilsson et al., 1993 To assess the polarized distribution of gold particles within the Golgi
apparatus, for every Golgi apparatus analyzed, the position of each gold
particle was calculated as a fraction of the distance across the Golgi apparatus as follows: d1 = distance from cis face, d2 = distance from trans face,
and d1/(d1 + d2) is the position within the Golgi apparatus. Thus, individual gold particles were assigned to one of ten equal fractions, and the sum
of gold particles of each size in each fraction was expressed as a percentage frequency of the total number of each size of gold (Rabouille, C., and
T. Nilsson, personal communication).
Comparison of Sequences Coding for the Endogenous
and Transfected Bases 1-278 of the Development of Stable Cell Lines Expressing
The cDNA encoding the human The cell line MTSV1-7 has many characteristics of normal mammary epithelial cells. It is nontumorigenic, forms
organized colonies in collagen gels (Berdichevsky and
Taylor-Papadimitriou, 1991 Expression and Activity of the Northern blot analysis revealed that all the selected cell
lines were expressing the transfected DNA (Fig. 2 a) and,
moreover, the expression level varied with the clone, with
the MTSV1-7 transfectants expressing more than the
T47D clones. The difference in molecular weight between
the message in T47D clones and the MTSV1-7 is due to
the
Western blotting with the 9E10 antibody showed a specific band in the region of 46-48 kD, which corresponds to
the myc-tagged
Table I.
Sialyltransferase Activity in Mammary Epithelial Cell
Lines Transfected with cDNA Encoding
Analysis of the O-Glycans on MUC1 Expressed
by the Transfectants
We have previously shown that most of the O-glycans
added to MUC1 produced by T47D cells have either the
core-1 structure (Gal Fig. 5 shows the HPAEC profiles of the reduced oligosaccharides released from the MUC1 immunoprecipitates. In wild-type T47D and T47D neo, the labeled oligosaccharides were equally distributed between peaks 1 and 2 (Fig. 5, a and b). We have previously shown that
peak 1 contains neutral oligosaccharides consisting mainly of Gal
Table II.
Relative Distribution of Radiolabeled
Oligosaccharides of MUC1 from T47D, T47D neo, and T47D
3STMYC Cell Lines
As for the T47D series, the binding of PNA seen in the
parental MTSV1-7 cell line and the puromycin transfectant (Fig. 6, e and f) was not evident in the MTSV1-7 transfectants (Fig. 6, g and h), but it could again be induced by
neuraminidase treatment. Thus, the sialyltransferase was
also active in the MTSV1-7 transfectants in increasing the
level of sialylated core-1. To test whether the
Intracellular Localization of the Immunofluorescence microscopy with
the 9E10 mAb that recognizes the myc-tagged
Thin-sections of pellets of
the transfected MTSV1-7 and T47D cells were labeled
with the myc antibody followed by goat anti-mouse coupled to gold particles. From visual observations of the immunoelectron micrographs of both cell types, the gold labeling appeared to be localized over the Golgi stack and
was polarized to one side (Fig. 9, a and b). Since the structure of the Golgi apparatus was more clearly defined in
MTSV1-7 cells and the expression of the transfected gene
was higher in their transfectants, further detailed analysis
of the Golgi apparatus localization was performed with
one of the MTSV1-7 3ST clones (3.14). Golgi localization, was confirmed unambiguously by counting gold particles
over 18 Golgi apparatus and an equivalent area of nucleus,
as this demonstrated a labeling ratio of 90:1 Golgi stack to
nucleus.
To orientate the Golgi apparatus, an antibody to the
The number of each size of gold particles in the TGN
was also estimated, and the distribution in the Golgi apparatus and TGN is shown in Table III. Quantitation of the
5-nm gold particles showed that the GalT was localized
late in the Golgi stack, corresponding to the trans cisternae, and in the TGN (Table III and Fig. 10) as previously
reported (Rabouille et al., 1995 Table III.
Quantitative Distribution of ;
Clausen and Bennett, 1996
). The oligosaccharide chains
are then built up by the sequential addition of individual sugars, each reaction being catalyzed by a specific enzyme
or enzymes (Brockhausen, 1996
). Thus, the final structure
of the O-glycans is strongly influenced, not only by the
level of activity of an enzyme but also by its position
within the Golgi apparatus.
). Of great interest is
the observation that, in carcinomas, the composition of the
O-glycans added to the glycoproteins produced by the tumor cells may be altered compared with those expressed in normal cells. This has a profound effect on the structure
of those glycoproteins that carry multiple O-linked glycans
such as the epithelial mucins. This change in glycosylation
pattern has been best documented in breast cancer where
the MUC1 mucin (Gendler et al., 1990
) has been shown to
carry shorter and less complex O-glycans than the mucin
produced by normal cells (Hanisch et al., 1989
; Hull et al.,
1989
; Lloyd et al., 1996
).
2,3 sialyltransferase
(EC 2.4.99.4), which adds sialic acid to Gal
1,3 GalNAc
(core-1), and the
1,6 N-acetyl-d-glucosamine (GlcNAc)
transferase (EC 2.4.1.102), which forms core-2 from core-1
and is crucial for chain branching (Fig. 1) (Kuhns et al.,
1993
). These enzymes use the same substrate, and their effect is to terminate or initiate chain branching leading to
extension, respectively. We have recently observed that
the activity of the chain terminating enzyme,
2,3 sialyltransferase, is increased 8-10-fold in some breast cancer
cell lines, while core-2
1,6 GlcNAc T activity is either lost
or reduced (Brockhausen et al., 1995
). The expression of
the MUC1 mucin is upregulated in breast cancers and the
difference in glycosylation pattern causes the cancer-associated mucin to be antigenically distinct from the normal
mucin. MUC1-based immunogens are therefore prime candidates for cancer vaccines and several formulations are
being tested in the clinic.
Fig. 1.
Alternative pathways for O-linked glycosylation of
MUC1 involving either chain branching via core-2 1,6 GlcNAc
transferase or chain termination by addition of sialic acid via
2,3
sialyltransferase.
[View Larger Version of this Image (21K GIF file)]
2,3 sialyltransferase in the Golgi
apparatus will determine to some degree whether it can
compete directly with core-2
1,6 GlcNAc T for the Gal
1,3 GalNAc substrate. It is therefore important to precisely map its distribution within the Golgi apparatus.
cDNAs coding for the
2,3 sialyltransferase have been isolated from porcine submaxillary glands (Gillespie et al.,
1992
), murine brain (Lee et al., 1993
), and more recently from human placenta (Chang et al., 1995
). It is therefore
now possible to directly locate the transferase by transfecting a cDNA tagged with a sequence encoding an immunoreactive epitope (Nilsson et al., 1993
). We have tagged
the cDNA coding for the human
2,3 SAT (O) with an
epitope from the myc gene, and the construct has been
used to transduce or transfect two mammary epithelial cell
lines, both of which express the MUC1 mucin. The T47D
cell line was derived from a metastatic breast carcinoma (Keydar et al., 1979
), does not express core-2
1,6 GlcNAc
T, even at the level of mRNA (Brockhausen et al., 1995
),
and produces MUC1 carrying short O-glycans (Lloyd et al.,
1996
). The MTSV1-7 cell line was derived from normal human milk epithelial cells (Bartek et al., 1991
) and shows
many characteristics of normal cells (Shearer et al., 1992
),
including the ability to add core-2-based O-glycans to the
MUC1 mucin (Lloyd et al., 1996
). In the MTSV1-7 cell
line, the transfected
2,3 SAT (O) has been localized to the medial- and trans-Golgi cisternae, with some enzyme
being detected in the TGN. We have also demonstrated
that transfection of a cell with the
2,3 SAT (O) results in
increased sialylation of the surface protein, the MUC1 mucin, thus allowing the manipulation of O-linked glycosylation. In the MTSV1-7 transfectants, the increase in sialylated core-1 is accompanied by a decrease in the GlcNAc content of the O-glycans attached to MUC1, suggesting a
reduction in the synthesis of core-2-based structures.
Materials and Methods
2,3 Sialyltransferase
myc Constructs
2,3 SAT (O) was cloned into the HindIII/
XbaI site of pBluescript (Stratagene, La Jolla, CA). This was digested with
MroI and XbaI, and two annealing oligos encoding the myc epitope were
inserted. The sequence of these oligos was as follows: 5
CCGGATCTTCAAGGGGAGACCTGAACAGAAACTGATCTCTGAAGAAGACCTGTGAT 5
CTAGATCACAGGTCTTCTTCAGAGATCAGTTTCTGTTCAGGTCTCCCCTTGAAGAT. Bases encoding the myc
epitope recognized by the 9E10 mAb (Evan et al., 1985
) are underlined.
2,3 sialyltransferase-expressing
retrovirus, the HindIII/XbaI fragment was excised from pBluescript, blunt
ended with Klenow, and subcloned into the SnaBI site of the pBabe puro
vector. The amphitrophic packaging cell line AM12 was transfected with
this construct using calcium phosphate-mediated transfection. Briefly,
AM12 cells were grown to 70% confluency in 10-cm tissue-culture dishes
and refed with fresh medium 1 h before transfection. 15 µg of pBabepuro3STMYC DNA and 25 µg of carrier salmon sperm DNA were coprecipitated with calcium phosphate at pH 7. After 6 h at 37°C, dishes were
rinsed five times with serum-free medium and then refed with fresh medium. 48 h after transfection, cells were split 1:10 into growth medium
containing 2 µg/ml puromycin for selection. Medium was changed every
3-4 d for 4 wk until selection was complete. The
2,3 sialyltransferase-retrovirus producer cell line was grown to 70% confluency, and the spent medium was replaced with half the volume of fresh medium. 3 d later, the
virus-containing medium was removed, filtered, quick frozen in dry ice,
and stored at
70°C.
2,3 sialyltransferase retrovirus, the cells were grown to 70% confluency in 10-cm dishes and infected with 3 ml of viral stock (with or without dilution) with 8 µg/ml polybrene. Infection proceeded for 3 h before virus was replaced with fresh
medium. 2 d later, the plates were split 1:10 into medium containing 2 µg/ml
puromycin, and selection continued until individual colonies could be
identified and expanded. Selected clones were referred to as MTSV1-7
3STMYC. Cells were also infected with a retrovirus derived from the
pBabe puro vector, and a clone MTSV1-7 pBpuro was isolated.
. 25 µg of RNA from each cell
line was denatured in 1× MOPS buffer, 0.66 M formaldehyde, and 50%
(vol/vol) formamide, and subsequently size fractionated on a 1.3% agarose-formaldehyde gel. The RNA was transferred and immobilized onto
Hybond-N membrane (Amersham Intl., Little Chalfont, UK). The membrane was hybridized with a 1.2-kb HindIII/XbaI cDNA fragment from
the
2,3 SAT (O) plasmid according to the method of Church and Gilbert
(1984)
and washed to highest stringency as described previously (Brockhausen et al., 1995
). To assess the efficiency of loading and transfer of the
RNA, the membrane was reprobed for 18S expression. For detecting the
overexpressed transfected
2,3 SAT (O), the hybridized blot was exposed
to film overnight. For detection of endogenous transcripts, blots were exposed for 6 d.
2,3 Sialyltransferase Activity
2,3 sialyltransferase activity was measured in the transfected or
transduced lines as described previously (Brockhausen et al., 1995
).
). Briefly, cells were metabolically labeled with
100 µCi/ml [3H]glucosamine-hydrochloride (Amersham Intl.) and MUC1
immunoprecipitated with CT1, an antibody to the cytoplasmic tail of
MUC1 (Pemberton et al., 1992
). The carbohydrate side chains were released by alkaline borohydride treatment. Samples containing 10,000 cpm
were then separated on a Carbo Pak PA1 column (Dionex Corp., Sunnyvale, CA) using a gradient of 0.2 M NaOH to 0.2 M NaOH-0.25 M sodium acetate at 1.0 ml/min over 30 min (Lloyd and Savage, 1991
). Collected radioactive fractions were neutralized with 1 M HCl before counting. For
hexosamine analysis, the immunoprecipitate was eluted in 2% SDS and
hydrolyzed in 2 N trifluoroacetic acid at 100°C for 3 h, and 5,000 cpm samples were analyzed by HPAEC on a CarboPak PA1 column by isocratic
elution with 0.01 M NaOH at 1.0 ml/min.
).
) was included as a positive control for staining the permeabilized cells.
), and binding was detected with
FITC-conjugated goat anti-mouse (diluted 1:40; Dako) or rhodaminelabeled swine anti-rabbit (diluted 1:40; Dako) secondary antibodies.
). Antibody details are as follows: the grids
were incubated overnight at 4°C with 9E10 supernatant, diluted 1:5, followed, after washing, for 30 min with goat anti-mouse Ig conjugated to
10-nm gold particles. Galactosyltransferase rabbit polyclonal antibody
(Watzele et al., 1991
) was diluted 1:50 in 1% BSA, and the grids were incubated for 30 min at room temperature before washing followed by a 30min incubation with protein A coupled to 5-nm gold. The grids were embedded in 1.8% methyl cellulose/0.4% uranyl acetate before examination using a Zeiss 10C (Oberkochen, Germany) or JEOL 1010 (JEOL USA, Peabody, MA) electron microscope.
). Briefly,
the trans cisternae were defined as the last continuous cisternae that labeled for Gal-T, and the TGN comprises the tubuloreticular network adjacent to the trans side of the Golgi apparatus stack. The boundary of the
Golgi cisternae and TGN (defined as the interface between the outermost
membranes of the tubular network and the adjacent cytoplasm) (Rabouille et al., 1995
) was drawn on each micrograph. Gold particles over the TGN and the Golgi cisternae were directly counted and included gold
over budding vesicles but not structures such as vacuolar endosomes,
which can be found in the TGN and were excluded.
2,3 SAT (O)
2,3 SAT (O) DNA were PCR amplified from T47D
and MTSV1-7 genomic DNA prepared as described previously (D'Souza
et al., 1993
), using the following oligonucleotides: 5
GAATTCGAATTCGGACTGCGAAGATG and 5
AAGCTTAAGCTTAAGAGCGCGTTCTGGGC. The PCR product was cloned into the HindIII/EcoRI sites
of pBluescript and sequenced using the ABI PRISM automated sequencing method (Perkin-Elmer Corp., Norwalk, CT), for comparison with the
2,3 SAT-expressing plasmid.
Results
2,3 Sialyltransferase
2,3 sialyltransferase tagged
with a 30-bp stretch of DNA encoding the 9E10 epitope of
the myc protein was cloned into a mammalian expression
vector (pcDNAI neo). At the COOH terminus of this type
II transmembrane protein, the 9E10 epitope is far away
from the membrane-spanning domain that has been
shown to be involved in the localization of glycosyltransferases to their correct compartment within the Golgi (Machamer, 1993
). The construct was transfected into the breast
carcinoma cell line, T47D, and two stable cell lines (designated T47D 3ST 2 and 3) were selected in the presence of
gentimycin. The MUC1 mucin produced by the parental
T47D cells carries short O-glycans that do not contain
core-2 structures (Lloyd et al., 1996
).
; Shearer et al., 1992
; Lu et al.,
1995
), and glycosylates MUC1 in a manner similar to that
of normal mammary epithelial cells in adding core-2-based
O-glycans (Lloyd et al., 1996
). MTSV1-7 has proven to be
difficult to transfect, and a retrovirus expressing the myctagged
2,3 sialyltransferase was constructed (see Materials and Methods) and used to transduce MTSV1-7. Five
stable cell lines (designated MTSV1-7 3ST 3.10, 3.11, 3.14, 4.1, and 5.2) were selected in the presence of puromycin.
2,3 Sialyltransferase in
the Transfectants
2,3 sialyltransferase RNA in the MTSV1-7 cells being
expressed within the context of the retrovirus vector. Fig.
2 b shows endogenous expression of
2,3 SAT (O) in
T47D and MTSV1-7 parental cell lines, where the Northern blot was exposed for a longer period of time.
Fig. 2.
Northern blot analysis of 2,3 sialyltransferase
expression in (a)
2,3 SAT
(O) transfectants and (b)
wild-type T47D and MTSV1-7 cells. Blots were prepared
and probed as described in
Materials and Methods. The
bands representing the transfected gene are shown in a
after overnight exposure of
the film. The difference in
size of the mRNA transcript
in T47D and MTSV1-7 transfectants is due to the use of
different vectors for introducing the gene. The blot in
b was exposed for 6 d to detect endogenous transcripts.
[View Larger Version of this Image (41K GIF file)]
2,3 SAT (O). Fig. 3 a shows the blots for the
highest expressing MTSV1-7 and T47D transfectants, indicating higher expression of the enzyme in the MTSV1-7
clone. Expression of the protein was also confirmed by
FACS® analysis of permeabilized cells. Fig. 3 b shows the
3STMYC clone of T47D, the MTSV1-7 3STMYC, and
the T47D neo and MTSV1-7 puro cell lines. As seen on the
Western blots, the expression of
2,3 SAT (O) by the T47D
clones was considerably weaker than by the MTSV1-7
clones. However, the positive shift in 9E10 staining observed in the T47D transfectants was consistent and significant as the expression of keratin 18 completely overlaps
in the T47D neo and T47D transfectants (Fig. 3 b). Functionality of the expressed enzyme was demonstrated by
showing an increase in the specific activity of the enzyme
in the transfected clones. Table I shows the activity of the
enzyme in cell extracts of the clones in comparison with
cells transfected or transduced with vector alone. In the T47D clones,
2,3 sialyltransferase activity was elevated
about sevenfold compared with T47D neo, whereas the
MTSV1-7 clones had increased activity ranging from 19-
30-fold in comparison with MTSV1-7puro or wild-type
cells. The levels of activity of the enzyme correlated with
the levels of expression, (Figs. 2 and 3; Table I). (The levels of enzyme activity of the T47D series of cell lines and
the MTSV1-7 series are not comparable since they were
done at different times and do not reflect the increased level of activity present in the T47D cells [Brockhausen et al., 1995
]). The overexpression of
2,3 sialyltransferase had
no obvious effect on the morphology of the Golgi apparatus as shown by electron micrographs from wild-type
MTSV1-7 cells (Fig. 4 a) and one of the MTSV1-7 3ST
clones (Fig. 4 b).
Fig. 3.
Expression of 2,3 SAT (O) protein. (a) Western blot
analysis of myc-tagged
2,3 SAT (O) expression in T47D and
MTSV1-7 3STMYC transfectants, carried out as described in Materials and Methods. The expected band was detected by the antimyc antibody (9E10) in the transfectants but not in the parental
cell lines. M, markers. (b) Permeabilized cells were stained with
antibodies 9E10 (to the myc epitope) or LE61 (to keratin 18) and
subjected to FACS®can analysis as described in Materials and
Methods. (Left) Staining of T47D transfectants. (Right) Staining
of MTSV1-7 transfectants. (Thin continuous lines) T47D neo or
MTSV1-7 puro stained with 9E10; (thick continuous lines) T47D
neo or MTSV1-7 puro stained with LE61; (dashed lines) T47D
3STMYC or MTSV1-7 3STMYC stained with 9E10; (dotted lines)
T47D 3STMYC or MTSV1-7 3STMYC stained with LE61.
[View Larger Version of this Image (27K GIF file)]
2,3 Sialyltransferase,
2,3 SAT (O), or with Vector Only
Fig. 4.
EM analysis of (a) MTSV1-7, (b) MTSV1-7 3STMYC, (c) T47D, and (d) T47D 3STMYC, showing that the structural morphology of the Golgi apparatus has not been obviously altered by overexpression of the 2,3 sialyltransferase. Both transfected and untransfected cell lines show well-stacked Golgi consisting of several cisternae.
[View Larger Version of this Image (225K GIF file)]
1,3 GalNAc) or sialylated core-1. On
the other hand, 64% of the O-glycans added to MUC1
produced by MTSV1-7 cells are disialylated core-2-based
structures, the rest being mainly unsialylated core-1 (Lloyd
et al., 1996
). The same method was now used to analyze the oligosaccharide side chains on MUC1 produced by
T47D transfectants. The cells were metabolically labeled
with [3H]glucosamine and MUC1 immunoprecipitated
with the polyclonal antibody, CT1 (Pemberton et al., 1992
).
Since CT1 reacts with the cytoplasmic tail of MUC1, its
binding is not influenced by the carbohydrate attached to
the extracellular domain, and therefore all the glycoforms
of MUC1 are precipitated.
1-3 GalNAc-ol with a small amount of GalNAc-ol,
whereas peak 2 contains monosialylated Gal
1-3 GalNAc-ol (Lloyd et al., 1996
). Strikingly, the
2,3 SAT (O)
transfected T47D clones show an increase in the proportion of the monosialylated peak (Fig. 5, c and d, peak 2;
Table II), and a corresponding reduction of the neutral
peak (Fig. 5, c and d, peak 1; Table II). These data indicate
that activity of the
2,3 sialyltransferase in the transfected cell lines has resulted in the increased sialylation of the
Gal
1,3GalNAc substrate. Further confirmation of the increase in sialylated core-1 comes from FACS® analysis using
PNA lectin, which binds to the unsialylated disaccharide (Gillespie et al., 1993
). Fig. 6 shows that, while the parental T47D (a) and T47D neo transfectant (b) bind the lectin, in
the transfected clones PNA binding is totally absent (c and
d). However, after removal of sialic acid with neuraminidase treatment, both the transfectants and the untransfected cells show equivalent binding of peanut lectin.
Fig. 5.
HPAEC analysis of
radiolabeled reduced oligosaccharides from MUC1. (a)
T47D, (b) T47Dneo, (c)
T47D 3STMYC 2, and (d)
T47D 3STMYC 3. MUC1
was immunoprecipitated from
[3H]glucosamine-hydrochloride-labeled cell lysates with
the polyclonal antibody CT1
(), or with preimmune serum (
). Peak 1 consists of
neutral oligosaccharides, peak
2 corresponds to monosialylated species, and peak 3 elutes with disialo oligosaccharides (Lloyd et al., 1996
).
The extra peak at 9 min in b
is free NeuAc released during the work up of the sample.
[View Larger Version of this Image (22K GIF file)]
Fig. 6.
FACS®can analysis of the effect of increased 2,3 SAT
(O) expression on Arachis hypogaea lectin (PNA) binding. (a)
T47D; (b) T47Dneo; (c) T47D 3STMYC 2; (d) T47D 3STMYC 3;
(e) MTSV1-7; (f) MTSV1-7 puro; (g) MTSV1-7 3STMYC 3.14;
and (h) MTSV1-7 3STMYC 4.1. Cells were incubated with media
only (thin continuous lines), or with PNA labeled with fluorescein
in the presence (dotted lines) or absence (thick continuous lines)
of neuraminidase. The x-axis shows cell number and the y-axis
shows log fluorescence intensity.
[View Larger Version of this Image (26K GIF file)]
2,3 SAT
(O) enzyme was affecting side chains that contain GlcNAc
(Fig. 1), the radiolabeled MUC1 precipitates from the
MTSV1-7 puro and MTSV1-7 3ST clones were analyzed
for hexosamine content. Fig. 7 a shows the elution profiles
of the puromycin clone, and Fig. 7 b shows the corresponding profile of one of the sialyltransferase transfectants. It can be clearly seen that, in the
2,3 SAT (O) transfectant, there is a marked increase in the proportion
of counts eluting in the first peak, which corresponds to
galactosamine, compared to the second peak, which corresponds to glucosamine. This reduction in GlcNAc content
clearly demonstrates a loss of chain branching and/or extension of the side chains on the MUC1 expressed by the
MTSV1-7 transfectants.
Fig. 7.
Hexosamine content of O-glycans attached to MUC1
produced by 2,3 SAT (O) transfected MTSV1-7 cells. HPAEC
analysis of radiolabeled hexosamines released after acid hydrolysis of MUC1 immunoprecipitates from (a) MTSV1-7 puro and
(b) MTSV1-7 3STMYC 5.2. Peak 1 contains GalN; peak 2 contains GlcN.
, CT1;
, preimmune serum.
[View Larger Version of this Image (15K GIF file)]
2,3 Sialyltransferase
2,3 SAT
(O) of the MTSV1-7 3ST and T47D 3ST cell lines gave the
expected pattern of staining. The
2,3 sialyltransferase gave a perinuclear staining, often located to one side of the nucleus, characteristic of Golgi apparatus staining (Fig. 8 a). No staining was observed elsewhere in the cell and staining of unfixed cells showed no positive surface staining
(data not shown). To confirm the localization in the Golgi
apparatus, the cells were double labeled using a polyclonal
antibody to mannosidase II, a resident of the Golgi medial
cisternae, and 9E10. Fig. 8 b shows that these enzymes are
coexpressed, as shown by the yellow staining resulting
from the overlapping of fluorescein and rhodamine second
antibodies, thus confirming localization to the Golgi apparatus of the recombinantly expressed
2,3 sialyltransferase.
Fig. 8.
Immunofluorescence microscopy
of an MTSV1-7 3STMYC cell line. (a)
MTSV1-7 3STMYC cells were fixed, permeabilized, and labeled with 9E10 for myctagged 2,3 SAT (O) (green). (b) MTSV1-7
3STMYC cells show double labeling of the
same cells with an antibody against a resident Golgi marker, mannosidase II, such
that
2,3 SAT (O) myc (green) and mannosidase II (red) show overlapping distribution (yellow) to a compact, juxtanuclear
reticulum. Bar, 20 µm.
[View Larger Version of this Image (23K GIF file)]
Fig. 9.
Distribution of the stably expressed myc epitope-tagged 2,3 sialyltransferase in the Golgi apparatus of T47D (a) and
MTSV1-7 (b-d) cell lines. In a and b, the sections were stained by single label immunogold microscopy using the 9E10 antibody and a
second antibody coupled to 10-nm gold particles (solid arrowheads). c and d show double labeling of MTSV1-7 3STMYC 3.14 with polyclonal anti-GalT and monoclonal 9E10 antibodies followed by protein A coupled to 5-nm gold (GalT; open arrowheads) or goat anti-
mouse coupled to 10-nm (
2,3 SAT [O]; closed arrowheads) gold.
[View Larger Version of this Image (134K GIF file)]
1-4 galactosyltransferase (GalT) operative in N-linked
glycosylation (Watzele et al., 1991
), which has been previously mapped to the trans cisternae and the TGN in HeLa
cells by immunoelectron microscopy (Nilsson et al., 1993
),
was used in double-labeling experiments (Fig. 9, c and d).
The relative distribution of the two enzymes was then determined by counting each size of gold particle across the
Golgi stacks, using a method developed by T. Nilsson and
C. Rabouille (personal communication). The distance between the beginning of the cis cisternae and the end of the
trans compartment was divided into 10 equal fractions and
converted to real distances (nm) using the magnification
factor of the micrograph. The number of gold particles of
each size, in each of the 10 fractions, was counted, converted to a percentage frequency, and plotted against distance as shown in Fig. 10. This method provides a more
detailed outline of the distribution of the GalT and
2,3
SAT (O) across the Golgi apparatus, rather than in each
cisternal profile, as the stack often consisted of more than
three cisternae, which could not therefore be simplified to
cis, medial, or trans.
Fig. 10.
Quantitation of the distribution of GalT and 2,3 SAT
(O) in MTSV1-7 cells. Proteins were localized as described in
Materials and Methods and illustrated in Fig. 9, and the distribution of the gold particles was expressed as relative percentage frequency across the Golgi stack (nm). 307 (Gal-T) and 354 (
2,3
SAT [O] ) gold particles were counted over 18 Golgi, and the results are expressed as the mean percentage frequency. Using the
Stratified Wilcoxon rank sum test, a Mann-Whitney statistic of
76820, P < 0.0001, shows that the difference in distribution between
2,3 SAT (O) and GalT is statistically significant.
[View Larger Version of this Image (18K GIF file)]
). The larger (10-nm) gold
particles identifying the
2,3 SAT (O) show that this enzyme is coexpressed in the trans cisternae but is equally
well expressed earlier in the stack in an area probably corresponding to the medial cisternae. 26% of the
2,3 sialyltransferase was also found in the TGN.
2,3 Sialyltransferase
and GalT in MTSV1-7 3STMYC 3.14
Sequencing the cytoplasmic tail, transmembrane domain,
and part of the stalk region of endogenous 2,3 sialyltransferase, prepared by PCR from T47D and MTSV1-7 (see
Materials and Methods), showed that the sequence was
identical to the transfected enzyme (data not shown).
Thus, the transfected enzyme contains the same sequence
that determines the residency of the endogenous
2,3 sialyltransferase in the Golgi apparatus.
The 2,3 sialyltransferase studied here is particularly relevant to the study of breast cancer since the activity of
the enzyme is elevated in breast cancer cell lines (Brockhausen et al., 1995
). Using in situ hybridization, we have
also recently noted elevation of expression of the mRNA
coding for the enzyme in some primary breast cancers,
with the less differentiated tumors showing a higher expression (Burchell, J., and R. Poulsom, manuscript in preparation). The data presented here show that increasing the expression of the
2,3 sialyltransferase in breast
epithelial cell lines results in increased sialylation of the
O-glycans added to the MUC1 glycoprotein, manifest as
an increase in sialylated core-1 structures. Moreover,
the enzyme may compete with the core-2
1,6 GlcNAc
transferase (when it is expressed) for the common core-1
substrate with the result that the GlcNAc content of the O-glycans is reduced (Fig. 7). Although the core-2
1,6
GlcNAc T is absent in some breast cancer cell lines, in others such as MCF-7, mRNA and enzyme activity have been
demonstrated (Brockhausen et al., 1995
). Our results indicate that the increase in the
2,3 SAT (O) activity seen
in the breast cancers could still inhibit chain extension
and influence the composition of the O-glycans added to a
tumor-associated antigen, even when the core-2
1,6 GlcNAc T is present and active. This in turn could affect
the behavioral properties of the tumor cell. As a result of
the complexity of the structure of the core-2-based oligosaccharides on MUC1 produced by MTSV1-7 (Lloyd
et al., 1996
), a detailed structural analysis of the side chains
is necessary to prove unequivocally that the decrease in
GlcNAc content is due to a reduction in core-2 branching
rather than a reduction in chain extension from galactose in core-1.
Since in O-glycosylation sugars are added individually
and sequentially, the position of an enzyme in the Golgi
apparatus relative to other enzymes active in the synthesis
of O-glycans will influence the final structure. The conclusion from the studies reported here is that the 2,3 sialyltransferase is located in the medial and trans cisternae of
the Golgi apparatus with some of this enzyme also being
present in the TGN. To localize the protein by immunoEM, the cDNA was tagged with an immunoreactive epitope (a sequence from the myc gene), and cells were transfected with the tagged gene. The inclusion of the myc
epitope (10 amino acids) at the extreme COOH terminus
(which in this case is in the lumen of the Golgi compartment) has previously been shown to have no effect on the
localization of glycosyltransferases (Rabouille et al., 1995
;
Nilsson et al., 1993
; Munro and Pelham, 1986
). In our studies, the tag clearly had no obvious effect on the folding of
the protein as the
2,3 SAT (O) was highly active, not only
in cell extracts (Table I) but also in the intact transfected cells, as shown by the increased sialylation of the MUC1
glycoprotein (Figs. 5, 6, and 7). Thus, anomalous localization
because of misfolding is unlikely. In addition, the sequences
that have been shown to be involved in the localization of
glycosyltransferases were identical in the endogenous and
transfected genes, making it likely that the coded proteins
were directed to the same compartment of the Golgi apparatus. The location of the sialyltransferase to within more
than one compartment of the Golgi apparatus supports
the view that the Golgi cisternae are characterized by their
different mixtures of these enzymes, not by discrete types
(Nilsson et al., 1993
; Rabouille et al., 1995
).
There is a formal possibility that overexpression of a glycosyltransferase may alter the fine localization of the enzyme. This has been suggested from data localizing the 1,2
N-acetyl glucosaminyltransferase (NAGT-1), where direct
immunolocalization of the endogenous enzyme showed localization to the medial compartment of the Golgi apparatus (Dunphy et al., 1985
), while the transfected tagged enzyme (fourfold increased expression) located to both the
medial and trans compartments (Nilsson et al., 1993
). It
must be noted, however, that the sensitivity of the method
used for quantitating the distribution throughout the Golgi
apparatus can affect the outcome, and with an increase in
the level of expression comes an increase in the sensitivity
of detection. The data of Rabouille and colleagues argue
against an effect of overexpression since the localization of
a tagged N-linked
2,6 sialyltransferase was not altered
when it was expressed at widely different levels (Rabouille
et al., 1995
). Even if overexpression of the enzyme does
result in a broadening of the distribution in the Golgi apparatus, this would also be expected to occur when the endogenous enzyme is overexpressed, as it is in breast cancers by 8-10-fold. Thus, any overlap or competition with
other glycosyltransferases such as is suggested from our
data would also be likely to occur in the malignant cells.
With the exception of the study of Roth et al. (1994),
who used an antibody to map the position of a GalNAc T
to the cis-Golgi apparatus, the positioning of enzymes involved in mucin-type O-glycosylation has relied mainly
on sucrose density gradient centrifugation (Chaney et al.,
1989
) or on following the glycosylation of marker proteins (Locker et al., 1992
). The work presented here represents
the first fine mapping of a sialyltransferase active specifically in O-glycosylation. Previous work by Locker and colleagues following the acquisition of oligosaccharides onto
the M protein of Coronavirus and the effects of brefeldin
A suggested that
2,3 SAT (O) is in an earlier compartment than the TGN (Locker et al., 1992
; LippincottSchwartz et al., 1990). The results presented here support
their interpretation of the data, as we place the
2,3 sialyltransferase in the trans and medial cisternae but also in the
TGN. However, with the cloning of genes encoding the
glycosyltransferases (Joziasse, 1992
; Clausen and Bennett,
1996
), it is becoming apparent that this group of enzymes
is extremely complex and that there is a large family of sialyltransferases (Tsuji, 1996
) that may be resident in different Golgi cisternae. Thus, it becomes imperative to localize each specific enzyme directly, which can be achieved
using tagged cDNA as described here, or by developing
antibodies to the expressed recombinant enzymes.
There is some evidence to suggest that increased sialylation of glycoproteins may be involved in malignant progression, and this question can now be addressed more directly by manipulating the composition of the O-glycans
synthesized by the tumor cells. In this context, it is significant that the product of the 2,3 SAT (O) enzyme,
NeuAc
2,3 Gal
1-3GalNAc, is the major ligand for sialoadhesin, a lectin expressed by macrophages (Kelm et al., 1994
), and cells expressing MUC1 carrying this O-glycan
are strongly bound by sialoadhesin (Crocker, P.R., personal communication). Whether increasing the level of
this glycan affects the macrophage infiltrate in tumors is a
specific question that can now be posed.
The change in glycosylation pattern seen in breast cancer also relates specifically to the studies on the MUC1
mucin as a potential target antigen in active specific immunotherapy of breast cancer. The extracellular domain of
the MUC1 glycoprotein is made up largely of tandem repeats of 20 amino acids: 25-100 depending on the allele
(Gendler et al., 1990), and each repeat contains potential
glycosylation sites (Nishimori et al., 1994
; Stadie et al., 1995
).
The antigenic profile of the mucin is therefore dramatically altered when the composition of the O-glycans added is changed from being core-2 based to the simpler, shorter,
and more heavily sialylated glycans found on the tumor
mucin. Indeed, both humoral and cellular responses to the
MUC1 mucin have been observed in breast cancer patients, who show some specificity for the aberrantly glycosylated mucin expressed by the tumor cells (von Mensdorff-Pouilly et al., 1996; Magarian et al., 1993
). Our findings
show that it may be possible to produce the appropriate glycoform of the mucin in recombinant form by manipulating the glycosyltransferases in the producer cell. CHO
cells, widely used for the production of recombinant proteins, do not in fact express the core-2
1,6 GlcNAc T (Li
et al., 1996
) and have been reported to synthesize short
O-glycans (Oheda et al., 1988
). It may then be relatively
simple to produce the MUC1 antigen in these cells with
minimal manipulation. Furthermore, although the results
presented here would support the hypothesis that the distributions of the core-2
1,6 GlcNAc transferase and the
2,3 sialyltransferase show some overlap, it will be important to confirm this by detailed mapping of the position of
the
1,6 GlcNAc transferase.
Received for publication 9 December 1996 and in revised form 3 April 1997.
1. Abbreviations used in this paper: GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; GalT,We thank Dr. J. Lau for his generous gift of 2,3 SAT (O) cDNA; Dr. E. Berger for the GalT antibody; Dr. G. Warren for the mannosidase II antibody; Jimmy Yang for carrying out the sialyltransferase assays; Mike
Bradburn for help with statistical analysis; and Drs. T. Nilsson (EMBL,
Heidelberg) and C. Rabouille (ICRF) for helpful discussion particularly
concerning the immunoquantitation.
This work was supported in part by a grant from the U.S. Public Health Service (National Institutes of Health CA 52477) and a travel grant to K.O. Lloyd from the Louisa Lewisohn Program of Memorial Sloan-Kettering/Imperial Cancer Research Fund.
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