(Received for publication, September 26, 1995; and in revised form, November 30, 1995)
From the
Treatment of mouse teratocarcinoma F9 cells with
all-trans-retinoic acid (RA) causes a 9-fold increase in
steady-state levels of mRNA for UDP-Gal:-D-Gal
1,3-galactosyltransferase (
1,3GT) beginning at 36 h. Enzyme
activity rises in a similar fashion, which also parallels the induction
of laminin and type IV collagen. Nuclear run-on assays indicate that
this increase in
1,3GT in RA-treated F9 cells, like that of type
IV collagen, is transcriptionally regulated. Differentiation also
results in increased secretion of soluble
1,3GT activity into the
growth media. The major
-galactosylated glycoprotein present in
the media of RA-treated F9 cells, but not of untreated cells, was
identified as laminin. Differentiation of F9 cells is accompanied by an
increase in
-galactosylation of membrane glycoproteins and a
decrease in expression of the stage-specific embryonic antigen, SSEA-1
(also known as the Lewis X antigen or Le
), which has the
structure Gal
1-4(Fuc
1-3)GlcNAc
1-R. However,
flow cytometric analyses with specific antibodies and lectins,
following treatment of cells with
-galactosidase, demonstrate that
differentiated cells contain Le
antigens that are masked by
-galactosylation. Thus, RA induces
1,3GT at the
transcriptional level, resulting in major alterations in the surface
phenotype of the cells and masking of Le
antigens.
Retinoids, which are natural and synthetic analogs of vitamin A,
alter in vitro growth and differentiation of a variety of
normal and neoplastic cell
lines(1, 2, 3, 4, 5) .
Profound changes in gene expression and pattern formation during
embryogenesis are regulated by retinoic acid (RA) ()via its
interactions with retinoic acid binding proteins and transcription
factors(6, 7, 8, 9, 10) . A
model system for studying such changes related to cellular
differentiation and development has been the mouse teratocarcinoma cell
line F9(11) . These cells exhibit characteristics of embryonic
cells from the inner cell mass of the blastula stage. When F9 cells are
treated with all-trans-retinoic acid, within 3 days they
differentiate into primitive endoderm-like cells, which begin to
synthesize basement membrane proteins, including type IV collagen and
laminin(11, 12, 13, 14, 15, 16) .
F9 cells contain many surface carbohydrate antigens that are shared
with those in early mouse
embryos(17, 18, 19, 20, 21, 22) .
Surface glycoconjugates in F9 cells contain the repeating disaccharide
[3Gal1-4GlcNAc
1]
or
poly-N-acetyllactosamine sequence (23, 24, 25) and the stage-specific embryonic
antigen SSEA-1 (also known as the Lewis X antigen or Le
),
which has the structure
Gal
1-4(Fuc
1-3)GlcNAc
1-R(17, 26) .
Differentiation of F9 cells induced by RA causes changes in expression
of surface glycoconjugates, and within 3 days following RA treatment
there is a marked decrease in expression of the Le
antigen(14, 19, 27) . However, the
mechanism by which RA/F9 cells express less Le
antigen is
unclear. Although it has been shown that 5 days following RA treatment
of F9 cells there is an 80% decrease in the level of an
1,3-fucosyltransferase activity that can synthesize the Le
antigen(28) , there is little change in this enzyme
activity following 3 days of RA treatment(29) . These changes
in Le
expression in differentiated F9 cells are relevant to
events occurring during early embryogenesis, since there are notable
changes in expression of Le
antigens during normal
differentiation of mouse embryos, and some studies have suggested that
Le
antigens play important roles in cell-cell adhesion and
development(30, 31, 32, 33) .
Cell extracts from both F9 and RA-treated F9 cells (RA/F9 cells)
contain a UDP-Gal:-D-Gal
1,3-galactosyltransferase
(
1,3GT) activity that synthesizes the terminal sequence
Gal
1-3Gal
-R(29, 34) . The presence of
this enzyme is consistent with studies suggesting that glycoproteins
from F9 and RA/F9 cells contain terminal
-galactosyl
residues(29, 35, 36) . However, the activity
of the
1,3GT is higher in RA/F9 cells, and there is an apparent
increase in
-galactosylation of surface glycoconjugates induced by
RA(29) . Little information is available about the induction of
the
1,3GT by RA, the glycosylation of proteins accompanying this
induction, and whether there is a relationship between changes in the
expression of terminal
-galactosyl residues on cellular
glycoproteins and the apparent decrease in expression of Le
antigens.
We have now examined the steady-state levels of the
1,3GT transcript during differentiation by RA and the enzymatic
activities in both cell extracts and cell culture media following RA
treatment. Our findings demonstrate that (i)
1,3GT is
transcriptionally regulated by RA treatment; (ii) differentiation of F9
cells results in increased activity of the enzyme and increased
expression of terminal
-galactosyl residues on selected cellular
glycoproteins; and (iii) the apparent loss of the Le
antigens on cell surface glycoconjugates in RA/F9 cells is in
part due to masking by terminal
-galactosyl residues.
Reaction mixtures for the 1,3GT routinely contained 30 mMN-acetyllactosamine, 20 mM MnCl
, 0.5
mM UDP-[
H]Gal (35,000 cpm/nmol), 5
mM ATP, 50 mMD-galactono-1,4-lactone, and
about 80 µg of cell extracts or 12.5 µl of 10-fold concentrated
cell culture media in a final volume of 25 µl. The reactions were
carried out at 37 °C for 3 h. The product of the enzyme reaction
was analyzed by Dionex high performance anion-exchange chromatography
using a Carbopac PA1 column (4
250 mm). The separation was
carried out in 160 mM sodium hydroxide for 15 min with a flow
rate of 1 ml/min. The fractions were collected and monitored by
scintillation counting. The radioactivity corresponding to the position
of standard Gal
1-3Gal
1-4GlcNAc was taken as the
product of
1,3GT. The values obtained from assays performed in the
absence of the acceptor were used as background. Assays for
UDP-Gal:GlcNAc
1,4-galactosyltransferase (
1,4GT) were
performed as described for
1,3GT, except that 20 mM GlcNAc was used as an acceptor, and cell culture media centrifuged
at 100,000
g for 1 h were used as an enzyme source
without concentration. The product N-acetyllactosamine was
identified by high performance anion-exchange chromatography, as
described above.
Figure 1:
Kinetics of
1,3GT mRNA induction in F9 cells by RA. A, total RNA was
isolated from F9 cells (0 h) and F9 cells exposed to RA for increasing
periods of time. 40 µg of total RNA from each time point was
isolated from the cells, electrophoretically separated on a 1% agarose
gel containing 7% formaldehyde, and transferred to nylon filters. The
immobilized RNAs were then hybridized with a
P-labeled
1,3GT cDNA probe, followed by stripping and reprobing with a
P-labeled mouse actin cDNA probe as a control. As negative
controls, RNAs isolated from CHO and COS7 cells were included. B, the magnitude of expression of
1,3GT was quantified by
densitometry, and the relative signal was normalized to the level of
actin message. The levels of expression of the
1,3GT transcript in
F9 cells were arbitrarily taken as 1 unit.
It has been reported that
dbcAMP enhances the transcriptional activation of genes by
RA(44) . Therefore, we tested the combined effects of RA and
dbcAMP on the changes induced in the transcript for the 1,3GT.
About 15 µg of total cellular RNA from untreated and treated cells
was analyzed by Northern blot analysis using a
P-radiolabeled murine
1,3GT cDNA in pCDM7. The
results demonstrate that after 3 days of treatment the inclusion of
dbcAMP and RA only slightly enhances the level of transcript over that
of RA alone (Fig. 2, A and B). Since the
levels of
1,3GT mRNA message of RA/F9 and that of RA/dbcAMP/F9 are
rather similar, we focused on the effect of RA alone in all other
experiments.
Figure 2:
Expression of 1,3GT mRNA in F9 cells
upon induced differentiation by RA or RA combined with dbcAMP. A, F9 cells were treated for 3 days with either
10
M RA alone or 10
M retinoic acid plus 10
M dibutyryl cyclic AMP (RA/dbcAMP). 15 µg of total RNA was
isolated from the cells and analyzed as in Fig. 1. B,
densitometry scans were used to quantify the relative signal in each
lane. Tubulin mRNAs were used to normalize for variations in the amount
and quality of RNA loaded in each lane. The level of expression of
1,3GT transcript in undifferentiated F9 cells was arbitrarily
taken as 1 unit.
Figure 3:
Nuclear run-on analysis of 1,3GT
transcripts in F9 cells and RA/F9 cells. Nuclei were isolated from F9
and RA/F9 cells, and transcription was performed in the presence of
[
-
P]UTP as described under
``Experimental Procedures.'' The labeled RNAs were hybridized
onto nylon filters containing pBluescript and
1,3GT, murine
-actin, and type IV collagen (
1) cDNA
fragments.
Figure 4:
Time course for RA-dependent induction of
1,3GT in F9 cells. F9 cells were exposed to RA for increasing
periods of time. A, approximately 80 µg of cell extracts
was assayed for
1,3GT activity by incubation at 37 °C for 3 h
using N-acetyllactosamine as an acceptor. The product was
isolated as described under ``Experimental Procedures.'' B, The culture media were concentrated 10-fold, and 12.5
µl portions were assayed for
1,3GT
activity.
In preliminary experiments, we found a considerable amount of
activity of the 1,3GT in the culture media from RA/F9 cells. We
therefore conducted a careful study of the enzymatic activity present
in the culture media at various times during differentiation. The total
enzyme activity was normalized to the amount of total cellular protein
on the dish. The
1,3GT activity in the cell culture media begins
to rise after 36 h of RA treatment and is approximately 4-fold higher
after 72 h of RA treatment (Fig. 4B). It can be seen
from the results in Fig. 4B that approximately of the
total
1,3GT enzyme activity detectable in the cultures of the
differentiated F9 cells is present in the culture media, and the
remaining is recoverable in cell extracts. These results demonstrate
that the
1,3GT is efficiently secreted by cells and that a
majority of the enzyme activity detected is present in a soluble form.
The increase in
1,3GT activity in media following RA addition
appears to rise with a time course slightly lagging behind the rise
observed in the cell-associated enzyme activity (Fig. 4, A and B).
The 1,3GT activity in culture media
arises by secretion from cells, since the growth media prior to
addition to the cultured cells has no detectable activity (data not
shown). In addition, the
1,3GT activity recoverable in the media
of RA/F9 cells cannot be pelleted by prolonged centrifugation at
100,000
g, although the cell-associated activity is
clearly microsomal (data not shown). This indicates that the enzyme in
culture media is soluble and is an active and probably a
proteolytically cleaved form, as has been observed for some other
glycosyltransferases(47, 48, 49, 50, 51, 52) .
To assess whether the increase in 1,3GT in F9 cells in response
to RA treatment is specific for this enzyme, we also measured the
activity of
1,4GT. It has been reported that
1,4GT activity
decreases to approximately one-third of control values during the first
3 days of differentiation of F9 cells following RA addition and then
begins to rise slowly on day 4 following RA treatment(53) .
Consistent with this, we found that the activity of the
1,4GT in
extracts of both F9 and RA/F9 cells is similar during the first 3 days
following RA treatment (data not shown). Our results are also
consistent with another recent study showing that there is little
change in either the
1,4GT activity or transcript levels within 3
days of treatment of F9 cells with RA(54) . The
1,4GT
activity is detectable in the cell culture media of both cell types,
but RA treatment causes only a small change (<2-fold increase) in
1,4GT activity in media after 72 h (data not shown). These results
demonstrate that RA treatment of F9 cells does not cause a general rise
in the activities of all galactosyltransferases but results in a
pronounced change in the expression and activity of the
1,3GT.
Figure 5:
Effects of RA on the production of laminin
in the F9 cell culture media. F9 cells were grown in the absence of RA (open circles) or in media containing 10M RA for 3 days (closed circles), and 100
µl of media was removed at each time indicated and coated onto
microtiter plates. The amount of laminin adsorbed onto the wells was
detected by anti-mouse laminin antibody, as described under
``Experimental Procedures.'' An arbitrary unit was
established by dividing the OD value (405 nm) by total cellular protein
amount in one plate of cells at different periods of RA treatment. The
results are the mean ± S.E. of triplicate
analyses.
Figure 6:
GS
I-B blotting of glycoproteins from F9 and RA/F9 cells. A, approximately 10 µg of microsomes from F9 and RA/F9
cells was separated by SDS-PAGE on a 10% polyacrylamide gel. The
proteins were transferred to nitrocellulose and incubated with 10
µg/ml of biotinylated GS I-B
in the presence or absence
of 200 mM raffinose followed by incubation with horseradish
peroxidase-conjugated streptavidin. The blots were developed using the
ECL chemiluminescence Western blotting kit. Mouse EHS laminin was
included as a positive control. B, serum-free culture media from each
cell line was also collected, and approximately 40 µg of soluble
protein was blotted with biotinylated GS I-B
. C,
RA/F9 media were prepared as described under ``Experimental
Procedures.'' Following immunoprecipitation with anti-laminin
antibody, bound proteins were subjected to SDS-PAGE on a 6%
polyacrylamide gel and blotted with biotinylated GS I-B
. D,
RA/F9 serum-free culture media and mouse EHS laminin were analyzed by
SDS-PAGE and immunoblotted with rabbit anti-mouse laminin antibody
followed by goat anti-rabbit IgG-peroxidase
conjugate.
Differentiation of F9 cells induced
by RA is also accompanied by a large increase in the terminal
-galactosylation of high molecular weight soluble glycoproteins
secreted into the cell media (Fig. 6B). A significant
band of the high molecular weight soluble glycoprotein in the range of
200-400 kDa reactive with GS I-B
is detectable in
RA/F9 serum-free cell culture media, whereas very little material in F9
serum-free cell culture media is reactive with GS I-B
. The
molecular weight of this GS I-B
-reactive material in the
growth media of RA/F9 cells suggested that it might be laminin. To
confirm the identity of this material, the RA/F9 culture media were
immunoprecipitated with anti-mouse laminin antibody, analyzed by
SDS-PAGE on 6% acrylamide gel, and then blotted with GS I-B
in the presence or absence of 200 mM raffinose. The
anti-mouse laminin antibody immunoprecipitates the major
-galactosylated components from RA/F9 cell culture media (Fig. 6C). As a control, the same RA/F9 culture media
and commercially purchased murine laminin were subjected to 6% SDS-PAGE
and analyzed by immunoblotting directly with rabbit anti-mouse laminin
antibody. The results obtained from immunoblotting the media of RA/F9
cells are similar to those obtained with commercial EHS laminin (Fig. 6D). Murine EHS laminin is known to contain
terminal
-1,3-galactosyl residues and to be reactive with GS
I-B
(62, 63) . Taken together, these data
demonstrate that the major
-galactosylated glycoprotein secreted
by RA/F9 cells is laminin.
To test for the possibility of masking of
Le determinants, the levels of Le
antigen on F9
and RA/F9 cells were analyzed by flow cytometry using the anti-Le
monoclonal antibody (anti-CD15 monoclonal antibody). As expected,
F9 cells express much higher levels of Le
antigen than
RA/F9 cells (Fig. 7, A and B). When F9 cells
are treated with
-galactosidase, there is no significant change in
the expression of the Le
antigen (Fig. 7A).
In contrast,
-galactosidase treatment of RA/F9 cells causes a
clear enhancement in the amount of surface Le
antigen (Fig. 7B). These results demonstrate that terminal
-galactosyl residues in differentiated F9 cells mask Le
determinants on RA/F9 cells generated by 3 days of RA treatment.
Figure 7:
Flow-cytometric analysis of F9 and RA/F9
cells for surface expression of Le antigens and terminal
-galactose residues. F9 cells (panels A and C)
and RA/F9 cells (panels B and D) were treated with
either
-galactosidase(+) or heat inactivated
-galactosidase(-) as described under ``Experimental
Procedures.'' The washed cells (5
10
) were
analyzed for surface Le
expression by direct immunostaining
with anti-CD15 monoclonal antibody (panels A and B)
and for surface expression of the terminal
-galactose residues by
indirect staining with biotinylated GS I-B
and
streptavidin-phycoerythrin conjugate (panels C and D). The numbers in each panel refer to the
experimental treatments as indicated on the right.
After treatment of F9 cells with -galactosidase, there is only
a slight decrease in GS I-B
staining (Fig. 7C). In contrast, treatment of RA/F9 cells with
-galactosidase causes a significant decrease in staining by GS
I-B
(Fig. 7D). These results demonstrate
that RA/F9 cells express more surface
-galactosyl residues than F9
cells and that many of the terminal
-galactosyl residues bound by
GS I-B
on RA/F9 cells are accessible to
-galactosidase. Taken together, these results support the
conclusion that RA treatment of F9 cells causes an increase in levels
of surface glycoconjugates containing
-galactosyl residues and
that these residues mask Le
determinants in RA/F9 cells.
To gain a full understanding of the roles of glycoconjugates
in development and differentiation and in disease conditions, it is
important to define the mechanisms regulating expression of
glycosyltransferases and their cognate glycoconjugate structures. In
many ways, F9 cells are ideal for studying these changes, since new
glycoconjugate expression is inducible by RA within 3 days in the
irreversibly differentiated cells. We previously reported that F9 cells
have an 1,3GT that appears inducible by
RA(29, 34) . Our studies now extend these findings to
show that differentiation of F9 cells is accompanied by a 9-fold
increase in steady-state levels of
1,3GT mRNA and that this
increase is transcriptionally regulated. This induction is associated
with secretion of the
1,3GT and enhanced
-galactosylation of
surface glycoproteins and secretion of an
-galactosylated
glycoprotein identified as laminin. Upon induced differentiation, there
is a marked decrease in expression of surface Le
determinants, but this decrease is due in part to masking of the
Le
determinants by
-galactosylation.
Although it is
appreciated that expression of glycosyltransferases in some cases is
regulated during cellular differentiation and transformation, little is
understood about the mechanisms controlling enzyme
expression(66, 67, 68) . Two of the best
studied glycosyltransferases in this respect are the sialyltransferases (69) and the 1,4GT. Within the
2,6-sialyltransferase
(ST6N) gene are at least four promoters, one of which is responsive to
liver-restricted transcription factors (70) and another which
appears to be B-cell specific and is regulated during B-cell
development(71, 72) . The
1,4GT is known to have
a regulatory element that controls transcript initiation (73) and may be related to hormone-dependent stimulation of the
enzyme in mammary glands(74) . Although the complete sequence
of the murine
1,3GT gene is known(75) , the cis-
and/or trans-acting elements that regulate the transcription
of
1,3GT are not yet understood.
There are several cases in
vitro where treatments of cells with various agents appears to
alter the levels of glycosyltransferases (76, 77, 78, 79, 80, 81, 82, 83) .
In regard to the 1,3GT, it has been observed that enzyme activity
is higher in mouse peritoneal macrophages elicited with
thioglycollate(84) , which may correlate with increased binding
to GS I-B
observed in stimulated macrophages compared to
resident mouse macrophages(85) . Other studies have examined
the effects of RA on glycosyltransferase activities in F9 cells, but
the results obtained are clearly different from those observed here for
the
1,3GT. Prolonged treatment of F9 cells with RA causes an
increase in activity for
1,6-N-acetylglucosaminyltransferase V and the core 2
1,6-N-acetylglucosaminyltransferase(86) . There
is little change of activity in these enzymes, however, after 3 days of
differentiation, but an increase in activity begins to occur after 4
days of differentiation.
The activity of some enzymes appears to
decline in F9 cells after RA treatment. The N-acetylgalactosaminyltransferase activity that converts
globoside to Forssman glycosphingolipid declines by approximately 70%
in F9 cells within 3 days following RA treatment (87) .
Treatment of F9 cells for 5 days with RA and dibutyryl cAMP results in
an approximately 80% decline in the activity of an 1,3FT (28) . However, using a different assay for fucosyltransferase
activity another group observed a slight increase in the activity of
fucosyltransferase upon RA treatment of F9 cells(88) . In the
case of
1,4GT, it was shown that
1,4GT activity in F9 cells
declines after 3 days of differentiation and then begins to rise around
5-6 days of differentiation, which correlates with increased
levels of transcript observed after 5-6 days of treatment with
RA(53) . Recently, it was reported that neither the
1,4GT
enzyme nor transcript levels change significantly in F9 cells treated
with RA alone, but treatment with RA and cAMP causes a 6.5-fold
induction of the transcript in 8 days(54) . Nuclear run-on
experiments demonstrated that this increase in
1,4GT was not due
to an increase in transcriptional rate, but it was due to
post-transcriptional regulation (54) . Induction of the
1,3GT by RA is clearly different from those other
glycosyltransferases cited above, in that elevated transcripts and
activity of the
1,3GT clearly begin to rise after 24-36 h
and are maximal at 3 days.
The time course of induction of
1,3GT transcripts by RA in F9 cells indicates that this gene is
induced like other known late or secondary genes in contrast to RA
induction of early or primary genes(44) . Examples of the early
inducible genes, which are transcriptionally activated within hours,
are ERA-1/Hox-1.6 (58) and the Hox 1.3(59) . Their
activation results from interaction of the RA-RA receptor complex with
their promoters(44, 89) . Examples of the late
inducible genes, which exhibit a delayed induction (24-48 h), are
laminin B1 and type IV collagen (
1)(55, 90) .
Whether the molecular mechanism for induction of the
1,3GT is
related to those for these other late inducible genes is currently
under investigation.
We observed that there is significant
1,3GT activity in the cell culture media and that
of the
total activity detectable in the cultured cells is recoverable in the
media. The amount of activity in the media increases in response to RA
treatment of the cells and is similar to the increase in activity
observed in the cell extracts. Soluble forms of some other
glycosyltransferases, such as
2,6-sialyltransferase and
1,4GT, have been found in various secretions and body fluids
including milk(91) , colostrum(92) , and
serum(48, 52, 93, 94) , and some
soluble glycosyltransferases have been purified from these
sources(91, 92, 95) . These soluble forms
appear to result from the proteolytic cleavage of the membrane-bound
forms of the enzymes(47, 48, 49) , and the
levels of some soluble enzymes are affected by disease status and
inflammation(50, 51) . Although the proteases
responsible for the solubilization of glycosyltransferases have not
been identified, there are suggestions that cathepsin D-like
proteases within the acidic trans-Golgi might be
involved(52) . The mechanism by which
1,3GT is secreted
into the culture media is not known, although we are currently
attempting to define the N terminus of the secreted
1,3GT to
identify the cleavage site. It would be expected that the cleavage
site(s) occur in the so-called ``stem-region,'' proximal to
the C-terminal catalytic domain(67) . The biological functions,
if any, for secreted glycosyltransferases are not known. It is
conceivable that the secretion of
1,3GT could be regulated and
purposeful and that the soluble enzyme could have a function during
differentiation of F9 cells. In preliminary studies, we have detected a
soluble and active form of the
1,3GT in mouse serum and media from
other cultured murine cells, indicating the secretion of soluble forms
of this enzyme may be a common occurrence in cells.
Although the
full-length transcript for the murine 1,3GT is 3.7 kilobases, four
different mRNA transcripts that differ in the length of the sequences
encoding the putative stem region have been detected by RNA-polymerase
chain reaction analysis(75) . It is thought that these
transcripts arise by alternative splicing of a pre-mRNA according to a
cassette model. Interestingly, although all four transcripts are
present in both F9 and RA/F9 cells, only a single transcript for bovine
1,3GT was observed in bovine thymus and MDBK cells(75) .
The splice variants generate enzymes predicted to vary particularly in
the stem region, which is predicted to contain the putative cleavage
site for proteases. Future studies are required to understand the
functions of different
1,3GT splice variants and whether these
isoforms exhibit differential cell and/or tissue expression.
There
are many studies demonstrating that expression of cell surface
carbohydrates is developmentally regulated, but the mechanisms of
regulation are largely unknown. Our finding that Le antigens may be masked in differentiated F9 cells is consistent
with some studies performed on mouse embryos. Both 8-cell mouse embryos
and embryonic ectoderm of 5- and 6-day-old embryos express Le
antigens, and
-galactosidase treatment enhances expression
of the antigen(96) . Although there is scant information about
antigen masking in vivo, such masking of carbohydrate
determinants has been performed in vitro using recombinant
glycosyltransferases. For example, transfection of cDNA for
2,6-sialyltransferase in Xenopus oocytes inhibits the
formation of polysialic acid onto neural cell adhesion
molecule(97) , and transfection of
2,3-sialyltransferase
into T lymphoblastoid cells converts the cells from a peanut agglutinin
positive (PNA
) phenotype to PNA
,
possibly caused by masking of the peanut agglutinin positive receptor,
Gal
1-3GalNAc
1-Ser/Thr, with sialic acid(98) .
Our study demonstrates, however, that antigen masking by
-galactosylation is a biochemical response of the embryonal
carcinoma cells to RA.
The observed masking of Le antigens on RA/F9 cells suggests that differentiated cells
synthesize the new sequence
Gal
1-3Gal
1-4(Fuc
1-3)GlcNAc
1-R
(
-galactosylated Le
). This possibility is supported by
recent evidence that Gal
1-3Gal
1-4GlcNAc can be
fucosylated in vitro using a partially purified
1,3-fucosyltransferase from human milk(64) . In contrast,
fucosylated oligosaccharides, such as
Gal
1-4(Fuc
1-3)GlcNAc
1-2Man, do not
serve as acceptors for
1,3GT(99) , indicating that there
must be an ordered addition of terminal sugars. Interestingly,
-galactosylated Le
structures occur normally in
oligosaccharides of mucins from cobra venom(65) . It is
conceivable that the carbohydrate phenotypes of F9 and RA/F9 cells are
reflective of a balance between competing glycosyltransferases for
available substrates. Competitive enzyme reactions are known to be
important in influencing the structures of newly synthesized
glycoconjugates(100-103). We previously demonstrated that
introduction of the murine
1,3GT into CHO cells results in
synthesis of glycoconjugates containing terminal
-galactosyl
residues and decreased levels of terminal sialic acid (45) .
Enzyme competition has been shown to be involved in the biosynthesis of
mucin oligosaccharides in porcine and ovine submaxillary glands (103, 104) and other mucin chains and blood group
antigens(100) .
The results of this study demonstrate that
differentiation of F9 cells into parietal endoderm-like cells is
accompanied by a profound increase in 1,3GT transcripts and enzyme
activity, resulting in changes in the surface carbohydrate phenotype
and masking of antigens. These changes may result in the synthesis of
carbohydrate structures containing
-galactosyl residues, which
could be functionally important for cellular interactions during murine
embryogenesis (30, 31, 32, 33) .
There is particular interest in Le
antigens or masked
Le
antigens because of evidence that they may participate
in intercellular adhesion events during early embryogenesis via direct
interactions with endogenous lectins or via direct
carbohydrate-carbohydrate interactions(33) . Although no
lectins have yet been found that exclusively prefer Le
itself or
-galactosylated Le
as a carbohydrate
ligand, many different animal lectins bind to oligosaccharides
containing the underlying Le
structure, such as sialyl
Le
and sulfated sialyl Le
(105) . Future
studies will be aimed at defining the mechanism by which the
1,3GT
gene is responsive to RA, the detailed structures of the masked
Le
antigens, and the possibility that murine cells express
lectins interactive with Le
-containing oligosaccharides.