(Received for publication, August 19, 1996, and in revised form, November 14, 1996)
From the Department of Pathology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
¶ Department of Physiology, University of Kansas Medical Center,
Kansas City, Kansas 66103
Successful pregnancy is dependent upon an array
of signaling proteins secreted by the trophoblast cells of the
placenta. Among these is a group of proteins related to pituitary
prolactin, known as the prolactin/growth hormone family. These proteins
are expressed at specific times during gestation and synthesized in
distinct trophoblast cell types in the rat placenta. We report here
that select members of this family, prolactin-like protein (PLP-A), PLP-B, PLP-C, decidual/trophoblast PRP, and placental lactogen I
variant, only which are expressed in the spongiotrophoblast, late in
rat placental development bear Asn-linked oligosaccharides terminating
with NeuAc2,6GalNAc
1,4GlcNAc
-R. This reflects the concurrent
expression of these prolactin/growth hormone family members with the
peptide-specific
1,4GalNAc-transferase and an
2,6-sialyltransferase, which can add sialic acid to terminal
1,4-linked GalNAc. We have determined that at least one of the prolactin-like proteins, PLP-A, is recognized by the
protein-specific GalNAc-transferase. The presence of
NeuAc
2,6GalNAc
1,4GlcNAc
-R on only a limited number of
glycoproteins synthesized by the spongiotrophoblasts between mid
gestation and birth reflects the need for both the GalNAc-transferase
and the peptide recognition determinant for efficient addition of
GalNAc. Thus, expression of the GalNAc-transferase and specific members
of the prolactin/growth hormone family is developmentally
regulated in the rat placenta, suggesting a physiological role for
the terminal NeuAc
2,6GalNAc
1,4GlcNAc
-R sequence on Asn-linked
oligosaccharides of these proteins.
The placenta is a complex organ that is essential for successful
growth and maturation of the fetus throughout pregnancy (1). In
addition to bringing oxygen and nutrients to the fetus, the placenta
produces a number of hormones, cytokines, and growth factors which
influence the endocrine, immune, and metabolic functions of the mother.
Among these is a group of closely related proteins synthesized by the
rat placenta, the prolactin/growth hormone family. We demonstrate here
that specific members of this family bear asparagine (Asn)-linked
oligosaccharides with NeuAc2,6GalNAc
1,4GlcNAc
. This is the
result of high expression levels of a protein-specific GalNAc-transferase in the rat placenta. Levels of the transferase increase from undetectable at mid gestation to maximal at day 18 of
gestation. This corresponds precisely with the expression of the
prolactin/growth hormone family members
PLP1-A, PLP-B, PLP-C, d/t PRP, and PL-Iv by
spongiotrophoblasts from the junctional zone of the placenta. At least
one of these family members, PLP-A, contains a recognition determinant
that can be utilized by this protein-specific GalNAc-transferase, which
we have previously shown (2-5) to mediate
1,4-linked GalNAc
addition to Asn-linked oligosaccharides on the pituitary glycoprotein
hormones lutropin and thyrotropin.
We have shown previously that unique Asn-linked oligosaccharides
terminating with the sequence
SO4-4-GalNAc1, 4GlcNAc
1,2Man, which is closely
analogous to the oligosaccharide structure on the placental prolactin
family members, are present on the pituitary glycoprotein hormones
lutropin and thyrotropin and are critical for the expression of full
biological activity in vivo (6-8). The sulfated
oligosaccharides determine the circulatory half-life of the
glycoproteins bearing them because they are recognized by a receptor,
the GalNAc-4-SO4 receptor, in hepatic endothelial cells,
which rapidly removes these glycoproteins from circulation (9-12). In
the case of lutropin, the short circulatory half-life is essential for
producing the episodic rise and fall of hormone in the blood throughout
the ovulatory cycle (13-15). The presence of terminal
GalNAc-4-SO4 on lutropin and thyrotropin reflects the
action of a protein-specific GalNAc-transferase and a
GalNAc4-sulfotransferase expressed in the gonadotrophs and
thyrotrophs of the pituitary (16). The peptide sequences recognized by
the GalNAc-transferase consist of a cluster of basic amino acids in the
subunit and a similar cluster of basic residues in the
subunit
of lutropin (4, 5). The recognition determinant on the
subunit, as well as GalNAc-4-SO4, is found on pituitary glycoprotein
hormones of all classes of vertebrates, indicating that these are
highly conserved and biologically important structures (17). In
addition, the levels of the GalNAc-transferase in the pituitary
gonadotroph rise and fall in parallel to the expression of lutropin in
response to changing estrogen levels thus assuring that the
oligosaccharides on lutropin are always fully modified with
GalNAc-4-SO4 (18).
A rapidly increasing number of glycoproteins that bear Asn-linked
oligosaccharides with 1,4-linked GalNAc, but which are unrelated to
the glycoprotein hormones, have subsequently been described. Like the
glycoprotein hormones, the
1,4-linked GalNAc on a number of these
glycoproteins has been shown to be modified with sulfate at the
4-hydroxyl (19-25). In other instances oligosaccharides have been
shown to bear terminal GalNAc (23, 25, 26), NeuAc
2,6GalNAc (25,
27-32), and/or GalNAc
1,4(Fuc
1,3)GlcNAc (25, 29, 32, 33). Each of
these structures is confined to a limited number of glycoproteins
because of the peptide specificity of the GalNAc-transferase. In
contrast to the sulfated oligosaccharides on lutropin and thyrotropin, biological significance of these structures remains to be established; however, the presence of such unique structures on limited numbers of
glycoproteins makes it highly probable that they too will be recognized
by specific receptors and play critical biological roles.
The concurrent expression in the rat placenta of the protein-specific
GalNAc-transferase and specific members of the prolactin/growth hormone
family which contain a peptide recognition determinant results in
nearly quantitative modification of their Asn-linked oligosaccharides with the termini NeuAc2,6GalNAc
1,4GlcNAc
. Taken together this suggests that these structures will play a role in
the expression of the biological activity of prolactin/growth hormone
family members during pregnancy.
[35S]PAPS was enzymatically synthesized as described previously (34) using 35SO4 from ICN. UDP-[3H]GalNAc and CMP-[3H]NeuAc were purchased from American Radiolabeled Chemicals, Inc., St. Louis. Wheat germ agglutinin (WGA), Wisteria floribunda agglutinin (WFA), and Clostridium perfringens neuraminidase were obtained from Sigma.
Animals, Tissue Preparation, and Tissue CultureTimed pregnant female Sprague-Dawley rats were obtained from Harlan Sprague-Dawley (Indianapolis). Animals were sacrificed by CO2 inhalation on days 9-21 of pregnancy (day 0 being the day rats were found to be sperm-positive). The placentas were removed and washed with ice-cold sterile 20 mM phosphate buffer containing 0.15 M NaCl (pH 7.4). Placentas were homogenized and used for glycosyltransferase assays, or tissue slices were made for isolation of secreted glycoproteins. Undifferentiated Rcho-1 trophoblast cells were obtained from subconfluent cultures in fetal bovine serum, and differentiated Rcho-1 trophoblast cells were obtained from confluent cultures maintained in donor horse serum as described in (35, 36).
Glycosyltransferase AssaysExtracts of whole rat placentas, isolated junctional and labyrinth zones, or Rcho-1 trophoblast cells were made as described previously (16). The protein concentration of the combined postnuclear supernatants was determined by the Bradford dye binding assay (Bio-Rad) using bovine serum albumin as a standard.
Transfer of GalNAc or Gal by the glycoprotein hormone
1,4GalNAc-transferase or the
1,4Gal-transferase, respectively, to Asn-linked oligosaccharides acceptors on hCG was compared using the
assay described previously (18).
[3H]GalNAc transfer to Asn-linked oligosaccharides of different protein acceptors was assessed as described previously (4). Recombinant prolactin-like protein A (PLP-A) and recombinant prolactin-like protein B (PLP-B) were isolated from Chinese hamster ovary culture medium (37, 38). Day 18 rat placenta postnuclear supernatant (100 µg) or partially purified bovine pituitary GalNAc-transferase (2.4 microunits) was incubated with 0.25 mM UDP-[3H]GalNAc (1 × 107 cpm) and either agal-Trf, agal-hCG, agal-rPLP-A, or agal-rPLP-B at either 4 µM or 7.5 µM concentrations under conditions used previously for the GalNAc-transferase assay (4). The enzyme reaction was terminated by the addition of 450 µl of 0.1 M Tris-HCl (pH 8.0), 0.02 M CaCl2 containing 1 mg of Pronase (Calbiochem), and [3H]GalNAc incorporation was determined as described previously (4).
Rat placenta postnuclear supernatants were assayed for
2,6-sialyltransferase activity using GalNAc-Trf (39), a modification of a method described previously (40). Reactions consisted of 100 µg
of postnuclear supernatant protein, 50 mM sodium cacodylate (pH 6.0), 0.5% (w/v) Triton X-100, 50 µg of bovine serum albumin, protease inhibitors described above for the GalNAc-transferase assay, 2 µM CMP-[3H]NeuAc (2 × 105
cpm), and 20 µM GalNAc-Trf in a total volume of 60 µl.
The enzyme reaction was terminated by the addition of 40 µl of 5 mg/ml bovine serum albumin and 100 µl of ice-cold 10% (w/v)
trichloroacetic acid, 4% (w/v) phosphotungstic acid. The precipitated
protein was pelleted by brief centrifugation, the supernatant
containing unincorporated CMP-[3H]NeuAc was discarded,
and the pellet was resuspended and washed three times with 5%
trichloroacetic acid. The final precipitated protein was solubilized
with 250 µl of 1 N NaOH, neutralized with 250 µl of 1 N HCl, and incorporated [3H]NeuAc was
determined by liquid scintillation counting.
Individual rat chorioallantoic placentas from mid to late pregnancy were sliced using a Stadie-Riggs slicer (A. H. Thomas Co., Philadelphia) and incubated in a 35-mm dish containing 2 ml of minimum essential medium/Earle's medium with penicillin (100 units/ml) and streptomycin (100 µg/ml). Alternatively, sliced placentas were incubated with sulfate-, cysteine-, and methionine-free minimum essential medium/Earle's containing 100 µCi/ml Trans35S-label (ICN Inc., Irvine, CA; 70% [35S]methionine, 1,173 Ci/mmol). In either case, medium was collected after 16 h of incubation at 37 °C under an atmosphere of 95% air, 5% CO2. Medium was separated from tissue debris by centrifugation at 10,000 × g for 20 min. Saturated ammonium sulfate was added to give a final concentration of 70% (v/v) and stirring for 1 h at 4 °C. Precipitated proteins were isolated by centrifugation at 10,000 × g for 20 min.
[35S]Cysteine/methionine-labeled ammonium sulfate pellets were resuspended in TBS (20 mM Tris-HCl (pH 7.5), 150 mM NaCl), and unincorporated 35S label was separated from the protein in 2-ml aliquots by gel filtration on a 45-ml bed volume column of Sephadex G-25 (Pharmacia Biotech Inc.) equilibrated in water. Protein-containing fractions were pooled and lyophilized. The proteins were resuspended in 200 µl of 20 mM sodium cacodylate (pH 6.0) and incubated with either C. perfringens or Newcastle disease virus neuraminidase as described below. The protein mixture was diluted to 1 ml with TBS and applied to a 1.5-ml bed volume column of WFA-agarose (EY-laboratories, Inc., San Mateo, CA) equilibrated in TBS. Following adsorption, the column was washed with TBS (~50-column volumes) until radioactivity was no longer detectable, and bound proteins were specifically eluted with TBS containing 50 mM GalNAc.
Nonradiolabeled ammonium sulfate precipitates were resuspended in TBS
and dialyzed against 3 × 2 liters of TBS and applied to 5-ml
columns of WGA-Sepharose equilibrated in TBS. The unbound fraction of
proteins was brought to 1 mM MnCl2 and 1 mM CaCl2 and adsorbed to 5-ml columns of
ConA-Sepharose (Pharmacia) equilibrated in TBS containing 1 mM MnCl2 and 1 mM
CaCl2. After washing with 25 column volumes of the
equilibration buffer, bound proteins were specifically eluted with TBS
containing 0.5 M -methyl mannoside.
WGA-Sepharose columns were washed with 25 column volumes of TBS, and bound proteins were specifically eluted with TBS containing 0.5 M GlcNAc. Following removal of GlcNAc by dialysis and lyophilization, the bound glycoproteins were resuspended in 200 µl of 20 mM sodium cacodylate (pH 6.0), treated with neuraminidase, and subjected to WFA affinity chromatography as described above.
Glycosidase DigestionsPeptide:N-glycanase F
digestions were carried out as described previously (10). Digestions
with C. perfringens neuraminidase, Newcastle disease virus
neuraminidase, or diplococcal -galactosidase were performed in 20 mM sodium cacodylate (pH 6.0). Digestion with jack bean
-hexosaminidase was carried out in 50 mM sodium citrate
(pH 4.5). All buffers contained protease inhibitors described above in
the GalNAc-transferase assay.
Glycoprotein fractions purified from rat placenta tissue slice medium (7.5 µg/well) were subjected to 12.5% SDS-PAGE and electroblotted on to Immobilon-P membranes (Millipore). Immobilized proteins were probed with either biotinylated WFA (20 ng/ml) (23) or with antisera directed against PLP-A (1:2,000), PLP-B (1:1,000), PLP-C (1:500), d/t PRP (1:2,000), or PL-Iv (1:1,000) (37, 38, 41-43).
In Vitro Incorporation of [35S]SO4Partially purified bovine pituitary GalNAc-4-sulfotransferase was incubated with 5 × 106 cpm of [35S]PAPS and 25 µg of potential glycoprotein substrates under conditions described previously for the GalNAc-4-sulfotransferase (17, 23). Enzymatic reactions were terminated by the addition of an equal volume of sample buffer (10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.003% bromphenol blue, and 62.5 mM Tris-HCl (pH 6.8)). 35S-Labeled proteins were separated by 10% SDS-PAGE and detected by autoradiography.
Extracts of placenta were
prepared at various times during gestation and tested for the presence
of the glycoprotein hormone-specific GalNAc-transferase and
GalNAc-4-sulfotransferase. GalNAc-transferase activity with the
expected properties was detected in placental extracts (Fig.
1), commencing at day 9 of gestation and increased 150-fold by day 18 before falling to lower levels just before parturition at day 21. The specific activity of the GalNAc-transferase on day 18 is 8-fold higher than that typical of rat pituitary extracts,
making late gestational rat placenta the richest source of glycoprotein
hormone GalNAc-transferase found to date. The placental
GalNAc-transferase displays the same protein specificity as the
pituitary enzyme transferring 78 pmol of GalNAc to the oligosaccharide
acceptor on agal-hCG compared with 1 pmol to the oligosaccharides on
agal-Trf at an acceptor concentration of 4 µM.
GalNAc-4-sulfotransferase was not detected in placental extracts at any
point during gestation (not shown). Furthermore, the specific activity
for 1,4Gal-transferase, which is not protein-specific (2), does not
change over the same time frame (Fig. 1). Thus, a GalNAc-transferase
with the same peptide and oligosaccharide specificity as the pituitary
GalNAc-transferase is expressed at high levels in the rat placenta
during pregnancy between days 9 and 21 of gestation.
The absence of detectable GalNAc-4-sulfotransferase activity suggested
that the oligosaccharides produced would either terminate with
1,4-linked GalNAc or be capped by a moiety other than sulfate. Oligosaccharides terminating with GalNAc
1, 4GlcNAc (23, 25, 26, 32), GalNAc
1,4(Fuc
1,3)GlcNAc (25, 29, 32, 33), or
NeuAc
2,6GalNAc
1,4GlcNAc (25, 27-32) have been described on
native and recombinant glycoproteins. Even though the high levels of
GalNAc-transferase activity suggested that large amounts of a
glycoprotein or glycoproteins bearing
1,4-linked GalNAc are produced
in the placenta during the late phases of gestation, affinity
chromatography on WFA-agarose did not reveal large amounts of
glycoprotein(s) with terminal
1,4-linked GalNAc following metabolic
labeling. This suggested that terminal GalNAc moieties were efficiently
capped with another group like sialic acid. Since the
2,6-sialyltransferase (EC 2.4.99.1) will transfer sialic acid to
either terminal
1,4-linked Gal or GalNAc (44), we tested for the
presence of
2,6-sialyltransferase in rat placenta membrane preparations from mid to late pregnancy using GalNAc-Trf, transferrin synthetically modified to contain Asn-linked oligosaccharides terminating with
1,4-linked GalNAc, as an acceptor substrate (Fig.
2). Sialyltransferase activity was detected beginning at day 12 and increased 5-fold by day 18 of pregnancy. Day 18 placental extracts were also able to transfer sialic acid to the hydrophobic substrate GalNAc
1,4GlcNAc
1,2Man-(CH2)8
COOCH3 (not shown). The incorporated
[3H] sialic acid was resistant to release by Newcastle
disease virus neuraminidase, which is specific for
2,3-linked sialic
acid, but was sensitive to release by C. perfringens
neuraminidase, which removes both
2,3- and
2,6-linked sialic acid
(not shown). The concurrent increase in
2,6-sialyltransferase and
the GalNAc-transferase activity suggested that the absence of
oligosaccharides containing terminal
1,4-linked GalNAc might reflect
efficient capping with
2,6-linked sialic acid to produce
NeuAc
2,6GalNAc
1,4GlcNAc
-R.
A Limited Number of Late Gestational Rat Placenta Glycoproteins Bear Asn-linked Oligosaccharides Terminating with NeuAc
To identify glycoproteins
bearing oligosaccharides with 1,4-linked GalNAc, tissue slices from
day 18 rat placenta were incubated with
[35S]cysteine/methionine for 16 h so as to
incorporate 35S label into the peptide backbone of newly
made glycoproteins. The medium containing secreted glycoproteins was
collected, gel filtered over Sephadex G-25 to remove unincorporated
35S label, and then incubated overnight in the presence or
absence of neuraminidase. These samples were then fractionated by
lectin affinity chromatography using immobilized WFA, which is specific for terminal
1,4-linked GalNAc (45). The bound
35S-labeled proteins were eluted and detected by
fluorography following separation by 10% SDS-PAGE (Fig.
3). Secreted placenta glycoproteins were not bound by
WFA prior to treatment with C. perfringens neuraminidase (Fig. 3, lane 3), whereas large amounts of a limited number
of glycoproteins were bound following digestion (Fig. 3, lane
1). Digestion with Newcastle disease virus neuraminidase did not
result in binding to the WFA column (Fig. 3, lane 2),
indicating that a major fraction of
1,4-linked GalNAc is substituted
with
2,6-linked sialic acid.
The secreted placenta glycoproteins recognized by WFA after
neuraminidase digestion are bound quantitatively by immobilized WGA.
Secreted, placental glycoproteins that are bound by immobilized WGA
represent 15% of the protein applied. The glycoproteins bound by WGA
were digested with neuraminidase, applied to WFA-agarose, and equal
amounts of protein from the unbound fraction (lane 1) and
bound fraction (lane 2) were separated by 12.5% SDS-PAGE
and transferred to polyvinylidene difluoride (Fig.
4A). Glycoproteins bearing terminal
1,4-linked GalNAc were visualized using biotinylated WFA. The
absence of glycoproteins reactive with biotinylated WFA in the unbound
fraction (Fig. 4A, lane 1) and their presence in the bound fraction indicated quantitative binding by WFA-agarose (Fig.
4A, lane 2). Digestion of glycoproteins eluted
from WFA-agarose with diplococcal
-galactosidase (Fig.
4A, lane 3) did not reduce their reactivity with
biotinylated WFA, whereas digestion with jack bean
-hexosaminidase
(Fig. 4A, lane 4), reduced the reactivity with
biotinylated WFA. Taken together these results indicate that glycoproteins bearing terminal
1,4-linked GalNAc are efficiently bound by immobilized WFA and that virtually all of the
1,4-linked GalNAc is capped by
2,6-sialic acid.
The GalNAc-4-sulfotransferase is highly specific for the terminal
sequence GalNAc1,4GlcNAc
-R (46). Incubation of the
neuraminidase-digested, WFA-bound fraction of glycoproteins from day 18 placenta with [35S]PAPS does not result in
35SO4 incorporation (not shown), confirming
that no endogenous GalNAc-4-sulfotransferase is present. The addition
of exogenous partially purified bovine pituitary
GalNAc-4-sulfotransferase results in 35SO4
incorporation into the oligosaccharides of multiple glycoproteins (Fig.
4B, lane 1) with mobilities similar to those
identified above by blotting with WFA (Fig. 4A, lane
2) and to those identified by affinity chromatography of
metabolically labeled glycoproteins on immobilized WFA (Fig. 3). The
labeled glycoproteins are of placental origin since only two bands are
radiolabeled if the partially purified GalNAc-4-sulfotransferase is
incubated with [35S]PAPS in the absence of added
placental proteins (Fig. 4B, lane 3). The
incorporated 35SO4 is quantitatively released
by digestion with peptide:N-glycanase F (lane 2),
indicating that terminal
1,4-linked GalNAc is present exclusively on
Asn-linked oligosaccharides of these placental glycoproteins.
The results presented above
indicated that only a select group of rat placenta glycoproteins bears
Asn-linked oligosaccharides modified with
NeuAc2,6GalNAc
1,4GlcNAc
. The time during which GalNAc-transferase activity increases in the rat placenta coincides with the expression of a subset of members of the prolactin/growth hormone family: PLP-A, PLP-B, PLP-C, d/t PRP, and PL-Iv (Fig. 5). d/t PRP and PLP-B are expressed in either the
decidua or the placenta from early pregnancy until term (38, 43, 47),
whereas PLP-A, PLP-C, and PL-Iv are expressed in the placenta beginning around gestational day 12-13 and increasing until parturition at day
21 (48). PLP-A and PL-Iv each has two electrophoretic forms of 33 and
29 kDa, whereas PLP-B has a single electrophoretic form of 30-31 kDa.
PLP-C and d/t PRP also each has a single electrophoretic form of 29 kDa. All are expressed in the junctional zone of the placenta which is
made up of trophoblast giant cells (TGC) and spongiotrophoblasts
at mid gestation (49). PLP-A, PLP-C, d/t PRP, and PL-Iv are expressed
in both these cell types, whereas PLP-B is expressed exclusively
in spongiotrophoblasts (49). Since a number of the rat placental
glycoproteins that are specifically bound by WFA-agarose have the same
apparent molecular weights as members of the prolactin/growth hormone
family and are expressed over the same time frame as the
protein-specific GalNAc-transferase, we examined this family of
glycoproteins for the presence of
1,4-linked GalNAc on their
Asn-linked oligosaccharides.
Secreted glycoproteins were isolated from gestational day 12, 15, 18, and 21 rat placental tissue slices by sequential lectin affinity
chromatography as described in Fig. 4. Glycoproteins bound by
WGA-Sepharose were digested with neuraminidase and separated into
fractions that were not bound () or bound (+) by WFA. These fractions
were separated by 12.5% SDS-PAGE, transferred to polyvinylidene difluoride, and probed with biotinylated WFA to determine their relative levels of
1,4GalNAc-containing oligosaccharides (Fig. 6). At day 12, when the transferase levels are low, no
glycoprotein binding to WFA-agarose is detected even though d/t PRP and
PLP-B are expressed at this time. At day 15, there is a 10-fold
increase in GalNAc-transferase activity (see Fig. 1) over day 12, and
there is a corresponding increase in binding of glycoproteins to
WFA-agarose (Fig. 6). Maximal levels of
1,4GalNAc-bearing
glycoproteins migrating with apparent molecular masses of 29 and 33 kDa
occurs on day 18 when the GalNAc-transferase levels are also maximal
(see Fig. 1). As the transferase levels decline on day 21, so does the
amount of WFA reactive material. Thus, the addition of
1,4-linked
GalNAc to rat placenta glycoproteins in the range of 29 and 33 kDa is proportionate to the levels of GalNAc-transferase during late gestation.
The WFA bound (+) and unbound () fractions were probed with
antibodies specific for PLP-A, PLP-B, PLP-C, d/t PRP, and PL-Iv (Fig.
7, WFA-agarose) to determine which members of this
family were modified with
1,4-linked GalNAc. PLP-A, PLP-B,
PLP-C, d/t PRP, and PL-Iv bound to WFA-agarose (Fig. 6), indicating
that they are all modified with
1,4-linked GalNAc.
The forms of PLP-A, PLP-B, PLP-C, d/t PRP, and PL-Iv which bear
NeuAc2,6GalNAc
1,4GlcNAc
are bound quantitatively by WGA and
WFA. In contrast, those glycoproteins not bound to WGA-Sepharose in the
isolation procedure do not bear
1,4-linked GalNAc since digestion
with neuraminidase does not result in binding to WFA-agarose or
incorporation of 35SO4 by exogenous
GalNAc-4-sulfotransferase (not shown).
The forms of PLP-A, PLP-B, PLP-C, d/t PRP, and PL-Iv present in the
unbound fraction from WGA-Sepharose were bound by ConA-Sepharose, indicating they are glycosylated (Fig. 7, ConA-Sepharose). The amount
of each placental prolactin family member in the WGA()/ConA(+) fraction was compared with that in the WGA(+)/WFA(+) fraction (Fig. 7)
to determine the proportion of each glycoprotein which is modified with
NeuAc
2,6GalNAc
1,4GlcNAc
. PLP-A is fully modified with GalNAc
as it is found almost exclusively in the WFA-bound fraction on days 15, 18, and 21 of gestation. In contrast, PLP-B, PLP-C, d/t PRP, and PL-Iv
are not fully modified with
1,4-linked GalNAc as they are present in
both the WGA(
)/ConA(+) and the WGA(+)/WFA(+) fractions. Only the 33 kDa form of PL-Iv is present in the WGA(+)/WFA(+) fraction, whereas
both the 33- and 29-kDa forms are present in the WGA(
)/ConA(+)
fraction. PL-Iv has two potential Asn glycosylation sites, and the 29- and 33-kDa forms are thought to arise from differential glycosylation
at one of these two sites. The presence of GalNAc exclusively on the
33-kDa form suggests that the less efficiently utilized glycosylation site on the 33-kDa form is a significantly better substrate for GalNAc
addition than the more efficiently modified glycosylation site found on
both the 33- and 29-kDa forms.
The results presented in Figs. 6 and 7 demonstrate that the proportion
of modification with terminal 1,4-linked GalNAc reflects the level
of GalNAc-transferase expressed; however, not all of the placental
prolactin family members are modified with the same efficiency (see
Fig. 7). Addition of
1,4-linked GalNAc to glycoproteins by the
peptide-specific GalNAc-transferase requires that the transferase and
target protein be expressed in the same cell type and that the target
protein contains a peptide determinant that is recognized by the
transferase. Differences in either the site of glycosylation and/or the
recognition determinant could account for this.
At mid gestation, dramatic changes occur in cell types that make up the rat uteroplacental unit (49). At day 9, decidualized uterus is abundant, and the developing placenta is made up of stem cells and TGC. By day 12 the decidua begins to regress, and spongiotrophoblasts develop in the junctional zone of the placenta. Expression of d/t PRP and PLP-B switches from anti-mesometrial cells of the decidua to spongiotrophoblasts of the placenta.2 Spongiotrophoblasts account for the bulk of PLP-A, PLP-C, and PL-Iv expression. The increase in number of spongiotrophoblasts that express placental prolactin family members coincides with the increase in expression of GalNAc-transferase activity, suggesting that GalNAc-transferase is expressed selectively by spongiotrophoblasts.
Homogenates were made from isolated day 10 decidua and either isolated
junctional or labyrinth zones from day 13, 16, and 19 of gestation.
1,4Gal-transferase activity, but no GalNAc-transferase activity, was
detected in decidua (not shown). In contrast, GalNAc-transferase was
present in both junctional (Fig. 8A) and
labyrinth (Fig. 8B) zones and showed the same pattern of
increasing and decreasing expression with time as whole placenta
homogenates. This is consistent with the absence of
1,4-linked
GalNAc on glycoproteins of decidual origin and their presence on
glycoproteins from the junctional zone. It is not clear which cell
types in the junctional zone express GalNAc-transferase, since at mid
gestation the junctional zone is made up of both spongiotrophoblasts
and TGC.
Although TGC cannot be separated from spongiotrophoblasts, the Rcho-1
trophoblast cell line can be induced to differentiate into TGC (35,
36). When cultured in the presence of fetal bovine serum under
subconfluent conditions, Rcho-1 trophoblast cells have the properties
of trophoblast progenitor cells, whereas Rcho-1 trophoblast cells
allowed to grow to confluence in the presence of horse serum
differentiate into TGC over a period of 12 days. Undifferentiated
Rcho-1 trophoblast cells contained 215 pmol/mg/h GalNAc-transferase
activity. Following induction to TGC, no GalNAc-transferase activity
was detected even though 1,4Gal-transferase activity was readily
detectable (not shown). These results suggest that GalNAc-transferase
is expressed exclusively in spongiotrophoblasts of the junctional zone
along with PLP-A, PLP-B, PLP-C, d/t PRP, and PL-Iv. Since these
placental prolactin family members are all expressed in
spongiotrophoblasts, the more efficient modification of PLP-A with
1,4-linked GalNAc than the other family members may reflect features
of the glycoproteins themselves, most likely recognition of the peptide
determinant.
We have shown that the glycoprotein hormone
GalNAc-transferase recognizes a peptide determinant as well as the
oligosaccharide acceptor (2-5). In the presence of this determinant
the catalytic efficiency for GalNAc addition to the same acceptor
oligosaccharide is increased by as much as 500-fold. The high levels of
transferase seen in near term rat placenta extracts could result in
modification of glycoproteins devoid of the recognition determinant.
This seems unlikely since even among the placental prolactin family
members examined (Fig. 7), significant amounts of PLP-B, PLP-C, d/t
PRP, and PL-Iv are not modified. Transfer of GalNAc to oligosaccharide acceptors on PLP-A and PLP-B were compared with transferrin, which does
not have a recognition determinant, and hCG, which has recognition determinants on its and
subunits (Table I).
PLP-A and hCG are both modified efficiently with GalNAc. PLP-B is
modified more efficiently than transferrin, 10-fold more GalNAc
incorporated, but is a poor substrate when compared with PLP-A (Table
I). PLP-A appears to have a recognition determinant that is recognized
with an efficiency similar to those on hCG. The recognition determinant on PLP-B, if present at all, is poorly recognized. The difference in
recognition of PLP-A and PLP-B by the GalNAc-transferase most likely
accounts for the lower amount of
1,4-linked GalNAc found on the
Asn-linked oligosaccharides of PLP-B even though PLP-A and PLP-B are
both synthesized in spongiotrophoblasts late in gestation.
|
The mammalian placenta is a complex tissue that performs a number
of essential functions, among them: 1) nutrient and waste exchange
between the fetal and maternal circulations; 2) modulation of the
maternal immune response to prevent rejection of the embryo; and 3)
transduction of fetal and maternal signals (1). The rat has proved to
be a useful model to study placental development (48, 49). Between
implantation and mid gestation the choriovitelline placenta, which
contains a single differentiated trophoblast cell type, the TGC, plays
the dominant role. From mid gestation onward the chorioallantoic
placenta, which consists of a junctional zone and labyrinth zone,
predominates. Of four differentiated trophoblastic cell types, TGC,
glycogen cells, and spongiotrophoblasts are present in the junctional
zone, whereas TGC and syncytial trophoblasts are present in the
labyrinth zone. Individual PLPs that are structurally and functionally
related to the prolactin and growth hormone produced by the pituitary
are expressed at characteristic times during gestation by cells within
the decidualized uterus, choriovitelline placenta, and chorioallantoic
placenta (see Fig. 5). The initial appearance and rapid increase in
glycoprotein hormone-specific GalNAc-transferase after day 13 of
gestation in extracts from both the junctional and labyrinth zones of
the chorioallantoic placenta suggested that specific trophoblast
lineages of the chorioallantoic placenta are induced to express high
levels of the GalNAc-transferase as they differentiate. The absence of
either GalNAc-transferase or glycoproteins bearing 1,4-linked GalNAc
on Asn-linked oligosaccharides in extracts from either decidualized
uterus or the placenta prior to day 13 and the direct correlation of
late developmental increases in GalNAc-transferase activity with
spongiotrophoblast development support this conclusion.
PLP-A, PLP-B, PLP-C, d/t PRP, and PL-Iv are among the glycoproteins
that we have shown bear Asn-linked oligosaccharides terminating with
NeuAc2,6GalNAc
GlcNAc
. PLP-A, PLP-C, and PL-Iv are expressed predominantly in spongiotrophoblasts from mid gestation to term. The
glycoforms of PLP-A, PLP-C, PL-Iv bearing
1,4-linked GalNAc are
virtually quantitatively modified with sialic acid since little if any
of these glycoproteins is bound by WFA prior to neuraminidase digestion, whereas each is essentially quantitatively bound following enzymatic removal of sialic acid. Thus, it is the concurrent expression of both the
2,6-sialyltransferase and the GalNAc-transferase in
spongiotrophoblasts over the same time frame as their glycoprotein substrates which accounts for the highly efficient modification of
their Asn-linked oligosaccharides with both GalNAc and sialic acid.
PLP-B and d/t PRP are also expressed in spongiotrophoblasts; however,
prior to day 13 of gestation they are expressed in cells of the
decidualized uterus (47). PLP-B and d/t PRP synthesized prior to mid
gestation are devoid of NeuAc2, 6GalNAc
GlcNAc
, whereas after
day 12 significant amounts of PLP-B and d/t PRP contain
NeuAc
2,6GalNAc
GlcNAc
, consistent with their synthesis in
spongiotrophoblasts. As a result of the change in the cell type
synthesizing these hormones, their glycoforms are altered.
We have shown previously that the glycoprotein-specific
GalNAc-transferase recognizes peptide as well as oligosaccharide
determinants (2-5). In the case of the glycoprotein hormone subunit, the peptide determinant consists of a cluster of basic amino
acids present in two turns of an
-helix. The crystal structure of
hCG led us to the conclusion that it is the proximity of these residues in three-dimensional space to the oligosaccharides rather than their
relationship within the linear amino acid sequence which is critical
for efficient transfer of GalNAc. PLP-A also contains a peptide
recognition determinant that is recognized by the GalNAc-transferase from bovine pituitary and is modified with nearly the same efficiency as hCG at a concentration of 7.5 µM. PLP-B, in contrast,
is modified almost 10-fold more efficiently than transferrin but at 1%
of the rate of hCG. Thus, even though PLP-B is expressed in
spongiotrophoblasts after day 12 of gestation, it is not modified to
the same extent as PLP-A. PLP-C and PL-Iv are also not completely
modified with GalNAc, suggesting that their peptide recognition
determinants are not as effective as that on PLP-A.
The rodent prolactin/growth hormone family members are homologous with
30-80% similarity in their amino acid residues (48). The crystal
structure of growth hormone is known; and based on similarities to the
placental prolactin family members, it has been predicted that each
prolactin family member consists of four tightly packed -helical
domains (51). There is little homology in the first three domains of
these proteins, but the fourth domain at the carboxyl terminus is
highly conserved. This domain contains a high number of basic amino
acids that may take on an
-helical conformation. Even though this
region is carboxyl-terminal to the Asn-linked oligosaccharides on each
of the placental prolactin family members, it is a good candidate
region for the peptide determinant recognized by the
GalNAc-transferase.
The terminal sequence NeuAc2,6GalNAc
1,4GlcNAc
has to date been
described on a number of glycoproteins. However, only a limited number
of the glycoproteins synthesized by the rat placenta between mid
gestation and birth contain this structure. Efficient modification of
these glycoproteins with
1,4-linked GalNAc requires that they
contain a peptide recognition determinant and be synthesized in cells
expressing the GalNAc-transferase. In the case of PLP-B and d/t PRP,
the mid gestational cell type switch from anti-mesometrial cells of the
decidua to spongiotrophoblasts of the placenta is accompanied by a
major change in their glycoforms.
What is the biological significance of oligosaccharides terminating
with NeuAc2,6GalNAc
1,4GlcNAc
rather than the more commonly encountered NeuAc
2,6Gal
1,4GlcNAc
? Our results demonstrate that the synthesis of the former structure is developmentally regulated and
that it is confined to a limited number of rat placenta glycoproteins. We are able to find PLP-A and other glycoproteins with the
NeuAc
2,6GalNAc
1, 4GlcNAc
structure in the maternal
circulation between mid gestation and birth, but not at other times.
Glycodelin (placental protein 14), a human decidual and placental
protein expressed in high levels early in pregnancy, is reported to
have immunomodulatory effects (52, 53). Glycodelin has recently been
shown to bear Asn-linked oligosaccharides terminating with
NeuAc
2,6GalNAc
1,4GlcNAc
(32). The authors suggest that this
oligosaccharide structure may have a role in immunomodulation; however,
direct evidence for this remains to be obtained.
The oligosaccharide structure terminating with
NeuAc2, 6GalNAc
1,4GlcNAc
may be highly characteristic of
pregnancy in a number of species including humans. There are a number
of potentially important roles for this structure. Receptors specific
for NeuAc
2,6GalNAc
1,4GlcNAc
could regulate circulating
half-life or direct glycoproteins bearing it to specific sites in the
mother or fetus. This structure could have an immunomodulatory role by
interacting with carbohydrate-specific receptors related to CD22 or the
selectins (50, 54). Receptors for many of the placental prolactin
family members are likely to be present in both the fetus and mother.
Specific glycoforms could direct the different members of this family
to specific sites and/or modulate the activation of their
receptors.
Clearly many potential biological roles for
NeuAc2, 6GalNAc
1,4GlcNAc
are possible. The highly regulated
expression of the GalNAc-transferase and
2,6-sialyltransferase at
the same time as select members of the placental prolactin family
strongly supports the view that this structure will prove biologically
important for these hormones.
We thank the National Hormone and Pituitary Agency for providing purified hCG.