56
Biochimica et Biophysica Acta, 966 (1988) 56-64
Elsevier
BBA 22932
S e c r e t i o n of a l a c t o s a m i n o g l y c a n - c o n t a i n i n g g l y c o p r o t e i n
by p e r i - i m p l a n t a t i o n s h e e p c o n c e p t u s e s
G a r y R. N e w t o n a n d P e t e r J. H a n s e n
Department of Dairy Science, University of Florida, Gainesville, FL (U.S.A.)
(Received 19 October 1987)
(Revised manuscript received 26 February 1988)
Key words: Lactosaminoglycan; Embryo; Glycoprotein; (Sheep)
Sheep conceptuses from day 16 of pregnancy were cultured in the presence of [3Hlglucosamine and
[14C]leucine and a high-molecular-weight glycoprotein (HMWG) secreted into the culture medium was
purified by a combination of anion-exchange and gel filtration chromatography. The HMWG was found to
have a molecular weight between 800 000 and 900 000 and to be highly resistant to digestion with pronase.
Characteristics of the carbohydrate portion of the purified glycoprotein were examined by selective chemical
and enzymatic digestions and lectin binding studies. Mild alkaline reduction was ineffective in disassociating
carbohydrate chains from the protein core. Furthermore, the protein was resistant to both O-glycanase and
peptide:N-glycanase F. Harsh alkaline reduction caused the release of carbohydrates, however. After
pronase digestion of these products, three molecular weight classes of carbohydrates were resolved by
Sephadex G-25 chromatography. Two lines of evidence indicate that the HMWG contains lactosaminoglycan components. The intact molecule and two of the molecular weight classes of carbohydrates resolved by
harsh alkaline reduction bind Datura stramonium lectin. Binding of HMWG to iectin could be partially
inhibited by N-acetyllactosamine and completely inhibited by a mixture of N,N'-diacetylchitobiose and
N,N',N"-triacetylchitotriose. Secondly, digestion with endo-fl-galactosidase causes the release of 16% of
the [3Hlglucosamine from the intact molecule. Therefore, the HMWG of the sheep conceptus is the first
reported example of secretion of lactosaminoglycan-containing glycoprotein by peri-implantation embryos.
Introduction
Lactosaminoglycans are polysaccharides characterized by Gal/31 ~ 4GlcNac/31 ~ 3 repeats
[1-3]. They exist as glycolipids or glycoproteins
and have a significant heterogeneity in structure
Abbreviation: HMWG, high-molecular-weight glycoprotein;
Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose;
Glcitol, glucitol.
Correspondence: P. J. Hansen, Department of Dairy Science,
University of Florida, Shealy Drive and Ritchey Road, Gainesville, FL 32611, U.S.A.
with respect to degree of branching and terminal
structures [4-6]. Lactosaminoglycans have been
found in a variety of cell types, frequently as cell
surface or extracellular glycoproteins [2,4,7-10].
They may also play a functional role in fertilization and early embryonic development. Lactosaminoglycans have been identified as membrane
components of sperm [11] and early embryonic
cells [4,11,12] and have been shown to participate
in cell adhesion of embryonic cells [12] and endometrial epithelium [13] by binding to cell-surface
galactosyltransferase.
The peri-implantation conceptus of the sheep is
one example of an embryonic tissue that is ac-
0304-4165/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
57
tively involved in the synthesis and secretion of
glycoproteins [14]. Placentation in the sheep does
not involve true implantation but rather interdigitation and interfolding of trophoblast and uterine
epithelium. Contact between the conceptus and
the caruncular surface of the endometrial epithelium is evident by day 14 of pregnancy and
adhesion develops between days 16 and 18 [15].
Interdigitation of microvilli has been observed as
early as day 18 [16] to day 20 [17] or early in the
fourth week of gestation [18].
Around this time the conceptus is secreting a
high-molecular-weight (M r > 660 000) glycoprotein (HMWG) that is approximately 50% carbohydrate by weight [14]. Gas-liquid chromatography indicated that the major carbohydrates in this
molecule are galactose and N-acetylglucosamine
in a 1.57:1 ratio. These results suggest that the
glycoprotein may carry lactosaminoglycan saccharides. Therefore, we examined the properties of
the carbohydrate chains of HMWG to test whether
this protein does indeed contain lactosaminoglycans.
Materials and Methods
Materials. All materials employed for tissue
culture were supplied by the vendors noted by
Godkin et al. [19]. Radionucleotides (D-[6-aH]glucosamine, specific activity = 40 Ci/mmol; L-[114C]leucine, specific activity = 56.6 mCi/mmol)
were supplied by ICN (Irvine, CA) and Amersham
(Arlington Heights, IL), respectively. Genzyme
Co., Boston, MA, was the supplier of N-glycanase
(peptide:N-glycanase F from Flavobacterium
meningosaminidase) and O-glycanase (Endo-ct-Nacetylgalactosaminidase from Diplococcus pneumoniae). ICN Immunochemicals supplied endofl-galactosidase from Escherichia freundii. Chromatography supplies were obtained from Pharmacia, while the Zorbax GF-450 HPLC gd filtration column was from DuPont (Wilmington, DE).
All other proteins and reagents were obtained
from Sigma or Fisher and were of the highest
grade available.
In vitro culture of conceptuses. Adult ewes, primarily of Rambouillet and Florida Native breeds,
were checked for estrus every morning with a
vasectomized ram. Ewes were bred to intact rams
on the morning and afternoon of detected estrus
and on the following morning. On day 16 postestrus (estrus = day 0), conceptuses were flushed
from uteri with sterile medium as previously described [19]. Conceptuses were individually cultured in 15 ml of a modified Eagle's minimum
essential medium (custom formula No. 86-5007;
Gibco Laboratories, Grand Island, NY; see Basha
et al. [20] for modifications) with the L-leucine
content reduced to one-tenth its normal level. One
hundred microcuries of [3H]glucosamine and 25
#Ci of [14C]leucine were added to each culture.
Incubations were at 37°C in an atmosphere of
50% 02, 45% N2, and 5% CO 2 (by volume). After
30 h, the medium was changed and the culture
continued for a second 30 h period. Using this
culture procedure, conceptuses continue to secrete
de novo synthesized proteins for at least 4 days.
Cultures were terminated by centrifugation at 12
000 x g for 10 min at 4°C. Culture supernatant
fractions were harvested and frozen at - 2 0 ° C.
Purification of dual-labelled HMWG. Conceptus-conditioned culture medium (15 ml) was
dialyzed against four changes (16 liters total
volume) of 10 mM Tris-HC1 buffer (pH 8.2). The
retentate was applied to an anion-exchange column of DEAE-Sepharose CL-4B (10 x 1.5 cm)
that had been previously equilibrated with 10 mM
Tris-HC1 (pH 8.2). Protein bound was eluted with
a linear salt gradient (300 ml; 0-0.5 M) in 10 mM
Tris-HC1 (pH 8.2). Fractions, usually 5 ml, were
collected and assayed for radioactivity using a
1218 Rackbeta scintillation counter (LKB) programmed to count dual-labelled preparations.
Counting efficiencies for dual-labelled preparations were 53% for 3H and 21% for 14C. The major
[3H]glucosamine-labelled peak was concentrated
using a Millipore immersible CX-10 ultrafiltration
unit (cutoff = 10 kDa) and applied to a Sepharose
CL-6B column (96 x 1.5 cm) with 10 mM Tris-HC1
(pH 8.2), containing 0.33 M NaC1 as eluent. The
single [3H]glucosamine-labelled peak was dialyzed
against distilled water, aliquoted and stored at
-20°C.
Molecular weight estimation. Molecular weight
of HMWG was estimated using Sepharose CL-6B
with thyroglobulin (M r = 660000), bovine serum
albumin (M r = 69 000), and ovalbumin ( M r =
45 000) as standards. Molecular weight was also
58
estimated by high performance liquid chromatography using a Zorbax GF-450 gel filtration column calibrated with sheep IgM (M r = 900000),
thyroglobulin (M r = 660000), bovine serum albumin ( M r = 69000) and ovalbumin ( M r =
45 000). Column buffer used was 0.2 M sodium
phosphate buffer (pH 8.0) with a flow rate of 0.7
ml/min.
Tube gel polyacrylamide electrophoresis was a
third method used to estimate molecular weight
and test for the presence of subunits. Samples of
HMWG (approximately 5000 dpm [3H]glucosamine and 600 dpm [14C]leucine) were solubilized
in 62.5 mM Tris-HC1 buffer (pH 6.8) containing
2% (w/v) sodium dodecyl sulfate (SDS), 15%
(w/v) sucrose and 5% 2-mercaptoethanol (v/v).
SDS-polyacrylamide electrophoresis Was performed in tube gels (3 mm, i.d.) using the buffer
system of Laemmli [21] with a 10 cm running gel
of 5% (w/v) polyacrylamide and a 1 cm stacking
gel of 4.5% polyacrylamide. Electrophoresis was
performed at 3 mA/gel for 1-1.5 h. Gels containing HMWG were removed from the tubes, sliced
in 2 mm sections using a gel slicer and solubilized
in 0.4 ml hydrogen peroxide by heating at 60 °C
for 2 h. Scintillation fluid (4 ml) was added and
samples were counted for radioactivity. Gels containing molecular weight standards were removed
from the tubes and stained with Coomassie Blue
R-250 (0.125%, w / v ) dissolved in acetic
acid/ethanol/water (7:40: 53; v/v).
Pronase digestion. Pronase (10 mg/ml in 0.1 M
sodium acetate buffer, pH 6.0) was self-digested
for 30 rain at 60°C [22]. Dual-labelled HMWG
(approximately 43000 dpm [3H]glucosamine and
1500 dpm [14C]-leucine) was lyophilized and reconstituted in 1 ml of 0.1 M sodium acetate buffer
(pH 6.0) containing 1 mg pronase. Incubation
continued for 72 h with addition of 1 mg pronase
every 24 h. Digestion products were separated by
gel exclusion chromatography using Sepharose
CL-6B (96 × 1.5 cm). The ehiting buffer was 10
mM Tris-HC1 buffer (pH 8.2) containing 0.33 M
NaC1.
N-glycanase digestion. Purified HMWG (approximately 43 000 dpm [ 3H]glucosamine and 1500
dpm [14C]leucine) was lyophilized and reconstituted in 20/zl 0.5% (w/v) SDS containing 0.1 M
2-mercaptoethanol. The sample was further di-
luted with 10.8 /~1 sodium phosphate buffer (pH
8.6), 3/~1 100 mM phenanthroline hydrate (in
methanol) and 5#1 of 7.5% (w/v) Nonidet P-40.
N-glycanase (0.5 unit) was added and the reaction
mixture incubated at 37°C for 16 h. Ovalbumin
and bovine serum albumin (20/~g) served as positive and negative controls, respectively. HMWG
digestion products were separated by chromatography on Sepharose CL-6B (96 × 1.5 cm). Control
reactions were monitored by SDS-polyacrylamide
slab gel electrophoresis using 12.5% (w/v) polyacrylamide gels and the buffer system of Laemmli
[21].
O-glycanase digestion. HMWG (approximately
43000 dpm [3H]-glucosamine and 1500 dpm
[14C]leucine) was lyophilized and reconstituted in
20 mM Tris-maleate buffer (pH 6.0) containing 1
mM calcium acetate and 10 mM D-galactonolactone. Neuraminidase (0.1 unit) was added and the
reaction incubated at 37°C for 60 min. Oglycanase (2.9 mU) was then added in some reactions and the incubation continued at 37 ° C for 16
h. Final reaction volume was 50/~1. Bovine serum
albumin and fetuin (20 /zg) were negative and
positive controls, respectively. HMWG digestion
products were separated by chromatography on
Sepharose CL-6B (96 x 1.5 cm). Control reactions
were monitored by polyacrylamide gel electrophoresis.
Endo-fl-galactosidase digestion. HMWG (approximately 65 000 dpm [3H]glucosamine and 2100
dpm [14C]leucine) was lyophilized and reconstituted in 10 mM sodium acetate buffer (pH 5.8)
containing 10 mM 7-galactonolactone. To this
preparation 0.1 U endo-fl-galactosidase was added. The final reaction volume was 200 #1. The
reaction proceeded for 48 h at 37°C and was
stopped by addition of 1 ml 10 mM Tris-HC1
buffer (pH 8.2) containing 0.33 M NaC1. A second
aliquot of HMWG (approximately 33000 dpm
[ 3H]glucosamine and 1050 dpm [14C]leucine) was
lyophilized and incubated as described above except no enzyme was added. Reaction products
were separated by gel exclusion chromatography
on Sepharose CL-6B.
Alkaline borohydride treatment. HMWG (approximately 65 000 dpm [ 3H]glucosamine and 2100
dpm [14C]leucine) was lyophilized and reconstituted in 1 ml of either 1.0 M NaBH 4 in 1.0 M
59
N a O H (mild alkaline reduction conditions) or 4.0
M N a B H 4 in 1.0 M N a O H (harsh alkaline reduction conditions). Mild alkaline reduction was carfled out at 4 ° C for 48 h. Harsh alkaline reduction
was performed at 60 o C for 48 h. The reaction was
terminated by the addition of 2 ml of 10 mM
Tris-HC1 buffer (pH 8.2) containing 0.33 M NaC1
and neutralization with glacial acetic acid. Dissolved gasses were removed under reduced pressure. The reaction products were separated by gel
chromatography on Sepharose CL-6B as described
before. Peaks of [3H]glucosamine were pooled,
lyophilized, reconstituted in distilled water (usually 2 ml) and digested with pronase as outlined
earlier. These products were separated by gel chromatography on Sephadex G-25 superfine (90 x 1.5
cm) using 0.1 M acetic acid as a eluent. The
colunm was calibrated with Man9Glcitol[3H]NAc
and Man6Glcitol[3H]NAc as standards [23,24].
Binding to Datura stramonium lectin. Lectin (1
mg) was coupled to Sepharose 4B by the cyanogen
bromide method [25]. Coupling efficiency, determined by measuring protein concentration [26]
in the supernatant following the coupling procedure, was greater than 75%. Intact H M W G was
loaded onto a 2.5 ml column of coupled lectin
previously equilibrated with 10 mM potassium
phosphate buffer (pH 7.4) containing 0.9% ( w / v )
NaC1 (PBS). The column was sequentially washed
with 10 ml each of PBS, 10 mM potassium phosphate buffer (pH 7.4) containing 1 m NaC1, and 8
M urea. A control column was prepared as described above except no lectin was coupled to the
Sepharose 4B. H M W G was loaded onto this column and eluted as described above. The ability of
various low-molecular-weight sugars to inhibit
binding of H M W G to D. stramonium lectin was
evaluated. Fifty microliters of D. stramonium lectin
coupled to Sepharose CL-4B (25% v / v in PBS)
was incubated with an equal volume of PBS containing N-acetyllactosamine (400 mM) or N , N ' diacetylchitobiose (24 mM) and N , N ' , N " - t r i acetylchitotriose (16 mM). H M W G was then added and incubated for 2 h at room temperature on
a tube turner. The reaction was terminated by
centrifugation at 12 000 x g for 4 min and separation of supernatant from precipitate. The precipitate and an aliquot of the supernatant were
counted for radioactivity. Counts bound to lectin
were corrected for nonspecific binding to uncoupled Sepharose CL-4B and percent bound calculated relative to H M W G bound to D. stramonium
without inhibitor.
Peaks of oligosaccharides isolated by gel filtration of harsh alkaline reduction products of
H M W G were pooled, lyophilized and tested for
ability to bind D. stramonium lectin. Pools of
oligosaccharides were reconstituted in 1 ml of
PBS. Two hundred microliters of a slurry of D.
stramonium lectin coupled to Sepharose CL-4B
was added contents rotated on a tube turner for 2
h at room temperature. Contents were centrifuged
at 12 000 x g for 2 min and radioactivity was
determined in both supernatant and precipitate
fractions.
Results
Purification of H M W G
Dialyzed conceptus-conditioned culture medium was subjected to anion-exchange chromatography using DEAE-cellulose. The major [3H] glucosamine-labelled peak eluted early in the salt gradient (Fig. 1A). When this fraction was separated by
gel exclusion chromatography on Sepharose CL6B, one major dual-labelled peak was resolved,
having a Kay of approximately 0.165 (Fig. 1B).
This was an elution volume prior to that of
thyroglobulin (Kav of 0.247) and yielded a molecular weight based on extrapolation of approximately 765 000.
High performance liquid chromatography was
also employed to generate an estimate of molecular weight. H M W G had a retention time of 10.66
min, a value intermediate to that of sheep IgM
(10.52 min) and thyroglobulin (11.6 min). The
calculated molecular weight was 896 000.
Molecular weight was also estimated using
polyacrylamide tube gel electrophoresis on 5%
polyacrylamide gels in the presence of SDS with
or without 2-mercaptoethanol. The majority of the
radioactivity failed to enter the gel under both
conditions, indicating that the glycoprotein was of
large molecular weight ( > 500 000) and was composed of a single polypeptide chain (data not
shown).
60
Pronase digestion of intact HMWG
A
/
.-
f
DPM
~
G
3
--(3--
14C.LI
--+--
HMWG
x
,,,w - 4
10
u •
10 -3
H-GleN
Z
-0
,
s
O
Digestion with endoglycosidases
DPM
-¢.)
CO
O
o--
- - + - -
3H-GleN
14C.Le
•
10 .3
u •
10 -2
"O
¢l
n"
C
3
-
-3
3
2
1
O O
-
-
H M W G was treated with self-digested pronase
and digestion p r o d u c t s separated by chromatograp h y on Sepharose CL-6B. While most (71.8%) of
the []4C]leucine was in the salt volume, all of the
[3H]glucosamine was partially excluded from the
column. Pronase digestion caused a shift in the
elution volume of [3H]glucosamine label associated with H M W G f r o m a Kay of 0.165 to 0.434,
indicating that large segements of the molecule are
resistant to pronase.
l50
Enzymatic hydrolysis of N- [27] and O-linked
[28] c a r b o h y d r a t e side chains was used to determine the type of linkages present on H M W G .
Neither N- nor O-glycanase (with or without prior
treatment with neuraminidase) had an effect on
the size or glycosylation of H M W G as determined
by gel filtration c h r o m a t o g r a p h y . There was no
shift in molecular weight caused by these digestions nor was there any disassociation of
[3H]glucosamine into the salt volume (results not
shown). Utilizing identical reaction conditions, a
decrease in the molecular weight of ovalbumin
(N-glycanase) and fetuin (O-glycanase plus neuraminidase) was d e m o n s t r a t e d b y polyacrylamide
gel electrophoresis, indicating that the enzymes
were active. Furthermore, bovine serum albumin,
a protein with no oligosaccharide side chains, was
unaltered b y these treatments.
Fraction number
Fig. 1 Isolation of HMWG. Conceptus-conditioned culture
medium (15 ml) was applied to an anion-exchange column of
DEAE-Sepharose CL-4B (10 × 1.5 cm) previously equilibrated
with 10 mM Tris-HCl buffer (pH 8.2). Protein bound was
eluted with a linear salt gradient (G = 300 ml; 0-0.5 M) in 10
mM Tris-HC1 (pH 8.2) (panel A; 5 ml/fraction). Aliquots (100
~1) from each fraction were analyzed for radioactivity. The
major [3H]glucosamine-labelled peak (HMWG) was applied to
a Sepharose CL-6B column (96 x 1.5 cm) with 10 mM Tris-HC1
buffer (pH 8.2) containing 0.33 M NaC1 as eluent (panel B; 3.7
ml/fraction). Aliquots (100/zl) from each fraction were counted
for radioactivity. The major dual-labelled peak (Kay = 0.165)
was identified and utilized in subsequent experiments. The
void volume (V0) and elution volumes of thyroglobulin (T),
bovine serum albumin (B) and ovalbumin (O) are shown by
arrows. Panel C represents HMWG (peak 1), purified as
described above, from a conceptus cultured with L-[4,53H]leucine (specific activity = 146 Ci/mmol). Only the CL-6B
profile is shown. HMWG eluted as a symmetrical peak with a
Kav of 0.17. GlcN, glucosamine.
Alkaline borohydride reduction
Since neither N- n o r O-glycanase was effective
in determining the type of carbohydrate linkages
of H M W G , b o r o h y d r i d e treatment was employed
[31,32]. Mild (1.0 M N a B H 4 in 1.0 M N a O H )
alkaline reduction, carried out at 4 ° C for 48 h,
caused only a slight decrease in molecular weight
of H M W G ( K a v = 0.376). Only a single peak of
[3H]glucosamine was detected, suggesting the absence of O-linked oligosaccharides. W h e n harsh
reduction (4.0 M N a B H 4 in 1.0 M N a O H ) was
carried out at 60 o C for 48 h, one to three peaks of
[3H]glucosamine were present near or at the salt
volume of the c o l u m n (Fig. 2A), indicating hydrolysis of A s n - G l c N A c b o n d s a n d / o r extensive
destruction of peptide bonds. These fractions were
pooled separately, digested with pronase to insure
61
.oj-A
Vo
r e s o l v e d at o r n e a r t h e v o i d v o l u m e o f t h e c o l u m n ,
o n e n e a r t h e salt f r o n t (Kay = 0.727), a n d t h e r e
w a s a n i n t e r m e d i a t e p e a k w i t h a Kay o f 0.323. T h e
r e l a t i v e size o f e a c h p e a k v a r i e d b e t w e e n dig e s t i o n s : t h e l a t e s t e l u t i n g p e a k was s o m e t i m e s
q u a n t i t a t i v e l y less t h a n the first p e a k (Fig. 2B)
while, for o t h e r d i g e s t i o n s , it w a s the m a j o r p e a k
resolved.
I
.ot
DPM
40
3
--0---
)t
1 4
~
H-GIcN
-1
x 10
-
20
._>
o
°
~
"'
soo DPM
300
H-GI©N
2OO
2OO
Peak
2
Z
1 oo
O
-rI
/
t
gl.
20
40
SO
Fraction number
Fig. 2. Harsh alkaline reduction of HMWG. The glycoprotein
was incubated for 48 h at 60 ° C in a mixture of 4.0 M NaBH 4
and 1.0 M NaOH. Reaction products were separated by gel
chromatography on Sepharose CL-6B (96 × 1.5 cm) with 10
mM Tris-HC1 (pH 8.2), containing 0.33 M NaC1 as eluent
(panel A; 3.7 ml/fraction). V0 represents the column void
volume and I the elution volume of intact HMWG. Aliquots
(400 #1) from each fraction were analyzed for radioactivity and
peaks of [3H]glucosamine were pooled, lyophilized, reconstituted in distilled water and digested with pronase. These
products were separated by gel chromatography on a column
of Sephadex G-25 superfine (90 × 1.5 cm) previously calibrated
with Man9Glcitol[3H]NAc (Mang) and Man6Glcitol[3H]NAc
(Man6). Eluent was 0.1 M acetic acid and aliquots (400 ~l)
from each fraction (3.7 ml) were counted for radioactivity.
Panel B illustrates a representative profile of elution from
Sephadex G-25. Three molecular weight classes of oligosaccharides with Kay values of 0.029, 0.323 and 0.727 were
resolved. Elution profiles of other digestions from Sepharose
CL-6B, as resolved by Sephadex G-25, gave similar results
except that the relative size of the latest eluting peak was
sometimes larger than shown here. GlcN, glucosamine.
t h a t all p r o d u c t s w e r e o l i g o s a c c h a r i d e s o r m o n o amino acid glycopeptides, and each separated by
Sephadex G-25. Three peaks were consistently
r e s o l v e d (Fig. 2B)~ O n e p e a k (Kav = 0.029) was
S
ve
Man 9 MIn 6
a
lO -4
4O-
20-
0
2O
4O
6O
Fraction number
Fig. 3. Digestion of HMWG by endo-fl-galactosidase. HMWG
was reacted with 0.1 U enzyme in 200/.tl sodium acetate buffer
(pH 5.8), containing 0.01 M 3,-galactonolactone. The reaction
proceeded for 48 h at 37 o C. Reaction products were separated
by gel exclusion chromatography on Sepharose CL-6B (96 × 1.5
cm; 3.7 ml fractions) with 10 mM Tris-HC1 (pH 8.2), containing 0.33 M NaC1 as eluent. V0 represents the column void
volume and I the elution volume of intact HMWG. Aliquots
(400 /tl) from each fraction were analyzed for radioactivity
(panel A). Endo-fl-galactosidase resulted in dissociation of
approximately 16.3% of the [3H]glucosamine from the protein
core. The carbohydrates released in this manner (peak 2) were
separated by gel chromatography on a column of Sephadex
G-25 superfine (90×1.5 cm; 2.5 ml/fraction) previously
calibrated with Man9Glcitol[3H]NAc (Man 9) and Man6Glcitol[ 3H]NAc (Man6). Eluent was 0.1 M acetic acid and aliquots
(400 /~l) from each fraction were analyzed for radioactivity.
Four peaks were resolved: one near the void volume of the
column (Kay = 0.025) and peaks at Ka~ values of 0.198, 0.372
and 0.447. GlcN, ghicosamine.
62
Digestion with endo-~-galactosidase
This enzyme hydrolyzes/3-galactosidic linkages
of various substrates [33-35]. Endo-/3-galactosidase resulted in dissociation of approximately
22.6% of the [3H]glucosamine from the protein
core (Fig. 3A). The major peak of [3H]glucosamine was shifted to a Kav of 0.30. This peak was
followed by a broad, smaller peak with a Kav of
approximately 0.80 (Fig. 3A). When corrected for
[3H]glucosamine present in the region of the
smaller peak following chromatography of nondigested HMWG, specific cleavage of 16.3% of the
[3H]glucosamine was attributed to endo-/3-galactosidase. The carbohydrates released in this
manner (peak 2) were separated by Sephadex G-25
column chromatography (Fig. 3B). Four peaks
were resolved: one at or near the void volume of
the column (Ka~ = 0.025) and peaks at Kav values
of 0.198, 0.372 and 0.447.
Lectin binding
All the radioactivity associated with intact
HMWG could be bound to a column of D. stramonium lectin coupled to Sepharose CL-4B (resuits not shown). Radioactivity could be eluted
with 8 M urea, but not with 1 M NaC1. Preincubation of lectin with equal volumes of N-acetyllactosamine (400 mM in PBS) or PBS containing
N, N '-diacetylchitobiose (24 mM) and N, N', N"triacetylchitotriose (16 mM) inhibited HMWG
binding by 30% and 100%, respectively. Results
suggest that each molecule of HMWG contains at
least one carbohydrate chain with repeating fll,4linked oligomers of N-acetylglucosamine.
Fractions isolated by Sepharose CL-6B following harsh alkaline reduction were digested with
pronase and separated by Sephadex G-25 chromatography. The three peaks isolated in this
manner (Fig. 2B) were evaluated for ability to
bind D. stramonium lectin. The majority of the
radioactivity present in peak 1 (78.9%) and peak 2
(84.6%) bound to lectin while very little of the
radioactivity present in peak 3 was bound (7.8%).
Discussion
Results presented here suggest that the major
secretory glycoprotein of day 16-17 ovine conceptus is of high molecular weight (765 000-896
000), highly resistant to chemical and enzymatic
degradation and bears N-linked polylactosamine
groups. The protein was fairly resistant to pronase, since only a small portion of the molecule
was cleaved, leaving the bulk of the glycoprotein
intact. In addition, N- and O-glycanase were unable to disassociate carbohydrate label from the
protein backbone, indicating that the active sites
for these enzymes may be masked by the extensive
glycosylation occurring on the protein. Though
mild alkaline reduction cleaved a considerable
portion of the molecule, no specific release of
carbohydrate was evident. Only harsh alkaline
conditions succeeded in disassociating carbohydrate moieties from the protein core, suggesting
that chains are N-linked. Data obtained from
Sephadex G-25 chromatography of products released by harsh alkaline reduction indicate the
presence of three molecular weight classes of
oligosaccharides. The size of fraction 1, which
eluted near the void volume, is not known but the
second fraction appeared to be of relatively small
size (approximately 7 units) and may represent
carbohydrates that were partially cleaved by the
digestion procedure [31]. Fraction 3, of lowest
molecular weight, probably represents extensively
digested saccharide chains, since the relative magnitude of radioactivity in this peak was variable
and the peak eluted near the salt volume.
Three lines of evidence suggest that the HMWG
contains lactosaminoglycan components. The
carbohydrate composition of HMWG, determined
by gas-liquid chromatography [14], indicates that
the major sugars present are D-galactose and Nacetyl-D-glucosamine in molar ratios of 1.57 : 1.00.
Furthermore, 16.3% of the carbohydrate on
HMWG was removed by endo-/3-galactosidase, an
enzyme that cleaves/3-galactosidic bonds on certain types of lactosaminoglycans. The portion of
carbohydrate released by endo-/3-galactosidase is
similar to results of Masters et al. [14]. That more
extensive cleavage was not observed may be further evidence that the sugar units are extensively
branched [35] or contain fucose near the cleavage
site [34]. Finally, the intact molecule and two of
the three molecular weight classes of oligosaccharides isolated by harsh alkaline reduction bind
D. stramonium lectin. D. stramonium lectin recognized oligomers of N-acetylglucosamine [36,37].
63
B i n d i n g of i n t a c t H M W G c o u l d b e i n h i b i t e d b y
p r e i n c u b a t i o n of lectin with N - a c e t y l l a c t o s a m i n e
(30%) or a m i x t u r e of N , N ' - d i a c e t y l c h i t o b i o s e
a n d N, N ' , N " - t r i a c e t y l c h i t o t r i o s e (100%). T h e r e fore, each m o l e c u l e of H M W G c o n t a i n s o l i g o m e r s
of N - a c e t y l g l u c o s a m i n e . T h e s u s c e p t i b i l i t y of
H M W G to e n d o - f l - g a l a c t o s i d a s e indicates that
s o m e of these o l i g o m e r s are s u b s t i t u t e d w i t h
galactose to f o r m N - a c e t y l l a c t o s a m i n e units.
T h e role of H M W G is u n k n o w n . It m a y p r o t e c t
the c o n c e p t u s f r o m the uterine i m m u n e s y s t e m
b e c a u s e M u r r a y et al. [38] have shown that
H M W G can i n h i b i t blastogenesis of m i t o g e n - a n d
m i x e d - l y m p h o c y t e - s t i m u l a t e d l y m p h o c y t e s . I n addition, p r o t e i n a s e s can b e p r e s e n t in u t e r i n e secretions [39] a n d the local presence of H M W G a r o u n d
the c o n c e p t u s m a y p r o t e c t it f r o m p r o t e o l y t i c actions of these enzymes. In o t h e r systems, lactosaminoglycan-containing glycoproteins participate
in cell a d h e s i o n in e m b r y o n i c c a r c i n o m a a n d
u t e r i n e epithelial cells t h r o u g h b i n d i n g to cell
surface g a l a c t o s y l t r a n s f e r a s e [12,13]. Sheep b l a s t o cysts are spherical b e t w e e n d a y 4 a n d 10 a n d then
e l o n g a t e to a f i l a m e n t o u s f o r m b y d a y 15 [40],
t h r o u g h processes that p r o b a b l y involve extensive
cell migration. Similarly, c o n t a c t b e t w e e n the conc e p t u s a n d the c a r u n c u l a r surface of the u t e r i n e
l u m e n has b e e n r e p o r t e d a r o u n d d a y 14, with
a d h e s i o n d e v e l o p i n g a r o u n d d a y 1 6 - 1 8 [15]. Perh a p s r e a r r a n g e m e n t of e m b r y o structure or u t e r i n e
e p i t h e l i u m - t r o p h o b l a s t i n t e r a c t i o n s l e a d i n g to
p l a c e n t a t i o n involve cell a d h e s i o n m e c h a n i s m s
m e d i a t e d b y e m b r y o n a l l a c t o s a m i n o g l y c a n s such
as H M W G .
Acknowledgements
R e s e a r c h s u p p o r t e d b y N I H g r a n t HD20671.
T h e a u t h o r s a c k n o w l e d g e the i n d i s p e n s i b l e c o o p e r a t i o n of F u l l e r W. Bazer in o b t a i n i n g sheep
e m b r y o s , J. Vallet for assistance with surgeries, J.
J o h n s o n a n d W . W . T h a t c h e r for r u n n i n g H P L C
analyses a n d S t u a r t K o r n f e l d , W a s h i n g t o n U n i versity, St. Louis, M O , for d o n a t i o n of gel filtration s t a n d a r d s . This is J o u r n a l Series N o . 8441 of
the F l o r i d a A g r i c u l t u r a l E x p e r i m e n t Station.
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