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INTRODUCTION |
The rapid clearance of glycoproteins from the blood following
removal of sialic acid (Sia)1
residues and exposure of underlying galactose (Gal) residues was first
reported by Ashwell and Morell in the early 1970s (1, 2) and led to the
discovery of the asialoglycoprotein-receptor (ASGP-R). The specificity
and biochemical features of this endocytic receptor have been
extensively investigated since that time (3, 4). Although it is clear
that the ASGP-R receptor binds oligosaccharides with terminal
-linked N-acetylgalactosamine (GalNAc) or Gal and can
mediate the rapid clearance of glycoproteins bearing these terminal
sugars from the circulation, endogenous ligands for this abundant
receptor have not yet been identified.
We first described N-linked oligosaccharides containing
1,4-linked GalNAc on lutropin (LH) and other members of the
glycoprotein hormone family of glycoproteins (5, 6). The GalNAc is
found almost exclusively in the form of GalNAc-4-SO4,
reflecting the sequential action of a protein-specific
1,4-N-acetylgalactosaminyltransferse (
1,4GalNAcT) and
a GalNAc-4-sulfotransferase (GalNAc-4-ST1) (7, 8). The terminal
GalNAc-4-SO4 is recognized by a receptor in hepatic
endothelial cells that regulates the circulatory half-life of LH
following its stimulated release into the blood (9-12). The control of
circulatory half-life is important for regulating estrogen production
in vivo during implantation of the embryo (13). We
subsequently described the presence of N-linked
oligosaccharides terminating with Sia
2,6GalNAc
on
prolactin/growth (PLP) hormone family members that are synthesized by
rat placenta spongiotrophoblasts between mid gestation and birth (14).
The levels of protein-specific
1,4GalNAcT activity in rat placenta
increase 150-fold between day 9 and 18 of gestation, whereas levels of
2,6sialyltransferase increase 5-fold during the same period (14). We
have found significant levels of PLP hormone family members bearing
terminal Sia
2,6GalNAc
in the circulation of the pregnant rat late
in gestation. Furthermore, the pregnancy-specific glycoprotein
glycodelin (placental protein 14) that is found in the amniotic fluid
of humans also bears this structure (15).
The selective addition of
1,4-linked GalNAc to the oligosaccharides
on prolactin-like protein (PLP)-A, PLP-A, PLP-B, PLP-C, PRP, and
placental lactogen I variant (14) as well as human glycodelin (15)
raised the possibility that a receptor specific for Sia
2,6GalNAc
is present and may regulate the circulatory half-life of glycoproteins
bearing these structures and/or direct them to specific locations such
as the amniotic fluid. We have examined this possibility in the
pregnant rat and report that neoglycoconjugates bearing multiple
tetrasaccharides terminating with the sequence
Sia
2,6GalNAc
1, 4GlcNAc
1,2Man are rapidly removed from the
circulation by the ASGP-R. Thus, the PLP hormone family members that
are released into the blood during pregnancy may represent examples of
endogenous ligands for the ASGP-R.
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MATERIALS AND METHODS |
Timed pregnant female CD® IGS rats, E16-E18 (E1 is the day
one of gestation), were obtained from Charles River Laboratories (Wilmington, MA). Bovine serum albumin (BSA) conjugated with an average
of 15 trisaccharides with the sequence GalNAc
1,4GlcNAc
1,2Man
(GGnM-BSA) or SO4-3-GalNAc
1,4GlcNAc
1,2Man
(S3GGnM-BSA) were provided by Dr. O. Hindsgaul, University of Alberta
(Edmonton, Canada). CMP-
-D-sialic acid and recombinant
rat
2,6-sialyltransferase (EC 2.4.99.1) were from
Calbiochem-Novabiochem Corp. (La Jolla, CA). Galactose-BSA was
purchased from EY Laboratories, Inc. (San Mateo, CA). GalNAc-
-BSA,
CNBr-activated Sepharose, fetuin (from fetal calf serum), and
neuraminidase-agarose (Clostridium perfringens) were
obtained from Sigma (St. Louis, MO). TRIzol® reagent and primers were
purchased from Life Technologies (Grand Island, NY).
Preparation of Sia
2,6GalNAc
1,4GlcNAc
1,2Man
-BSA
(SiaGGnM-BSA)--
SiaGGnM-BSA was prepared using a modification of
the procedure described by van Seeventer et al. (16).
GGnM-BSA (700 µg) was incubated at 37 °C in a 330-µl reaction
containing 1.57 mg of CMP-
-D-sialic acid, 50 mM cacodylate buffer at pH 6.5, 0.5% Triton X-100, 3 units
of alkaline phosphatase, and 3.4 milliunits of recombinant rat
2,6-sialyltransferase. Additional CMP-
-D[R]-sialic acid (1.57 mg) was added to the reaction after 36, 60, and 96 h of
incubation. At 96 h, 0.85 unit of
2,6-sialyltransferase and 1 unit of alkaline phosphatase were added. The reaction was terminated at
136 h and stored at
20 °C. SiaGGnM-BSA was separated from the
reaction products using a Microcon filtration unit.
Radiolabeling--
25 µg of SiaGGnM-BSA, S3GGnM-BSA, and
Gal-BSA were dissolved in 95 µl of 20 mM Tris-HCl, 150 mM NaCl, pH 7.4, and incubated on ice for 15 min with one
IODO-BEAD (Pierce, Rockford, IL) and 5 µCi of 125I.
SiaGGnM-[125I]BSA and Gal-[125I]BSA was
separated from free 125I by gel filtration on column
containing 1 ml of Sephadex G-25 eluted with Buffer A (25 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 1 mg/ml BSA. The 125I-labeled neoglycoconjugates were stored
at
20 °C.
Clearance and Tissue Distribution Studies--
Clearance studies
were performed as previously described (11). Rats were anesthetized by
intraperitoneal injection of ketamine (87 mg/kg) and xylazine (13 mg/kg). The left jugular vein was cannulated, and the rats were
heparinized prior to injection of 4 × 106 cpm of
SiaGGnM-[125I]BSA in 100 µl of saline. Blood was
withdrawn at designated times, and the amount of
SiaGGnM-[125I]BSA or S3GGnM-[125I]BSA
present per milligram of blood was determined by
-counting. Rats
were euthanized at the termination of the clearance study by metofane
inhalation. Organs were removed and weighed, and the amount of
SiaGGnM-[125I]BSA present was determined by
-counting.
Preparation of Rat Liver Membranes--
Rat livers were
homogenized with a Polytron homogenizer (Brinkmann Instruments,
Westbury, NY) in 5 volumes of buffer containing 25 mM
HEPES, 50 mM KCl, 2 mM magnesium acetate, 1 mM dithiothreitol, and 10% (w/v) sucrose. Homogenates were
sedimented at 1,500 × g for 5 min, and the resulting
supernatants were layered over a 65% (w/v) sucrose cushion prior to
sedimentation at 100,000 × g at 4 °C for 75 min.
The membrane fraction at the interphase and the soluble fraction in the
upper phase were collected separately and stored at
80 °C. Protein
concentrations were determined using the Bradford method (Bio-Rad,
Richmond, CA).
Binding Assays--
Binding studies were performed in 100 µl
of Buffer A containing 0.5% Triton X-100, 2 mM
CaCl2, 2-5 × 104 cpm of
SiaGGnM-[125I]BSA, and 200 µg of membrane or soluble
liver protein. Following incubation for 30 min at 25 °C, 1.5 ml of
ice-cold 10% (w/v) PEG 8000 in Buffer B (25 mM Tris-HCl,
150 mM NaCl, 2 mM CaCl2, pH 7.4)
was added, and the reaction was vortexed for 2 s. Precipitated ligand-receptor complexes were collected after incubation for 30 min at
4 °C by vacuum filtration on Whatman GF/C filter discs that had been
incubated in Buffer B containing 5 mg/ml BSA. The filtrates were washed
three times with 1 ml of ice-cold 10% (W/V) PEG 8000 in Buffer B and
counted in a
-counter. Assays were also performed using a 96-well
MultiScreen® GF/C filter plate (Millipore, Bedford, MA); however, the
binding reaction was reduced to75 µl, and the ligand-receptor
complexes were precipitated by adding 75 µl of 20% (w/v) PEG 8000 in
Buffer B prior to filtration. Individual filters were removed using a
MultiScreen® punch tip (Millipore) and counted in the
-counter.
Receptor Purification--
Rat liver membrane proteins
(400 mg) prepared as described above were resuspended in 50 ml
of 50 mM Tris-HCl at pH 7.4 with 150 mM NaCl, 2 mM CaCl2, and 3% Triton X-100 using a Dounce
homogenizer. Insoluble material was removed by sedimentation for 2 min
at 700 × g. The supernatant was incubated with 1 ml of
GalNAc-
-BSA-Sepharose (5 mg of GalNAc-
-BSA/ml of Sepharose) for
16 h at 4 °C. After washing five times with 5 ml of Buffer B
containing 1% Triton X-100, the bound protein was eluted with Buffer A
containing 100 mM GalNAc. Fractions containing SiaGGnM-BSA
binding activity were pooled, dialyzed against Buffer B, concentrated,
and stored at
20 °C.
Western and Ligand Blots--
Proteins were separated in the
presence of SDS without reduction on NuPAGE® Bis-Tris gels and
electrophoretically transferred to Immobilon-P (Millipore). Duplicate
gels were stained with Coomassie Blue or silver. For two-dimensional
electrophoresis, isoelectric focusing was performed with PROTEAN® IEF
system (Bio-Rad) using pH 3-10 ReadyStrip IPG strips. Separation in
the second dimension was performed without reduction on 10%
CriterionTM Tris-HCl gels as recommended by the
manufacturer. Rabbit anti-rat ASGP-R (1:10,000) and mouse anti-rabbit
IgG:horseradish peroxidase (1:100,000) were used for Western analysis.
For ligand blots, the Immobilon-P membranes were blocked with 5% milk
protein for 30 min, washed once with Buffer A containing 25 mM EDTA, and washed three times with Buffer A containing
0.5% Triton X-100 and 2 mM CaCl2. The washed
Immobilon-P membrane was incubated overnight at 4 °C in the presence
of 2 × 105 cpm of SiaGGnM-[125I]BSA in
Buffer A containing 0.5% Triton X-100 and 2 mM
CaCl2, washed three times with Buffer A containing 0.5%
Triton X-100 and 2 mM CaCl2, and exposed to
film for 7-15 days. Proteins corresponding to the region reactive with
SiaGGnM-[125I]BSA were excised and examined by MALDI-TOF
at the Johns Hopkins University Mass Spectrometry Facility.
Cloning and Expression of Rat ASGP-R Subunit 1 and Subunit
2--
Total RNA was isolated from rat liver with TRIzol® reagent
per the manufacturer's instructions. First-strand cDNA was
synthesized using oligo(dT12) and SuperscriptTM
II RNase H
reverse transcriptase (Life Technologies). Rat
ASGP-R subunits 1 and 2 were amplified using KlenTaq LA DNA polymerase
mix (Sigma Chemical Co., St. Louis, MO) and the gene specific primers
RHL1-F (CGG GAT CCC ATC ATG ACA AAG GAT TAT CAA GAT TTC C) and
RHL1-
R (5'-CCC ATT GGC CTT GCC CAA CTC TG), RHL2-F (CGG GAT CCC ATC
ATG GAG AAG GAC TTT CAA GAT ATC C), and RHL2-
R (5'-CCC GTA GGT GAT GTC CCG TTT G), respectively. Amplified products were subcloned into
pcDNA3.1/V5His-TOPO© and sequenced. The cDNAs were designated as RHL1V5His and RHL2V5His, respectively.
CHO/Tag and 293/Tag cells grown on a 100-mm plate were transfected with
13 and 6 µg, respectively, of RHL1V5His or RHL2V5His using 35 µg of
LipofectAMINE (Life Technologies) in serum-free medium for 5 h
according to the manufacturer's protocol. The
pcDNA3.1/V5His-TOPO© cloning vector was used as a control for
negative expression. Forty-eight hours after transfection, the cells
were washed with PBS (8 g of NaCl, 0.2 g of KCl, 1.44 g of
Na2HPO4, and 0.24 g of
KH2PO4, pH 7.4) and incubated for 10 min at
20 °C. Cells were thawed and scraped off in 1 ml of Buffer A
containing 50 mM EDTA. Cells were pelleted by
centrifugation for 2 min at 12,000 × g and washed
three times with 1 ml of Buffer A. Cells were incubated with 250 µl
of Buffer A containing 1% (w/v) Triton X-100 per 100-mm diameter
culture plate for 30 min at 4 °C with mixing. The mixture was
centrifuged at 12,000 × g for 10 min at 4 °C. The
supernatant was collected and store at
20 °C until needed.
 |
RESULTS |
Sia
2,6GalNAc
1,4GlcNAc
1,2Man
-BSA Is Rapidly Removed from
the Circulation by the Liver--
We had previously observed that PLP
hormones bearing oligosaccharides terminating with the unique sequence
Sia
2,6GalNAc
are present in the blood of pregnant rats between
mid gestation and birth. We introduced SiaGGnM-[125I]BSA
into the blood of pregnant rats between days 16 and 18 of gestation to
determine if a receptor that recognizes this carbohydrate structure is
expressed. We found that the injected SiaGGnM-[125I]BSA
was cleared from the circulation in less than 1 min (Fig. 1), suggesting the presence of a receptor
specific for terminal Sia
2,6GalNAc
. In contrast,
S3GGnM-[125I] BSA, which bears the identical
trisaccharide with a SO4 linked to the C-3 hydroxyl of the
GalNAc, was cleared from the blood at a much slower rate.

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Fig. 1.
SiaGGnM-BSA is rapidly cleared from the
circulation. SiaGGnM-[125I]BSA was injected through
the carotid artery of timed pregnant rats. Aliquots of blood were
withdrawn at the times indicated, and the cpm/mg amounts were
determined. The rats were euthanized after 10 min (Rat #1,
E16, open squares) or 45 min (Rat #2, E16,
filled circles; and Rat #3, E17, open
circles). Rat #4, closed triangles, was
injected with S3GGnM-[125I]BSA and euthanized after 45 min. The animals were autopsied, and the cpm/organ was determined as
summarized in Table I.
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The rats were autopsied to determine the tissue distribution of the
SiaGGnM-[125I]BSA. Essentially all of the radiolabel
(>90%) was present in the liver when rats were sacrificed 10 min
after injection of the SiaGGnM-[125I]BSA (Table
I). The cpm/mg of tissue in the liver,
kidneys, and lungs was 47-, 6-, and 5-fold greater (not shown),
respectively, than that in the large intestine, indicating that the
SiaGGnM-[125I]BSA was concentrated in the liver as
compared with other highly vascular tissues. Although the amount of
label in the liver had declined by 45 min as compared with 10 min
following injection, the amount of label in the kidneys had increased.
This suggested that SiaGGnM-[125I]BSA was taken up
exclusively by receptors in the liver and subsequently degraded,
resulting in the release of radiolabel into the blood and its
appearance in the kidneys and urine. There was no evidence of binding
to or uptake by the uterus, placenta, or pups.
Cation-dependent Binding of SiaGGnM-BSA by a Receptor
in Rat Liver--
The rapid and efficient clearance of SiaGGnM-BSA by
the liver indicated that a receptor specific for terminal
Sia
2,6GalNAc
was present in either hepatocytes or hepatic
endothelial cells. The membrane fraction from liver, but not the
soluble fraction, displayed cation-dependent binding of
SiaGGnM-[125I]BSA (Fig. 2).
Binding was pH-dependent, reaching a maximum between pH 5.5 and 7.0, and declining markedly below pH 5.0 (Fig.
3A). Binding did not occur in
the absence of cations (Fig. 3B). Whereas Cd2+
or Co2+ enhanced SiaGGnM-[125I]BSA binding as
compared with Ca2+, other cations with the exception of
Ni2+and Mn2+ were significantly less effective.

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Fig. 2.
Rat liver membranes contain a receptor that
binds SiaGGnM-[125I]BSA in the presence of calcium.
Total rat liver membrane (M) and soluble (S)
protein fractions were prepared as described under "Materials and
Methods" and tested for SiaGGnM-[125I]BSA specific
binding activity. Each 100-µl assay contained 5 × 104 cpm of SiaGGnM-[125I]BSA, 0.05% TX-100,
200 µg of soluble or membrane protein, and 2 mM
CaCl2 or 4 mM EDTA.
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Fig. 3.
Properties of the SiaGGnM-BSA specific
binding activity. Each 100-µl binding reaction contained 5 × 104 cpm of SiaGGnM-[125I]BSA, 0.05%
TX-100, and 200 µg of membrane protein. A, buffers with
the indicated pH were prepared using 25 mM NaAc (open
circles), MES (filled circles), and Tris-HCl
(open squares) and contained 2 mM
CaCl2. B, the indicated divalent cations at a
concentration of 10 mM were added to the binding assay. A
binding assay containing 1 mg/ml BSA but not membrane protein was used
to control for nonspecific binding. C, binding assays were
incubated in the presence of the indicated monosaccharides and
disaccharides at a concentration of 20 mM. The amount of
SiaGGnM-[125I]BSA bound in the presence of each
saccharide is expressed relative to that obtained in the presence of
mannose, which did not inhibit binding.
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A range of monosaccharides were tested as inhibitors of
SiaGGnM-[125I]BSA binding (Fig. 3C). At a
concentration of 20 mM, GalNAc was the most potent
inhibitor of binding (3% of control). Lactose (Gal
1,4Glc) and Gal
both were good inhibitors, although not as potent as GalNAc, indicating
the binding site can accommodate either Gal or GalNAc. Free sialic acid
was also an inhibitor (36% of control) suggesting that the binding
site accommodated both the terminal sialic acid and the
-linked
GalNAc. The lack of inhibition by Man, GlcNAc, and
-methyl-Man
indicated the receptor was not likely the
Man/GalNAc-4-SO4-receptor found in hepatic endothelial cells.
Isolation of a Sia
2,6GalNAc
-specific Receptor--
The
marked inhibition of binding seen with GalNAc (Fig. 3C)
suggested that an affinity matrix containing
-linked GalNAc could be
utilized for purification of the Sia
2,6GalNAc
-specific receptor. Rat liver membrane proteins were solubilized using Triton X-100 and
incubated with GalNAc
-BSA-Sepharose. The
SiaGGnM-[125I]BSA specific binding activity was retained
by the affinity matrix and was eluted with 100 mM GalNAc.
When examined by SDS-PAGE on a 10% gel under non-reducing conditions,
the GalNAc-eluted material contained two major protein species with
mobilities equivalent to 33 and 25 kDa when stained with Coomassie Blue
(Fig. 4, lane 3). Because we
expected the ASGP-R to be present in the material that was bound by
GalNAc
-BSA-Sepharose and eluted with 100 mM GalNAc,
Western blot analysis with rabbit anti-rat ASGP-R antibody was used to
identify the ASGP-R-derived proteins. Proteins with mobilities
equivalent to 33, 70, 130, and >170 kDa reacted with anti-ASGP-R
antibody in both the unfractionated Triton X-100 solubilized membrane
fraction (Fig. 4, lane 4) and the affinity purified fraction (Fig. 4, lane 5). The same protein species bound
SiaGGnM-[125I]BSA when a ligand blot was performed on the
affinity-purified fraction (Fig. 4, lane 6). We also
examined the proteins in the affinity-purified fraction by
two-dimensional electrophoresis (Fig. 5).
A ligand blot using SiaGGnM-[125I]BSA (Fig.
5B) indicated that proteins migrating with pI values between
4.6 and 5.6 and molecular masses of ~33, 75, and 150 kDa (Fig.
5A) accounted for the Sia
2,6GalNAc
specific binding
activity. The material migrating at 33 kDa was visualized by silver
staining (Fig. 5A) and identified as the ASGP-R by MALDI-TOF
analysis of tryptic fragments. Six out of a total of 31 potential
tryptic peptides for subunit 1 of the ASGP-R were identified,
accounting for 17% the subunit 1 sequence. The ASGP-R represents the
highest non-trivial protein detected in the sample with a molecular
weight search (MOWSE) score of 197 for subunit 1. The only other
non-trivial protein detected was keratin. Because the
SiaGGnM-[125I]BSA used for the binding assays and the
ligand blots does not contain any terminal GalNAc, the results
indicated that the rat ASGP-R is capable of binding saccharides with
terminal Sia
2,6GalNAc
.

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Fig. 4.
Affinity purification of the SiaGGnM-BSA
specific binding activity. A detergent extract containing 400 mg
of protein was prepared from a rat liver membrane fraction and passed
over an affinity column containing 1 ml of GalNAc- -BSA-Sepharose.
The bound proteins were eluted with 100 mM GalNAc contained
the SiaGGnM-BSA binding activity. The bound and unbound fractions were
resolved by 10% SDS-PAGE and stained with Coomassie blue (lanes
1-3) or electrophoretically transferred to polyvinylidene
difluoride membranes for Western and ligand blotting studies.
Lane 1, molecular mass standards with the indicated kDa;
lane 2, 200 µg of total rat liver membrane protein;
lane 3, 3.5 µg of the proteins eluted from
GalNAc- -BSA-Sepharose with 100 mM GalNAc; lane
4, Western blot of 120 µg of unfractionated rat liver membrane
proteins probed with rabbit anti-rat ASGP-R antibody; lanes
5 and 6, 3.5 µg of the proteins eluted from
GalNAc- -BSA-Sepharose with 100 mM GalNAc probed with
rabbit anti-rat ASGP-R antibody (lane 5) and 200,000 cpm/ml
SiaGGnM-[125I]BSA (lane 6),
respectively.
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Fig. 5.
Analysis of affinity-purified SiaGGnM-BSA
specific binding activity by two-dimensional gel electrophoresis.
The proteins eluted from the GalNAc- -BSA-Sepharose column with 100 mM GalNAc were separated by two-dimensional gel
electrophoresis. A, silver stain of 3.5 µg of eluate that
was separated in the first dimension using ReadyStrip IPG, pH 3-10
(Bio-Rad Laboratories, Hercules, CA) and in the second dimension using
a non-reducing Criterion 10% Tris-HCl gel (Bio-Rad Laboratories).
B, a duplicate two-dimensional gel that was
electrophoretically transferred to a polyvinylidene difluoride membrane
and probed with SiaGGnM-[125I]BSA (2 × 103 cpm/ml). The material in A indicated by the
white box corresponds to the ~33-kDa region that bound
SiaGGnM-BSA in B and was excised for analysis by MALDI-TOF
following trypsinization.
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Subunit 1 Accounts for Sia
2,6GalNAc
-specific Binding by the
Rat ASGP-R--
cDNAs encoding subunit 1 (RHL1) and subunit 2 (RHL2) of the rat ASGP-R were amplified from rat liver mRNA. The
stop codons were mutated to permit expression as chimeric proteins
containing the V5 epitope followed by six His residues at the carboxyl
terminus. CHO-Tag and 293-Tag cells were transfected with
pcDNA3.1-RHL1V5His or pcDNA3.1-RHL2V5His. Equal fractions of
the transfected cells were examined by Western blot analysis with
anti-V5 antibody following SDS-PAGE on a 10% gel under reducing
conditions. RHL1V5His is expressed at higher levels than RHL2V5His in
both cell lines. In addition RHL1V5His is more heterogeneous with major
bands migrating with molecular masses of 52 and 57 kDa, whereas
RHL2V5His has a single band migrating with a molecular mass of 57 kDa
(Fig. 6). Small amounts of what are
likely homodimeric species of RHL1V5His and RHL2V5His were detected at
105 and 115 kDa, respectively.

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Fig. 6.
Recombinant rat AGSP-R subunit 1 binds both
Sia 2,6GalNAc - and
Gal -containing ligands. CHO-Tag
(top panel) or 293-Tag HEK (lower panel) cells
were transfected with pcDNA3.1-RHL1V5His, pcDNA3.1-RHL2V5His,
or pcDNA3.1V5His vector without any insert. Following
solubilization of membrane proteins with Triton X-100, equal aliquots
of each extract were incubated with 3 × 104 cpm of
SiaGGnM-[125I]BSA (open bars) or 4 × 104 cpm of Gal-[125I]BSA (closed
bars). Equal aliquots of the detergent extracts were analyzed by
Western blot following separation by SDS-PAGE using anti-V5 antibody.
Lane 1, contains RHL1V5His; lane 2, contains
RHL2V5His from CHO-Tag (upper panel) and 293-Tag
(lower panel) cells.
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RHL1V5His and RHL2V5His were solubilized from CHO-Tag and 293-Tag cells
using Triton X-100, and their ability to bind
SiaGGnM-[125I]BSA and Gal-[125I]BSA was
compared as shown in Fig. 6. RHL1V5His was able to bind and precipitate
both SiaGGnM-[125I]BSA and Gal-[125I]BSA
when expressed in either CHO-Tag or 293-Tag cells. No evidence of
binding activity was seen in extracts from cells transfected with the
pcDNA3.1V5His vector alone. In addition, no binding activity for
either SiaGGnM-[125I]BSA or Gal-[125I]BSA
was associated with RHL2V5His. This is consistent with the observations
of other groups that lectin activity is primarily associated with RHL1
(17-19). Nonetheless, it is clear that recombinant RHL1V5His is able
to bind both SiaGGnM-BSA and Gal-BSA as was seen with the
affinity-purified ASGP-R prepared from rat liver above.
 |
DISCUSSION |
The ASGP-R was originally identified on the basis of its ability
to mediate the rapid clearance of glycoproteins such as ceruloplasmin from the blood following removal of sialic acid and exposure of underlying Gal moieties. Numerous studies have supported this conclusion, and sialylated glycoproteins are typically used to demonstrate the specificity of the ASGP-R for terminal Gal and GalNAc
moieties. Our observation that the rat ASGP-R is able to bind
saccharides with the terminal sequence Sia
2,6GalNAc
is both
unexpected and remarkable. The binding activity we have observed does
not reflect the presence of terminal GalNAc on the SiaGGnM-BSA glycoconjugate, because less than 1% of the
SiaGGnM-[125I]BSA preparation was bound by Wistaria
fluoribunda agglutinin-agarose (not shown), a lectin that
would detect the presence of saccharides with a single terminal
-linked GalNAc (20). Furthermore, the same amount of
SiaGGnM-[125I]BSA was precipitated by the ASGP-R before
and after neuraminidase digestion to remove the sialic acid (not
shown), indicating that removal of the sialic acid did not result in a
significant increase in binding by the ASGP-R.
The terminal sequence Sia
2,6GalNAc
1,4GlcNAc
1,2Man that we
described on PLP hormones produced by rat spongiotrophoblasts from mid
gestation to birth is a unique structure that is present on only a
limited number of glycoproteins (14, 15, 21). It is not yet clear if
the terminal sequence Sia
2,6Gal
1,4GlcNAc
1,2Man that is
present on large numbers of glycoproteins will also be recognized by
the ASGP-R. Our observation that SiaGGnM-[125I]BSA
binding by the ASGP-R is partially inhibited by 20 mM
sialic acid suggests that the sialic acid may actively participate in binding to the RHL1 subunit. The crystal structure of the Gal/GalNAc binding site of subunit 1 of the human ASGP-R has been resolved (22).
It will be of interest to determine how Sia
2,6GalNAc
1,4GlcNAc
is bound as compared with either GalNAc
1,4GlcNAc
or
Gal
1,4GlcNAc
. Because the pH and calcium dependence for
binding are similar for binding Sia
2,6GalNAc
1,4GlcNAc
and
Gal/GalNAc terminal ligands, it is likely that GalNAc coordinates with
the same calcium (site 2) and other contact residues in the presence or
absence of the
2,6-linked sialic acid.
The rapid clearance of the neoglycoconjugate SiaGGnM-BSA by the ASGP-R
was unanticipated, because the ASGP-R has long been assumed to exist
for the clearance of ligands whose terminal sialic acid has been
removed to expose underlying Gal or GalNAc moieties. Because
SiaGGnM-BSA is multivalent, we have not yet been able to establish the
affinity of RHL1 for Sia
2,6GalNAc
1,4GlcNAc
as compared with
GalNAc
1,4GlcNAc
or Gal
1,4GlcNAc
. The
Kd values for glycoproteins bearing 1, 2, 3, or more
terminal Sia
2,6GalNAc
1,4GlcNAc
sequences will have to be
determined to establish how rapidly such glycoproteins would be cleared
from the circulation. The results we have obtained with the
Man/GalNAc-4-SO4-receptor are, however, instructive.
The Man/GalNAc-4-SO4-receptor is found predominantly in the
form of a dimer at the surface of hepatic endothelial cells (9). Each
cysteine-rich domain at the amino terminus of the receptor is able to
engage a single terminal GalNAc-4-SO4 (23), and two terminal GalNAc-4-SO4 moieties on separate
N-linked oligosaccharides must be engaged simultaneously to
form a stable complex with the affinity seen for binding bovine LH to
isolated endothelial cells through its sulfated carbohydrate chains.
The neoglycoconjugate SO4-4-GalNAc
1,4GlcNAc
1,2Man
-BSA, which like
SiaGGnM-BSA has a high density of conjugated saccharides, is cleared
from the circulation of the rat in less than 2-3 min, whereas LH with
three N-linked oligosaccharides bearing terminal
GalNAc-4-SO4 moieties is cleared with a half-life of 7.5 min (11, 12). The clearance rate is consistent with the
Kd of 1.6 × 10
7 M
for binding to the Man/GalNAc-4-SO4-receptor; however, LH
in the circulation never exceeds a concentration of 1 × 10
9 M (12).
The clearance rate seen for LH in vivo reflects the enormous
amount of Man/GalNAc-4-SO4-receptor that is expressed in
the liver. With 4 × 108 endothelial cells in the
liver, >600,000 receptors at the surface of each cell, and a rapid
rate of endocytic uptake (t1/2 of 20 s) there
is sufficient capacity to clear all of the LH from the blood even if
only 0.5% of the receptor is occupied. Furthermore, the rate of
clearance will remain constant at all concentrations of LH below its
Kd of 1.6 × 10
7 M
for Man/GalNAc-4-SO4-receptor.
Based on the example of LH and the
Man/GalNAc-4-SO4-receptor, our studies suggest that a
different group of circulating glycoproteins may be the endogenous
ligands for the ASGP-R in vivo. Like the Man/GalNAc-4-SO4-receptor, ASGP-R is highly expressed
at the cell surface (500,000 receptors/cell) (24, 25) of an abundant
cell and the hepatocyte (6-7 × 108 cells per rat
liver) (26) and is rapidly endocytosed (27-29). Thus, if glycoproteins
with multiple terminal Sia
2,6GalNAc
moieties can be engaged with
affinities in the range of 1 × 10
7 M or
below, they would likely be removed from the blood with half-lives of
5-20 min that would not change with the changing concentration of each
glycoprotein below its Kd. Because
1,4-linked Gal
and GalNAc are both recognized by the ASGP-R, it is possible that
Sia
2,6Gal
may also be recognized. This raises the possibility
that the ASGP-R may function to actually clear glycoproteins with
terminal Sia
2,6GalNAc
or Sia
2,6Gal
moieties in
vivo rather than ones with terminal Gal or GalNAc.
Our current studies have revealed that glycoproteins bearing terminal
Sia
2,6GalNAc
are recognized by the rat ASGP-R. Thus, the PLP
hormones that bear these structures may represent examples of
endogenous ligands for the ASGP-R. Notably, in mice the levels of
ASGP-R have been reported to increase between mid gestation and birth
and return to the levels seen in non-pregnant animals within 24 h
after delivery (30). This pattern of expression further supports a
potential relationship between the addition of these carbohydrate
structures to PLP hormones and the regulation of their circulatory
half-life by the ASGP-R.
The specificity of the ASGP-R may also provide insight into other
observations. For example, terminal Sia
2,6GalNAc
is found on
glycodelin, a human pregnancy-specific glycoprotein with potent immunosuppressive and contraceptive activities that is found in the
amniotic fluid (15, 31). The immunosuppressive effects seen with
glycodelin have been attributed to the Sia
2,6GalNAc
-bearing oligosaccharides that may specifically block adhesive and
activation-related events mediated by CD22, the human B
cell-associated receptor. Because the ASGP-R is present on late
stage spermatids (32), the contraceptive activity associated with
glycodelin may be mediated through the carbohydrate recognition via the
ASGP-R subunit 1.
Our observations clearly demonstrate that the rat ASGP-R is capable of
binding oligosaccharides terminating with Sia
2,6GalNAc
as well as
ones terminating with GalNAc or Gal. Furthermore, glycoproteins bearing
oligosaccharides terminating with Sia
2,6GalNAc
are rapidly removed from the blood by the ASGP-R. Although PLPs bearing 2-4 termini with the sequence Sia
2,6GalNAc
would not likely be
removed from the circulation as rapidly as SiaGGnM-BSA, their
half-lives, like that of LH, may reflect clearance by the ASGP-R. Our
observations raise the possibility that oligosaccharides bearing sialic
acid in
2,6-linkage to GalNAc and possibly also to Gal may represent endogenous ligands for the ASGP-R.