From the Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, February 2, 2001, and in revised form, March 5, 2001
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ABSTRACT |
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Recognition of carbohydrates on
glycosylated molecules typically requires multivalent interactions with
receptors. Monovalent forms of terminal saccharides engaged by the
receptor binding sites typically display weak affinities in the
mM range and poor specificity. In contrast,
multivalent forms of the same saccharides are bound with strong
affinity (10 We previously demonstrated that glycoproteins such as
LH1 and thyrotropin
bearing N-linked oligosaccharides terminating with The latter observations were unexpected because we had reported that
bovine serum albumin substituted with 6-8 molecules of SO4-4-GalNAc Materials--
Man/GalNAc-4-SO4-Fc(Mu0) (the
transmembrane and cytosolic domains are replaced by the hinge,
CH2, and CH3 domains of human IgG1),
Man/GalNAc-4-SO4-Fc(Mu10) (deletion of the fibronectin type
II repeat and carbohydrate recognition domains (CRDs) 1-3 from Mu0),
and Man/GalNAc-4-SO4-Fc(Mu11) (deletion of the fibronectin type II repeat and CRDs 1-8 from Mu0) were prepared as described (5). 3'-SO4-LewisX
(3'-O-SO3Gal Surface Plasmon Resonance Analysis--
Surface plasmon
resonance (SPR) analyses were performed on an Amersham Pharmacia
Biotech BIACORE 2000 instrument as described previously (8). For
studies using immobilized the Cys-rich domain, a predetermined amount
of Man/GalNAc-4-SO4-Fc(Mu11) was allowed to bind to protein
A (Pierce), which had been covalently attached to a CM5 sensor chip.
The amount of Mu11 bound was monitored by SPR. No detectable
dissociation of Mu11 from protein A was observed under the conditions
of the assay. For inhibition studies the bovine LH
Binding of Mu11 to immobilized S4GGnM-BSA and S3GGnM-BSA was also
monitored using SPR. Similar amounts of S4GGnM-BSA and S3GGnM-BSA were
attached covalently to a BIACORE CM5 sensor chip through primary amine
groups using the amine coupling kit provided by the manufacturer. The
amount of S4GGnM-BSA and S3GGnM-BSA conjugated was determined from the
increase in response units. Mu11 was passed over immobilized BSA
conjugates at a flow rate of 5 µl/min for 10 min. The amount of Mu11
bound at equilibrium was used to generate saturation curves that were
analyzed by nonlinear regression using PRISM software (version
2.0).
Mole ratios were determined by dividing the change in response unit
values obtained for immobilized or bound glycoproteins by their
respective molecular weights. The molecular weights utilized were:
LH Equilibrium Dialysis
Studies--
3'-SO4-LewisX and a glucose
pentasaccharide (Glc5) prepared from dextran by mild acid hydrolysis
(10) were derivatized with 9-aminopyrene-1,4,6-trisulfonic acid (APTS)
by reductive amination as described (11). Binding was performed in a
two-chamber Teflon Micro-Equilibrium dialyzer (Amika, Inc.) utilizing
ultrathin 10,000-dalton cut-off membranes (The Nest Group, Inc.). One
chamber contained either 10 µM Mu11 (based on
Mr = 98,100) or BSA (1 mg/ml) in 25 µl of TBS
buffer. The second chamber contained 0.1-10 nmol of 3'-SO4-LewisX-APTS and Glc5-APTS in 25 µl of
TBS buffer. Dialysis was allowed to proceed for 96 h at 4 °C.
Thereafter, 2-µl aliquots were removed from each side of the
membrane, and the amounts of 3'-SO4-LewisX-APTS
and Glc5-APTS were quantitated by capillary electrophoresis using an
N-CHO-coated capillary column on a Beckman P/ACE 5000 and
laser-induced fluorescence for detection as described by the manufacturer. The Glc5-APTS was used to normalize the amount of 3'-SO4-LewisX-APTS because it did not
demonstrate any binding to Mu11 or BSA.
Monosaccharide Composition Analysis of BSA
Conjugates--
Quantitative analysis of the sugar constituents of the
sulfated glycoconjugates was performed according to the method
developed by Chen et al. (12). S4GGnM-BSA and S3GGnM-BSA
were hydrolyzed with 2.0 N trifluoroacetic acid to
release their constituent sugars. After amino sugar
N-acetylation with acetic anhydride in sodium carbonate, the
released monosaccharides were derivatized with APTS by reductive
amination. Capillary electrophoresis was performed using a fused silica
column in 120 mM MOPS buffer, pH 7.0, on a Beckman P/ACE
5000 (13). The APTS monosaccharides were identified and quantitated
using laser-induced fluorescence and comparison with monosaccharide standards.
Solution Binding Assays--
Affinity-purified
Man/GalNAc-4-SO4-Fc(Mu10) was incubated with
125I-labeled S4GGnM-BSA (1 nM) in 150 µl of
TBS buffer containing 1% (w/v) Triton X-100 and 0.1% (w/v) bovine IgG
in the presence of increasing concentrations of unlabeled
S4GGnM-BSA or S3GGnM-BSA. Mu10-S4GGnM-125I-BSA
complexes were precipitated by the addition of 1.5 ml of ice-cold 10%
polyethylene glycol-8000 (Sigma) and collected on Whatman GF/C filter
discs as described (3).
Specificity of the Cys-rich Domain for Monovalent
Ligands--
We have characterized the affinity and stoichiometry of
binding for ligands bearing different numbers and/or configurations of
terminal
We also determined Kd and
Bmax values for
3'-SO4-LewisX binding to Mu11 using equilibrium
dialysis. Preliminary experiments established both the conditions and
the time required for equilibrium to be reached (data not shown).
Glc5-APTS did not bind to Mu11, indicating no interaction between APTS
and the Cys-rich domain (data not shown).
3'-SO4-LewisX-APTS bound to Mu11 with a
Kd of 18.5 µM and a
Bmax of 1.02 mol of
3'-SO4-LewisX-APTS bound/mol of Cys-rich domain
(Fig. 2). The Kd
obtained by equilibrium dialysis is in good agreement with the
Ki of 29.7 µM obtained by inhibition
studies on the BIACORE as described above (Table I). Furthermore, in
agreement with our previous studies (8) and the crystallographic
analysis (6), each Cys-rich domain has a single binding site for a
sulfated saccharide.
Specificity of the Cys-rich Domain for Multivalent Ligands--
We
previously determined the dissociation constant for S4GGnM-BSA is 0.013 µM for Man/GalNAc-4-SO4-Fc(Mu0) and
Man/GalNAc-4-SO4-Fc(Mu11) (5). The assay utilized for these
determinations relied on polyethylene glycol-mediated precipitation of
S4GGnM-125I-BSA complexed to Mu0 or Mu11. We determined
inhibition constants for S4GGnM-BSA and S3GGnM-BSA using
S4GGnM-125I-BSA and Man/GalNAc-4-SO4-Fc(Mu10)
(Fig. 3). Mu10 was used because it is
secreted more efficiently after transfection than Mu0 and is more
efficiently precipitated by 10% polyethylene glycol-8000 than Mu11.
S4GGnM-BSA has a Ki of 0.013 µM,
whereas S3GGnM-BSA has a Ki of 0.170 µM (Table I). This results in a 2000-fold change in the
affinity for S4GGnM-BSA as compared with monovalent S4GGnM (0.013 versus 25.8 µM) and a 95-fold change in
affinity for S3GGnM-BSA as compared with the monovalent S3GGnM (0.170 versus 16.2 µM). Even though the
Ki for S4GGnM is 1.6-fold greater than the
Ki for S3GGnM, the Ki for
S3GGnM-BSA is 13-fold greater than the Ki for
S4GGnM-BSA. Thus, the Man/GalNAc-4-SO4 receptor displays
significantly greater specificity for multivalent than for monovalent
forms of the same ligand.
Kinetics for Binding S4GGnM-BSA and S3GGnM-BSA--
The above
inhibition studies alone did not provide insight to the molecular basis
for the difference in specificity for monovalent and multivalent forms
of the same trisaccharides. We therefore used SPR to examine the
kinetics of S4GGnM-BSA (Fig.
4A) and S3GGnM-BSA (Fig.
4B) binding to immobilized Mu11. At a concentration of 400 nM, the binding of S4GGnM-BSA rapidly reaches saturation.
Elution with buffer devoid of S4GGnM-BSA beginning at 700 s
results in little or no dissociation. The highest amount of S4GGnM-BSA
bound yields a mole ratio of 0.34 mol S4GGnM-BSA/mol of Mu11 or 0.17 mol S4GGnM-BSA/mol of Cys-rich domain. This is equal to a Cys-rich domain/S4GGnM-BSA mole ratio of 5.9, which is a value close to the
theoretical maximum of 6.7 based on the number of trisaccharides conjugated to the BSA (see below) and a single binding site for terminal GalNAc-4-SO4/Cys-rich domain. Further, this
indicates that the slow dissociation rate is a result of multivalent
binding. At a concentration of 8 nM S4GGnM-BSA (Fig.
4A) the amount of S4GGnM-BSA bound increases throughout the
binding phase of the experiment. Because of the lack of significant
dissociation of the bound ligand, if given sufficient time the amount
of S4GGnM-BSA bound to Mu11 would attain the same maximum as was seen
with 400 nM S4GGnM-BSA. Further evidence for the
contribution of multivalency to the slow dissociation rate was obtained
by co-injection of 1 mM GalNAc-4-SO4 during the
dissociation phase (Fig. 4C). The S4GGnM-BSA was rapidly and
completely dissociated, indicating that the equilibrium at individual
GalNAc-4-SO4 binding sites is rapid. The dissociation rate
in the presence of 1 mM GalNAc-4-SO4 is so
rapid that the dissociation rate could not be determined by SPR.
S3GGnM-BSA was also bound by immobilized Mu11 (Fig. 4B). The
amount of S3GGnM-BSA bound was less than the amount of S4GGnM-BSA bound
at the same concentration (compare 400-nM concentrations in
Fig. 4, A and B). However, the amount of
S3GGnM-BSA bound to Mu11 continues to increase with time.
Furthermore, little or no dissociation of S3GGnM-BSA is observed
when elution with buffer free of S3GGnM-BSA is initiated at 700 s.
Thus, as was seen for S4GGnM-BSA, complexes formed with S3GGnM-BSA are
multivalent and highly stable. Furthermore, bound S3GGnM-BSA is rapidly
dissociated from Mu11 in the presence of 1 mM
GalNAc-4-SO4 (Fig. 4C). Given sufficient time
and/or increased concentrations of S3GGnM-BSA, maximal amounts of
binding equal to those obtained with S4GGnM-BSA can be attained (data
not shown).
Because neither S4GGnM-BSA nor S3GGnM-BSA dissociates at a significant
rate from the immobilized Mu11, the results shown in Fig. 4,
A and B, indicate that at identical
concentrations the association rate for S3GGnM-BSA is considerably
slower than for S4GGnM-BSA. A difference in the ratio of trisaccharide
conjugation to BSA was excluded as an explanation for the different
rates by determining the mole ratio of trisaccharide to BSA for
each conjugate. After acid hydrolysis, the released monosaccharides were derivatized with APTS and quantitated by capillary electrophoresis using a laser-induced fluorescence detector (data not shown). The mole
ratio of trisaccharide to BSA was 6.73 for S4GGnM-BSA and 6.75 for
S3GGnM-BSA, which is in agreement with the analyses performed at the
time of synthesis. Thus, the different characteristics seen for
S4GGnM-BSA and S3GGnM-BSA binding to immobilized Mu11 reflect a
significantly slower association rate for S3GGnM-BSA than for
S4GGnM-BSA.
The multivalent nature of the binding of S4GGnM-BSA and
S3GGnM-BSA to immobilized Mu11 and the consequent lack of dissociation prevent kinetic analyses. We therefore used SPR to characterize the
binding of Mu11 to identical concentrations of immobilized S4GGnM-BSA
and S3GGnM-BSA. Although the immobilized BSA conjugates are
multivalent, the Man/GalNAc-4-SO4-Fc(Mu11) chimera is
bivalent and can only engage two terminal
sulfated GalNAc moieties. The results of
these analyses are shown in Figs. 5 and 6 and summarized in Table II.
Mu11 reaches equilibrium rapidly when
binding to either immobilized S4GGnM-BSA or S3GGnM-BSA (Fig. 5).
Mu11 bound to immobilized S4GGnM-BSA with
a Kd of 3.90 µM and bound to
immobilized S3GGnM-BSA with a Kd of 3.93 µM. However, Mu11 bound to S4GGnM-BSA with a
Bmax of 0.74 mol/mol and bound to S3GGnM-BSA
with a Bmax of 0.09 mol/mol (see Fig. 6 and
Table II). Thus, even though Mu11 binds to both conjugates with a
similar affinity under these conditions, there is an 8-fold difference in the amount of Mu11 bound at saturation. This is true even though the
number and concentration of terminal sulfated saccharides are
essentially identical.
Our analyses demonstrate that inhibition constants obtained
with monovalent ligands do not necessarily predict the
properties of multivalent forms of the same ligands. This study
provides new insights into the basis for the remarkable difference in
specificity displayed by the Man/GalNAc-4-SO4 receptor for
multivalent as compared with monovalent ligands. The clearance studies
we performed in the rat (1), as well as subsequent analyses with the
isolated hepatic endothelial cells (2) and the purified
Man/GalNAc-4-SO4 receptor (3), indicated that this receptor
displays specificity for the location of the sulfate on terminal
Each Cys-rich domain is able to engage a single terminal sulfated
monosaccharide. In agreement with the studies of Leteux et
al. (7) and Liu et al. (6, 14), monovalent forms of the
trisaccharide GalNAc7-10
9 M) and
significantly greater specificity. Although multivalency can readily
account for increased affinity, the molecular basis for enhanced
specificity is not well understood. We have examined the specificity of
the cysteine-rich domain of the mannose/GalNAc-4-SO4 receptor using monovalent and multivalent forms of the trisaccharide GalNAc
1,4GlcNAc
1,2Man
(GGnM) sulfated at either the C4
(S4GGnM) or C3 (S3GGnM) hydroxyl of the terminal GalNAc. Monovalent
S4GGnM and S3GGnM have Ki values of 25.8 and 16.2 µM, respectively. Multivalent conjugates of the same
GalNAc-4-SO4- and GalNAc-3-SO4-bearing trisaccharides (6.7 mol of trisaccharide/mol of bovine serum albumin) have Ki values of 0.013 and 0.170 µM,
respectively. The 2000-fold versus 95-fold change in
affinity seen for the multivalent forms of these 4-sulfated and
3-sulfated trisaccharides reflects a difference in the impact of
conformational entropy. A large fraction of the
SO4-3-GalNAc structures exists in a form that is not
favorable for binding to the Cys-rich domain. This reduces the
effective concentration of SO4-3-GalNAc as compared with
SO4-4-GalNAc under the same conditions and results in a
markedly lower association rate. This difference in association rate
accounts for the 12-fold difference in the rate of clearance from the
blood seen with S4GGnM-BSA and S3GGnM-BSA in
vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-linked GalNAc-4-SO4 are recognized by a receptor
located in hepatic endothelial cells, the Man/GalNAc-4-SO4
receptor, and are rapidly removed from the circulation (1-4). Rapid
clearance in conjunction with stimulated release from dense core
storage granules in LH-producing cells located in the anterior lobe of the pituitary produce the episodic rise and fall in serum LH levels that is important for the expression of LH bioactivity in
vivo. A novel feature of the Man/GalNAc-4-SO4 receptor
is its capacity to bind carbohydrate moieties terminating with Man and
GalNAc-4-SO4 at physically distinct domains that are not
structurally related. The binding site for GalNAc-4-SO4 is
located in the cysteine-rich domain found at the N terminus of the
Man/GalNAc-4-SO4 receptor (5). The Cys-rich domain is a
member of the
-trefoil family of proteins that includes proteins
such as acidic fibroblast growth factor. The binding site is a
neutral pocket that accommodates the sulfate group, which accounts for
the major interactions with the protein (6). Inhibition and
modeling studies with monovalent ligands have indicated that the
binding site in the Cys-rich domain can also accommodate saccharides
with terminal Gal or GalNAc when the sulfate is located at C3 rather
than C4 (6, 7).
1,4GlcNAc
1,2Man
(S4GGnM-BSA) is
removed from the circulation at a rate that is at least 12-fold greater
than that seen for bovine serum albumin substituted with 6-8 molecules
of SO4-3-GalNAc
1,4GlcNAc
1,2Man
(S3GGnM-BSA).
Furthermore, isolated hepatic endothelial cells do not bind and
internalize S3GGnM-BSA, and the purified Man/GalNAc-4-SO4
receptor does not display significant binding of S3GGnM-BSA in
precipitation assays (2, 3). We have recently determined that the
Man/GalNAc-4-SO4 receptor must be dimeric and engage at
least two terminal GalNAc-4-SO4 moieties on separate
oligosaccharides to mediate uptake by hepatic endothelial cells with a
Kd of 1.63 × 10
7 M
(8). These observations raise the possibility that the
Man/GalNAc-4-SO4 receptor may display significantly greater
specificity when binding ligands with multiple terminal sulfated
saccharide moieties than when binding monovalent forms of the same
ligands. We have examined this possibility using well characterized
ligands of known valency and structure. Our studies demonstrate that
the Man/GalNAc-4-SO4 receptor displays greater specificity
for multivalent forms of the same structures than for monovalent forms
and that this can be explained best by the presence of favorable and
unfavorable conformations of the trisaccharides for binding to the
Cys-rich domain. Remarkably, inhibition constants obtained with the
monovalent forms of these trisaccharides are not predictive of the
properties of multivalent forms of the same structures.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4(Fuc
1,3)GlcNAc) was from
Calbiochem. S4GGnM-MCO, S3GGnM-MCO, and their BSA conjugates,
S4GGnM-BSA and S3GGnM-BSA, were prepared as described (9). The LH
subunit was supplied by the National Hormone and Pituitary Program
(NIDDK, National Institutes of Health) and A. F. Parlow.
subunit (685 nM) was passed over the chip at a flow rate of 5 µl/min
for 300 s in the presence of increasing concentrations of
saccharide inhibitors. The amount of LH
bound at equilibrium was
used to generate inhibition curves that were analyzed by nonlinear
regression using PRISM software (version 2.0). Ki
values were calculated from the concentration of inhibitor that
effected 50% inhibition. For glycoconjugate binding studies, differing
concentrations of S4GGnM-BSA and S3GGnM-BSA were passed over
immobilized Mu11 at a flow rate of 5 µl/min for 10 min. Dissociation
was initiated at 10 min by elution with TBS buffer (20 mM
Tris·HCl, pH 7.4, 150 mM NaCl) + 0.005% (w/v) surfactant P20 or TBS buffer + 0.005% surfactant P20 containing 1 mM
GalNAc-4-SO4.
, 14,600; Mu11, 98,100; S4GGnM-BSA, 73,100; and S3GGnM-BSA, 73,100. The values calculated for S4GGnM-BSA and S3GGnM-BSA reflect the
presence of 7 mol of sulfated trisaccharide conjugated/mol of BSA and
are consistent with their mobility when examined by SDS-polyacrylamide
gel electrophoresis (data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-linked GalNAc-4-SO4 moieties utilizing SPR.
The competitive inhibition studies shown in Fig.
1 were performed by passing the LH
subunit over Man/GalNAc-4-SO4-Fc(Mu11) bound to immobilized
protein A in the presence of increasing amounts of monovalent
inhibitors. GalNAc-4-SO4,
3'-SO4-LewisX, S4GGnM-MCO, and S3GGnM-MCO have
Ki values of 28.2, 29.7, 25.8, and 16.2 µM, respectively (Table I).
The similar Ki values obtained for
GalNAc-4-SO4 and S4GGnM indicate that recognition is
directed almost exclusively at the terminal sulfated monosaccharide.
The Ki value determined for S3GGnM is half that
obtained for S4GGnM. Thus, for the monovalent trisaccharide
SO4-GalNAc
1,4GlcNAc
1, 2Man
-MCO location of the sulfate at the C3 hydroxyl as compared with the C4 hydroxyl of the
terminal, GalNAc enhances interaction with the binding site in the
Cys-rich domain.
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Fig. 1.
Inhibition of LH
binding to immobilized Man/GalNAc-4-SO4-Fc(Mu11) by
monovalent sulfated saccharides. LH
was passed over immobilized
Mu11 in the presence of the indicated concentrations of
SO4-3-GalNAc
1,4GlcNAc
1,2Man
-MCO (
),
SO4-4-GalNAc
1,4GlcNAc
1,2Man
-MCO (
),
GalNAc-4-SO4 (
), or
3'-SO4-LewisX (
). The amount of LH
bound
at equilibrium in the presence of inhibitor was used to generate the
curves shown. Ki values were calculated using the
inhibitor concentration that effected 50% inhibition.
Binding constants for monovalent and polyvalent ligands sulfated at
either the C3 or C4 hydroxyl of terminal GalNAc or Gal
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Fig. 2.
Determination of the stoichiometry and
dissociation constant for 3'-SO4-LewisX binding
to the Cys-rich domain of the Man/GalNAc-4-SO4
receptor. Increasing amounts of
3'-SO4-LewisX-APTS and Glc5-APTS were dialyzed
against 10 µM Man/GalNAc-4-SO4-Fc(Mu11) for
96 h at 4 °C. The amount of
3'-SO4-LewisX-APTS and Glc5-APTS in each
25-µl chamber was determined by capillary electrophoresis. A
Scatchard analysis of the results is shown and yields a
Kd of 18.5 µM and a
Bmax of 1.02 mol of
3'-SO4-LewisX-APTS bound/mol of Cys-rich domain
(N) at saturation. B/F, bound/free
ligand.
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Fig. 3.
S4GGnM-BSA and S3GGnM-BSA have different
Ki values. S4GGnM-125I-BSA
was incubated with Man/GalNAc-4-SO4-Fc(Mu10), and complexes
were precipitated by the addition of 10% polyethylene glycol-8000.
Increasing amounts of either S3GGnM-BSA ( ) or S4GGnM-BSA (
) were
added to generate the inhibition curves shown. Ki
values were determined as summarized in Table I.
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Fig. 4.
S4GGnM-BSA and S3GGnM-BSA display markedly
different rates of binding to immobilized
Man/GalNAc-4-SO4-Fc(Mu11). Mu11, 4180 response units,
was bound to the protein A that had been coupled to a CM5 biosensor
chip. Concentrations of 400, 80, and 8 nM S4GGM-BSA
(A) and S3GGM-BSA (B) were injected at a flow
rate of 5 µl/min, and the amount of binding was monitored by SPR.
After 700 s, buffer without S4GGnM-BSA or S3GGnM-BSA was passed
over the chip to monitor dissociation. C, S4GGnM-BSA (80 nM) and S3GGnM-BSA (400 nM) were injected as
above except that 1 mM GalNAc-4-SO4 was
co-injected during the dissociation phase.
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Fig. 5.
Analysis of
Man/GalNAc-4-SO4-Fc(Mu11) binding to immobilized S4GGnM-BSA
and S3GGnM-BSA using surface plasmon resonance. Increasing
concentrations of Mu11, ranging from 2 to 500 µg/ml (0.02-5
µM), were injected over a surface with immobilized
S4GGnM-BSA (A) or S3GGnM-BSA (B) for 10 min. at a
flow rate of 5 µl/min. The surfaces contained 6267 and 5769 response
units of immobilized S4GGnM-BSA and S3GGnM-BSA, respectively. No
significant change in the refractive index was observed in the presence
of 1 mM GalNAc-4-SO4 or when Mu11 was injected
over a surface with immobilized Man-BSA (data not shown).
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Fig. 6.
Man/GalNAc-4-SO4-Fc(Mu11) binds
to immobilized S4GGnM-BSA and S3GGnM-BSA with the same
Kd but a different
Bmax. The equilibrium binding data
from the experiments shown in Fig. 5 were used to generate saturation
curves for Mu11 binding to immobilized S4GGnM-BSA ( ) and S3GGnM-BSA
(
). The results of regression analyses are summarized in Table II.
RU, response units
Binding of increasing concentrations of Mu11 to equal amounts of
immobilized S3GGnM-BSA and S4GGnM-BSA was monitored by SPR
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4-linked GalNAc in the context of the sequence
GalNAc
1,4GlcNAc
1,2Man
. The recent report (7) that monovalent
saccharides with a sulfate located at the C3 hydroxyl of Gal have
Ki values similar to those of monovalent structures
with the sulfate located at the C4 hydroxyl of Gal or GalNAc seemed to
contradict these observations. The studies we have presented here
demonstrate that the Man/GalNAc-4-SO4 receptor displays far
greater specificity for multivalent ligands than would be predicted on
the basis of binding constants obtained for monovalent forms of the
same ligands.
1,4GlcNAc
1,2Man
-MCO sulfated at either the
C3 or C4 hydroxyl of the terminal GalNAc are bound with similar affinities of 16.2 and 25.8 µM, respectively. How can we,
therefore, account for the dissociation constants of 0.170 and 0.013 µM obtained for S3GGnM-BSA and S4GGnM-BSA, respectively?
Investigators have previously observed greater specificity on the part
of lectins for polyvalent ligands as compared with their monovalent
counterparts (15-22). The specificity for multivalent ligands
has been attributed to a requirement for unique spacing patterns
(15-18), secondary protein-protein and protein-carbohydrate
interactions (19, 20), and amplification in the polyvalent state of
small differences in the affinities between monovalent forms (21). One
prediction of a model in which small differences in affinity of
monovalent ligands are amplified in the polyvalent state is that the
affinities obtained for Mu11 binding to immobilized S4GGnM-BSA and
S3GGnM-BSA would differ but that the Bmax
obtained at saturation would be the same. This is not the case because
Mu11 binds to immobilized S4GGnM-BSA and S3GGnM-BSA with the same
affinity but with an 8-fold difference in Bmax
at saturation. The behavior seen in Fig. 6 is explicable if the
trisaccharide S3GGnM exists in two distinct conformations, only one of
which is favorable for binding (see Fig.
7A). The crystal structures of
the Cys-rich domain binding 3'-SO4-Lewisa
(3'-(SO4-Gal
1,3(Fuc
1,4) GlcNAc)) and
3'-SO4-LewisX indeed display significant
differences in the structure of the carbohydrate, suggesting that one
or both of the 3'-sulfated oligosaccharides undergo a conformational
change when binding to the Cys-rich domain (14). The
N-acetyl present on the GalNAc-3-SO4 may require
an even greater change in conformation to allow binding. Formation of
stable complexes requires the simultaneous engagement of two terminal
GalNAc-3-SO4 moieties by the bivalent Mu11. If only a small
portion of the S3GGnM is in a form favorable for binding at any point
in time, and all of the S4GGnM is in a favorable form for binding, the
effective concentration of S3GGnM-BSA would appear to be a fraction of
that of S4GGnM-BSA. However, when binding does occur to the favorable
form of S3GGnM, it would have the same properties as the binding to
S4GGnM-BSA. Hence the two would have the same Kd but
different Bmax values.
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Fig. 7.
A model for how the Cys-rich domain of the
Man/GalNAc-4-SO4 receptor interacts differently with
S4GGnM-BSA and S3GGnM-BSA. A, bivalent Mu11
(tulip shape) binds to both immobilized S4GGnM-BSA and
S3GGnM-BSA. Equilibration between conformations favorable and
unfavorable for Mu11 binding reduces the effective concentration of
S3GGnM termini as compared with S4GGnM termini. B, Mu11
immobilized on a surface (dark line) will bind multivalent
forms of S4GGnM-BSA (filled triangles,
GalNAc-4-SO4) and S3GGnM-BSA (open triangles,
GalNAc-3-SO4). Because S3GGnM exists in both favorable and
unfavorable forms for binding to Mu11, it takes longer to form multiple
interactions with the immobilized Mu11. S4GGnM and S3GGnM are both in
rapid equilibrium at individual sites but do not dissociate because of
the inability to simultaneously release all of the bound termini. In
the presence of free GalNAc-4-SO4 dissociation is rapid
because no rebinding can occur.
Differences in the contribution of conformational entropy (23), defined as the contribution of entropy arising from a difference in the number of conformations available before complexation and after complexation, are the most likely basis for the 13-fold difference in the Ki values obtained for S4GGnM-BSA and S3GGnM-BSA. When S3GGnM-BSA and S4GGnM-BSA bind to immobilized Mu11, this difference is manifested as a slow association rate for S3GGnM-BSA as compared with S4GGnM-BSA (Fig. 4). Once a sufficient number of terminal sulfated saccharides are engaged, the multivalent interaction results in a negligible dissociation rate for both S3GGnM-BSA and S4GGnM-BSA (see Fig. 7B). As a result, if given sufficient time S3GGnM-BSA will reach the same maximal level of binding as S4GGnM-BSA when examined on the BIACORE. Equilibration at individual binding sites is rapid, however, because both S3GGnM-BSA and S4GGnM-BSA dissociate from the immobilized Mu11 in the presence of 1 mM GalNAc-4-SO4 at a rate that is too rapid to determine the koff (Figs. 4 and 7B). Rapid equilibration at individual binding sites in conjunction with rapid equilibration between conformations that are favorable and nonfavorable for binding may account for the similar Ki values obtained for monovalent forms of these sulfated trisaccharides.
The different rates of binding to immobilized Mu11 and the different Ki values obtained for S4GGnM-BSA and S3GGnM-BSA mirror the behavior seen in vivo (2). Radiolabeled S4GGnM-BSA is rapidly removed from the blood (t1/2 = 1.3 min), whereas S3GGnM-BSA is removed slowly (t1/2 = 15.3 min). Thus, the 12-fold difference in clearance rate correlates well with the 13-fold difference in Ki. Furthermore, S4GGnM-BSA but not S3GGnM-BSA is bound and internalized by isolated hepatic endothelial cells (2). Because the GalNAc-4-SO4-specific form of the Man/GalNAc-4-SO4 receptor in hepatic endothelial cells exists as a dimer (8), binding two terminal GalNAc-3-SO4-moieties is not likely to produce a sufficiently stable complex to mediate internalization. In addition, the interaction with a greater number of terminal GalNAc-3-SO4-moieties by bridging between dimeric GalNAc-4-SO4 receptors is likely a slow process relative to the rate of receptor internalization. As a result ligands terminating with Gal or GalNAc sulfated at the C3 hydroxyl are not likely to represent ligands for the Man/GalNac-4-SO4 receptor in vivo.
Soluble forms of the Man/GalNAc-4-SO4 receptor have been reported in the circulation and are thought to arise as a result of proteolytic digestion (24). These soluble forms of the Man/ GalNAc-4-SO4 receptor would not be likely to bind terminal GalNAc-4-SO4, GalNAc-3-SO4, or Gal-3-SO4 unless they remain dimeric or are in some manner multimerized. The amount of GalNAc-3-SO4 or Gal-3-SO4 that would be required would be at least 8-fold higher than GalNAc-4-SO4. Whether the soluble forms of the Man/GalNAc-4-SO4 receptor in serum exist as dimeric or monomeric molecules remains to be addressed.
Our studies demonstrate that although the binding site of the Cys-rich
domain of the Man/GalNAc-4-SO4 receptor can accommodate a
number of different sulfated monosaccharides as monovalent ligands, it
displays much more highly restricted specificity when simultaneously engaging two or more terminal sulfated GalNAc or Gal moieties. That
this specificity is attained through differences in conformational entropy is remarkable and may apply to other instances in which multivalent ligands are recognized by lectins with greater specificity than the same monovalent ligands. The affinity and specificity of the
Man/GalNAc-4-SO4 receptor for oligosaccharides terminating with 1,4-linked GalNAc-4-SO4 are essential for
regulating the half-life of LH in the circulation throughout the
ovulatory cycle including the preovulatory surge. The ability to ignore
other ligands whether monovalent or multivalent assures that the
clearance of LH will be maintained even if other sulfated ligands are
present in the blood. Precisely how multivalent forms of S3GGnM are
excluded from binding while multivalent forms of S4GGnM are not
excluded will require real-time analysis of such ligands in the
presence of dimeric Cys-rich domains. Models of oligosaccharides
terminating with GalNAc-4-SO4 and GalNAc-3-SO4
reveal marked differences in their three-dimensional structures that
would potentially have a major impact on access to the binding site on
the Cys-rich domain. This represents a highly novel mechanism to
dictate the specificity of carbohydrate receptors for complex
multivalent saccharide structures in vivo.
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ACKNOWLEDGEMENT |
---|
We thank Nancy Baenziger for critical comments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R37-CA21923 and R01-DK41738.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 314-362-8730;
Fax: 314-362-8888; E-mail: Baenziger@Pathology.WUSTL.EDU.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M101027200
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ABBREVIATIONS |
---|
The abbreviations used are:
LH, lutropin;
Man, mannose;
S4GGnM, SO4-4-GalNAc1,4GlcNAc
1,2Man
;
S3GGnM, SO4-3-GalNAc
1,4GlcNAc
1,2Man
;
BSA, bovine serum
albumin;
MCO, O-(CH2)8OOCH3;
3'-SO4-LewisX, 3'-O-SO3Gal
1,4(Fuc
1,3)GlcNAc;
Mu0, Man/GalNAc-4-SO4-Fc(Mu0), transmembrane and cytosolic
domains of Man/GalNAc-4-SO4 receptor replaced with the Fc
refion of human IgG1;
Mu10, Man/GalNAc-4-SO4Fc(Mu10),
fibronectin type II repeat and carbohydrate recognition domains 1-3
deleted from Man/GalNAc-4-SO4Fc(Mu0);
Mu11, Man/GalNAc-4-SO4-Fc(Mu11), fibronectin type II repeat and
carbohydrate recognition domains 1-8 deleted from
Man/GalNAc-4-SO4-Fc(Mu0);
SPR, surface plasmon
resonance;
APTS, 9-aminopyrene-1,4,6-trisulfonic acid;
MOPS, 3-[N-morpholino]propane sulfonic acid.
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