Sugar Chip: A Novel Method for Immobilization of Oligosaccharides for Surface
Plasmon Resonance Analysis of Heparin-Protein Interactions
Yasuo Suda1 and Michael Sobel2
Department of Nanostructure and Advanced Materials, Graduate School of Science and Engineering,
Kagoshima University and Japan Science and Technology Agency,
2
Department of Surgery, University of Washington and VA Puget Sound HCS
1
ABSTRACT
We report here a novel method for the
immobilization of oligosaccharides, and its
application to the analysis of heparin-protein
interactions with Surface Plasmon Resonance (SPR).
By the optimized reductive amination reactions and
well-designed linker molecules, a structurally
defined disaccharide unit of heparin was
immobilized on a Au coated chip, and its binding
interaction with known heparin-binding domains of
von Willebrand factor (vWF) were quantitatively
analyzed by SPR. The prepared chip (named
“sugar chip”) bound the heparin-binding domain of
vWF highly specifically with affinity comparable to
that previously reported by other methods.
This
novel technique for oligosaccharide immobilization
in SPR studies is accurate, specific, and easily
applicable to both synthetic and naturally derived
oligosaccharides.
INTRODUCTION
Oligosaccharides are increasingly being recognized
as important partners in receptor-ligand binding and
cellular signaling1.
In biological systems, the
oligosaccharides are usually clustered to exert their
full biological activity, because the activity of an
isolated oligosaccharide is usually not sufficient.
The challenges of studying glycosaminoglycan
(GAG)-protein interactions at the molecular level
have included the heterogeneity of polysaccharides,
and difficulties in their labeling and immobilization.
SPR technology permits the real-time analysis of
molecular binding using microgram quantities of
materials. It can measure the binding affinity, on
and off rates, and is useful for the high throughput
screening of new drug targets. So far, however, no
simple method has been available to immobilize
oligosaccharides directly on the SPR chip without
interference with the biological activity of the
oligosaccharide.
Current methods were only
applicable to limited synthetic oligosaccharides
without sulfate or phosphate groups2,3. To this end,
we pursued novel immobilization strategies for
oligosaccharides, and demonstrated their utility in
the study of heparin binding to proteins.
In previous work we had shown that a specific
disaccharide
unit
in
heparin,
O-(2-deoxy-2-sulfamido-6-O-sulfo--D-glucopyrano
syl)-(1-4)-2-O-sulfo--L-idopyranosyluronic
acid
(abbreviated as GlcNS6S-IdoA2S), was a key unit
responsible for the heparin’s binding interaction with
human platelets and von Willebrand factor (vWF)4-7.
In the present work, this GlcNS6S-IdoA2S unit was
conjugated with a newly developed linker molecule,
and ligand-conjugate was then immobilized on the
gold sensor chip through a gold-sulfur (Au-S)
permanent bond to prepare the sugar chip. The
application of the sugar chip for SPR analyses are
described.
EXPERIMENTAL
A linker molecule and ligand-conjugates were
prepared as illustrated in Scheme 1. By soaking the
gold-coated chip in a 0.1 mM methanol solution of
the ligand-conjugate 6, which contains the
GlcNS6S-IdoA2S unit and a disulfide moiety, 6 was
directly immobilized on the chip. A permanent
Au-S bond was formed spontaneously to yield an
sugar chip (SPR sensor chip), in which the
GlcNS6S-IdoA2S units are expected to be
two-dimensionally clustered on the surface of the
chip.
The hydrophilic conjugate 5 was also
immobilized on the sensor chip similarly.
RESULTS AND DISCUSSION
In SPR, the change of the surface plasmon resonance
at the interface of the coated gold layer are
proportional to the number of molecules that bind the
ligand immobilized on the surface of the chip8.
Using an SPR670 apparatus (NLE, Nagoya, Japan),
binding to the ligand moiety on the chip was
observed when 2 M of the synthetic peptide
KDRKRSELRRIASQVK (a heparin binding domain
of human vWF9) was injected over the surface of the
chip (figure 1).
Because almost no binding
interaction was found when 0.1 mg/ml of a control
protein, bovine serum albumin (BSA), was run on
the same chip, it was not necessary to subtract a
non-specific binding value.
When a control
conjugate 5 was immobilized using the same method,
no significant increase in resonance units was
observed even though a high concentration of
synthetic vWF-peptide was used. To estimate the
KD, binding to the sugar chip (using 6) was measured
over a range of concentrations, and the data
presented as a binding plot. The estimated KD
value (220 nM) was very close to that reported
previously9. The same chip was used to study the
binding of a larger portion of the vWF protein that
still encompassed the same heparin-binding domain:
a recombinant fragment of the entire A1 domain of
vWF10. Figure 2 summarizes the binding data of
this recombinant vWF A1 domain. The estimated
KD of this protein/oligosaccharide interaction was 1.2
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National Institutes of Health (HL 39903, MS) and
the Department of Veterans Affairs Research Service
(MS).
M.
CONCLUSION
The current results illustrate three advantageous, and
complementary aspects of this approach to the study
of heparin-protein interactions. First, SPR directly
measures equilibrium binding, with no modification
of the binding proteins. Second, using reductive
amination techniques, the structurally defined
oligosaccharide can be easily incorporated, without
significant modification of their functional groups,
into novel conjugates for their immobilization on
sensor chips. Finally, this method facilitates the use
of small quantities of structurally defined molecules
to elucidate the specific structure-function relations
of GAG-protein binding.
Any oligosaccharide
molecule with a reducing end (which typically can be
obtained
from
natural
sources
using
endo-glycosidases) should thus be eligible for this
linkage strategy. SPR, combined with this novel
method for the immobilization of oligosaccharides,
has the potential to elucidate a range of fundamental
structure-function relations and biological effects of
oligosaccharides at the molecular level.
REFERENCES
1. Varki, A., In Essentials of Glycobiology. (eds.
Varki, A., et al.) p. 57-68, and references therein
(Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, New York, NY, 1999).
2. Fazio, F., et al., J. Am. Chem. Soc.,124, 14397
(2002)
3. Park, S., et al., J. Am. Chem. Soc.,126, 4812
(2002)
4. Suda, Y., et al., Thromb. Res. 69, 501 (1993).
5. Poletti, L.F., et al., Arterioscler. Thromb. Vasc.
Biol. 17, 925 (1997).
6. Koshida, S., et al., Tetrahedron Lett. 40, 5725
(1999).
7. Koshida, S., et al., Tetrahedron Lett. 42, 1289
(2001).
8. Liedberg, B., et al., Sensors and Actuators 4,
299 (1983).
9. Sobel M., et al., J. Biol. Chem. 267, 8857(1992).
10. Cruz, M.A., et al., J. Biol. Chem. 268, 21238
(1993).
ACKNOWLEDGEMENTS
This work was supported in parts by grants from
Japan Science and Technology Agency (YS), the
BocHN
H2N
a
NH2
1
H
N
R HN
b
NH2
S
S
O
2
3 R = Boc
4R=H
c
HO
O
4
+
OH
OH
d
OH
OH
HO
HO
OH
OSO3Na
O
+
OH
O
S
S
O
5
O
O
O
CO2Na
OH
e
OH
OH
HO
OH
NHSO3Na
OSO3Na
O
OSO3Na
OH
O
O
CO2Na
OH
OH
OH
O
HO
HO
OH
NHSO3Na
OSO3Na
HO
H
N
OH
OH
4
H
N
H
N
H
N
S
S
O
6
Scheme 1 Reagents and conditions: a, (Boc)2O, Et3N, MeOH, 0 °C then rt,
19 h, 68%; b, Thioctic acid, HOBT, EDC-HCl, rt, 14 h, 86%; c, TFA,
dioxane, -10 °C then 0 °C, 17 h, 85%;d, NaBH3CN, AcOH-H2O, 37 °C, 4 d,
92%; e, NaBH3CN, AcOH-H2O-MeOH, 37 °C, 3 d, 36%
600
1000
800
Buffer
400
2 M vWF-Peptide
Injection
Response (RU)
Resposnse (RU)
500
300
Equilibrium
Binding
200
600
400
100
0
0
200
400
600
800
1000
1200
1400
1600
1800
Estimated
KD = 1.16 M
200
0.1 mg/ml BSA
0
2000
0
Time (sec)
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Concentration (nM)
Fig. 1 Binding of vWF-peptide and BSA to ligand-conjugate 6 containing
GlcNS6S-IdoA2S on the chip was observed (Flow rate = 5 ml/min, Temp = 25
°þC, pH 7.4 PBS);
Fig. 2 The equilibrium binding data were used to generate a saturation curve
for the binding of recombinant human vWF A1 domain to the ligandconjugate 6 on the chip.
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