Biological Evaluation of Multivalent-Type
N-Acetyl-D-Glucosamine (GlcNAc) Conjugates
for Wheat Germ Agglutinin (WGA) by the
Surface Plasmon Resonance (SPR) Method
Amrita Kumari1, Tetsuo Koyama1, Ken Hatano1 and Koji Matsuoka*
1Division of Material Science, Graduate School of Science and Engineering, Saitama University, Sakura, Saitama 338-8570,
Koji Matsuoka, Professor of Graduate School of Science and Engineering, Division of Functional Material Sciences,
Saitama University, Shimookubo, Sakura-Ku, Saitama 338-8570, Japan, Tel. No: +81-48-858-3099, Fax: +81 48 858 3099; E-mail:
Received: 08 December, 2016; Accepted: 20 December, 2016; Published: 30 December, 2016
Kumari A, Koyama T, Hatano K, Matsuoka K (2016) Biological Evaluation of Multivalent-Type N-Acetyl-D-Glucosamine
(GlcNAc) Conjugates for Wheat Germ Agglutinin (WGA) by the Surface Plasmon Resonance (SPR) Method. SOJ Biochem 2(3), 7. DOI: http://dx.doi.org/10.15226/2376-4589/2/3/00118
Analysis of the interaction of synthetic avidin-biotin-GlcNAc
(ABG) glycocluster complex with a well-known lectin, wheat
germ agglutinin (WGA), was performed with a biosensor based
on surface plasmon resonance (SPR). In the SPR measurements,
WGA was covalently coupled to the gold surface using the
amine-coupling method. Artificial glycopolymers of N-acetyl-Dglucosamine,
polystyrene-based linear-type glycoclusters with a
polymeric backbone of acrylamide, were used as controls. Three
glycopolymers, including tetrameric ABG complex, glycopolymer 1
with a monomer and acrylamide ratio of 1:10 and glycopolymer 2
with a ratio of 1:4, were used as analytes. The SPR method was used
for the analysis of the interactions that covered a high affinity range;
namely, the strong binding of KA ~ 6.45 x 107 M-1 for ABG compared
with glycopolymers 1 and 2, which show binding of KA ~ 3.41 x 10
5 M -1 and KA ~ 3.30 x 105 M-1 respectively. SPR measurements
confirmed that WGA has higher affinity toward the tetrameric ABG
complex than toward the linear-type glycopolymers 1 and 2, and the
usefulness of these synthetic glycopolymers as tools in the study of
sugar-lectin interactions has been proved due to the very well-known
Keywords: Surface plasmon resonance; Glycocluster effect;
Polystyrene; Analyte; Amine coupling; Multivalent
GlcNAc: N-Acetyl-D-glucosamine, GPC: Gel Permeation
Chromatography, WGA: Wheat Germ Agglutinin, SPR: Surface
Plasmon Resonance, ABG: Avidin-Biotin-GlcNAc, RU: Resonance
Units, PL: Photo Luminescence, PBS: Phosphate Buffer Saline,
ELLA: Enzyme-Linked Lectin Assay
Carbohydrates present on the cell surface play a vital role in
cell recognition, and they help to protect the cell from the outside
world and provide biological information. The phenomena of cell
adhesion and cell activation prompted by carbohydrate-protein interaction are among the recent topics of active research.
Synthetic polyvalent glycoconjugates that imitate the cell
surface glycocalyx have been focused on due to their fascinating
biological properties [1-2]. In contrast to the weak and poorly
specific interactions that arise between individual carbohydrates
and proteins, the multivalent display of carbohydrates at the
surface of a molecular scaffold is currently used to boost the
binding avidity and selectivity toward a target protein [3, 4]. This
phenomenon is known as “cluster glycoside effect” [5-7].
Lectins are inherently proteins of a non-immune source
that recognize and bind to specific saccharide structural
epitopes present on the surface of a cell membrane . Many
of the proteins that participate in multivalent interactions are
oligomers such as the lectin wheat germ agglutinin (WGA), an
N-acetyl-D-glucosamine (GlcNAc)-specific plant lectin that is
extensively used in model studies with glycopolymers to monitor
their binding selectivity. WGA is a 36-kDa dimer protein with
eight specific binding sites for GlcNAc that are separated by a
distance of 14 Å [9, 10]. GlcNAc is important in several biological
systems. It has been proposed as a treatment for autoimmune
diseases, and recent tests have claimed some success . The
most important thing about GlcNAc is that it is highly specific
towards binding with the plant lectin WGA . This is the
reason we have chosen WGA as a model lectin and GlcNAc as a
sugar model to do further biological research in this project
The difficulty in studies on carbohydrate-protein interactions
is that binding affinities are very weak, usually with dissociation
constants in the millimolar range . This limitation is
repeatedly overcome by derivatization of carbohydrates to obtain
higher binding values due to the phenomenon of multivalency or
Koivula and co-workers confirmed by using SPR technology
that WGA has higher affinity toward self-assembled monolayers(GlcNAc-SAM) than toward free GlcNAc monosaccharide
. Wittman and co-workers developed a library of
cycloheptapeptides exhibiting up to six GlcNAc moieties through
a urethane spacer. Enzyme-linked lectin assay (ELLA) with WGA
has enabled the identification of tetra-, penta-, and hexavalent
glycocluster exhibiting higher binding affinity than the
monomeric GlcNAc control . On the basis of structural data
and recent progress made in the understanding of multivalent
effects, a large variety of synthetic glycoclusters based on
peptide dendrimers , carbosilanes , and nanostructures
 have been developed as ligands for studying biologically
relevant targets or providing compounds with anti-pathogenic
and anti-tumoral properties.
The availability of radical polymerization approaches, mild
reaction conditions and facile purification steps has enabled
the synthesis of glycopolymers 1 and 2 in an aqueous solution
. Moreover, a tetrameric ABG complex could be prepared by
glycosylation steps followed by coupling reaction with biotin and
finally avidin-biotin conjugation . Such synthetic multivalent
glycopolymers can bind specifically to WGA with enhanced
affinity compared to GlcNAc only.
We present here data on the interaction between the plant
lectin WGA and synthetic GlcNAc polymers [19, 20] by finding
the kinetics/affinity using surface plasmon resonance (SPR).
SPR is a highly sensitive and powerful technique that has been
used in mechanistic studies of carbohydrate-protein interactions
at interfaces in real time and in a quantitative manner [21, 22].
SPR biosensing can provide data with desirable reproducibility
and therefore offers the possibility of detailed computational
Synthesis of glycopolymers
To obtain multivalent carbohydrate-protein interactions,
we synthesized a glycopolymer of a tetrameric structure ABG
complex by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMT-MM) as a coupling reagent
followed by biotin-avidin complexation [Figure 1], and then
structurally examined by NMR, IR, MS and elemental analysis
The other two glycopolymers, 1 and 2, were synthesized
by the use of 4-(chloromethyl) styrene, and further azidation
followed by reduction reactions were able to yield amino
styrene. Condensation of amine with GlcNAc monomer was
accomplished and it was polymerized with acrylamide to
yield the corresponding water-soluble glycopolymers. These
glycopolymers were structurally examined by NMR, IR, MS and
gel permeation chromatography (GPC) .
Surface Plasmon Resonance Studies
Despite the fact that SPR has been used for the analysis of
carbohydrate-lectin binding, lectin was often immobilized on
the sensor surface instead of the glycan moiety  because
of the low sensitivity inferred by the low molecular weight of the glycan. In order to compare the bindings of different
carbohydrate derivatives containing N-acetyl-D-glucosamine to
WGA, we immobilized lectin on the sensor chip.
The surface concentration of the ligand at pH 5.5 was higher
(RU 29783.4) than that at pH 5.0, 4.5 or 4.0, and pH 5.5 therefore
appeared to be the best choice [Figure 2]. Thus, for immobilization
of WGA, pH 5.5 (a value less than pI 8.7 ± 0.3 of WGA) [24, 25] was
chosen for further modification of the sensor chip.
Immobilization of WGA on a CM5 chip
The experimental sensor chips (CM5 chips) were modified
with a covalently bound ligand (WGA) via amine coupling by
0.4 M [N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide
hydrochloride] (EDC) and 0.1 M N-hydroxysuccinimide (NHS)
derivatization. After activation, WGA was immobilized by
injection of solutions of 36 μg/ mL and 720 μg/ mL to the
respective sensor surfaces, followed by deactivation of residual
NHS esters with 1 M ethanolamine. Upon immobilization of
Figure 1:Model representation of the synthetic assembly of avidin-biotin-
GlcNAc (ABG) complex . Four units of biotin-GlcNAc conjugate
are stirred with avidin for 1 h at room temperature in the presence of
Figure 2:“pH scouting” of WGA Sensorgram showing a relative response-
time graph in real time
approximately 50 RU, the final response achieved was 68.50 RU
on one chip, and another chip was targeted with 1000 RU, the
final response achieved being 695.6 RU [Table 1].
Sensorgrams in Figure 3 show the immobilization of WGA on
the flow channel of the CM5 sensor chip via amine coupling at pH
5.5 with 10 mM sodium acetate buffer. The procedure includes
the following steps. (A) A “pre-concentration” test is carried
out to determine the appropriate ligand concentration to inject
in order to reach the targeted level of response (RU, response
units). Here, RU are 50 in the case of Figure 3a and 1000 in the
case of Figure 3b The injection period ends after acquisition and
completion of pre-concentration analysis above the baseline of
approximately 23,000 [Figure 3a] and 16,500 [Figure 3b]. (B)
“Rinse with buffer” involves washing the non-covalently bound
ligand with 50 mM NaOH to completely remove the ligand and
obtain the baseline. (C) “Activation” involves injection of the
coupling reaction mix (NHS/EDC) onto the surface, activating the
carboxymethyl group by forming a highly reactive succinimide
ester, followed by termination of the injection. (D)There is a
slight increase in RU reflecting the activation, compared to the
baseline between B and C. (E) Surface activation is followed by
injection of a ligand sample diluted in 10 mM sodium acetate pH
5.5 buffer, with continuation of injection resulting in covalent
binding of the ligand to the reactive surface to yield the targeted
RU of 50 [Figure 3a] and 1000 [Figure 3b]. These values are the
difference between the achieved constant level just before F
and the baseline at D. (F) The remaining non-reacted activated
carboxymethyl groups are blocked by injection of ethanolamine,
followed by cessation of injection. (G) Immobilization of the
ligand is achieved.
Various sensorgrams can be acquired at different
concentrations of the injected compound and they can
be simultaneously used to obtain precise kinetic (k) and
equilibrium (K) constants. Equilibrium constants can be derived
independently from ratios of rate constants or by fitting the
Table 1: Kinetic parameters obtained from the interactions of lectin
(WGA) with artificial GlcNAc polymers by use of a biosensor SPR
technique. The closeness of fit is indicated by the value of χ2
Molecular weight (Da)
Immobilized ligand (RU)
1.55 x 10-8
2.99 x 10-6
3.05 x 10-6
6.45 x 107
3.41 x 105
3.30 x 105
Ka [M-1 s-1]
1.85 x 105
0.70 x 103
0.80 x 103
2.86 x 10-3
2.05 x 10-3
2.44 x 10-3
Figure 3:WGA immobilization via amine coupling steps: (A) pre concentration,
(B) Washing with 50 mM NaOH, (C) NHS/EDC activation,
(D) RU reflecting the activation, (E) amount of ligand bound, (F) Ethanolamine
injection and (G) immobilized ligand
steady-state response versus the concentration of the binding
molecule in the flow solution over a range of concentrations.
SPR signal alteration is a wonderful method to conclude
binding stoichiometry, since the refractive index change in SPR
experiments produces basically the identical response for each
bound molecule and depends on the molecular weight of the
binding molecules. Kinetic analysis was performed by injection
of analytes of ABG complex and GlcNAc polymers 1 and 2
dissolved in HEPES buffer at different concentrations on a WGAimmobilized
chip. Between binding cycles, the WGA surface was
regenerated with a 30 s pulse of 200 mM EDTA [26, 27]. Maximum
responses of the glycopolymers to the surface-bound WGA were
analyzed and plotted against glycoside concentrations [Figure 4].
All of the three glycopolymers showed binding to WGA. Figure
4a shows that the bound relative response (RU) of ABG complex
is high even at low concentrations of (4.30 – 340 nM) because
of its strong binding to WGA due to its symmetrically arranged
tetrameric structure. However, the binding of glycopolymers 1
and 2 was small even at high concentrations (~100 μM) compared
to ABG. The binding of tetravalent ABG complex is approximately
190-times higher than that of glycopolymers 1 and 2, and this
difference is explained by the “glycocluster effect”.
Some strange inflections can be seen in Figure 4, and they
may have been due to the bulk effect [28, 29]. Such effects
basically occur if the running buffer and analyte dilution bufferare not the same. The response in Biacore is the extent of
refractive index change at the surface of the sensor chip. The
variation in refractive index between the dilution buffer and the
baseline buffer (running buffer) is called the “bulk effect” and is
induced by the existence of dissolved material including buffer
components, biomolecules and salt. Samples should be prepared
in running buffer to avoid bulk effects during the injections due
to differences in the refractive index between the running buffer
and sample. The ABG analyte stock was prepared in 10 mM PBS
pH 7.4 containing ~ 137 mM NaCl, while stocks of the other two
analytes, glycopolymers 1 and 2, were prepared in MilliQ and
then three-fold dilutions were made in running buffer (10 mM
HEPES, pH 7.4 containing 500 mM NaCl), though the dilution
buffer and running buffer we used were the same. However,
the first higher concentration we used for ABG during the assay
contain a mixture of PBS and HEPES buffer and we therefore
found some spikes at the end of injection as the buffers contain
different salt concentrations.
The affinity between immobilized lectin and glycoclusters
used in this study seems to be higher (~ 107 and 105 M-1) than the
monosaccharide’s affinity (~103 M-1), and this strong binding was
dissociated by a special regeneration solution (10 mM HEPES
with 200 mM EDTA, pH 7.4 to chelate bivalent Ca2+) [26, 27] to
obtain an accurate dissociation rate constant. Regeneration at
30 μL/ min was performed after every cycle of analyte bound
to the surface and efficacy of the surface was maintained. The
Regeneration process allows the sensor chip to be reused, thus
reducing the cost of SPR analysis. However, the lectin surface
should be intact without any damage or inactivation of the surface.
The regeneration step shortens the analysis procedure as the
chip can be used several times without a further immobilization
Figure 4: Sensorgrams show interactions of synthetic glycopolymers
with lectin WGA. Sensorgrams of the interactions show each association
and dissociation phase as a relative response of SPR against time. (a)
ABG tetramer complex (4.30-340 nm), (b) glycopolymer 1 (0.153-113
μM), (c) glycopolymer 2 (0.23-54 μM)
We calculated dissociation constants KD, which indicate
the concentration of the analyte in the equilibrium state. We
performed this calculation by fitting each measured value to a
single site interaction model, which is a common procedure used
in this type of experiment . The results were analyzed by BIAevaluation
software and are summarized in Table 1. As can be
seen, different KD and KA values were obtained for different glycol
conjugates. Interestingly, higher binding constants were recorded
with KA ~ 107 M-1 in the case of ABG complex, its tetrameric known
structure afforded the glycocluster effect in comparison with KA
~ 105 M-1 for the other two linear-type polymers used as controls.
The WGA lectin is a dimer of two identical 18-kDa subunits,
each consisting of four homologous domains of 43 amino
acids. WGA specifically recognizes GlcNAc. Eight independent
carbohydrate-binding sites are present per lectin molecule [9,
10] and it is therefore interesting to use such a lectin model.
During the pre-concentration procedure performed for WGA,
the optimum pH of sodium acetate buffer was 5.5. This bufferwas therefore used for immobilization of the lectin in the
working channel, resulting in an SPR baseline rise by ~ 700
RU (target of 1000 RU)on one sensor chip and ~ 70 RU (target
of 50 RU) on another sensor chip. In the binding experiments
with WGA, the ABG glycopolymer was used as an analyte at a
concentration of 340 nM, which was diluted three fold, and at
such low concentrations, it gave higher binding and higher SPR
response (118 RU) than those of the other two glycopolymers.
The corresponding glycopolymers, 1 and 2 showed lower SPR
responses, which were comparable to the tetrameric ABG
complex. Although the valency is four, the avidin-biotin scaffold
of the ABG complex enhances binding affinity ~ 6380 times than
GlcNAc only and ~ 190 times higher than control glycopolymers
1 and 2 and it is assumed that this exceptionally strong effect is to
be due to the phenomenon of chelate binding approach [31-33].
The distance between binding sites of WGA is 13-14 Å (distances
between anomeric oxygen’s of two bound GlcNAc residues) [34,
35] and the shortest distance between binding sites appears
to be as small as 13 Å. For this reason WGA binds strongly to
most multivalent analogs that can execute an assured degree of
chelation and augment the binding with every valency . The
other two glycopolymers (1 and 2) are products after radical
means of polymerization. These polymers are linear type with
pendant top GlcNAc residues and they were used as controls in
this study which showed binding affinity towards the WGA ~ 34
times than GlcNAc only.
From the results obtained in this study, it can be concluded that
the use of the biosensor BIAcore with SPR as a detection method
is a powerful method for investigating the interaction between
lectins and glycoproteins. We have already demonstrated that
tetravalent ABG complex could be easily prepared using avidinbiotin
complexation . Experiments with WGA showed
that GlcNAc conjugates after organic modifications had higher
protein-carbohydrate binding affinity. However, all the synthetic
polymers used in this study showed specific and strong KA than
their monomeric glycosides, but the ABG complex is more in
the favor of glycocluster effect due to its tetrameric structure
~ KA= 6.45 x 107 M-1. The binding of tetravalent ABG complex
is approximately 190-times higher than that of glycopolymers1
and 2 and ~ 6380-times higher than its monomer, which can
be explained by the glycocluster effect. Glycopolymers 1 and 2
showed ~ KA= 3.41 x 105 M-1and ~ KA= 3.30 x 105 M-1, respectively,
as measured by SPR. We compared the kinetic affinity calculated
by SPR data with previously determined photoluminescence (PL)
or fluorospectrophotometry data for the same sugar conjugates
and both methods (PL & SPR) are almost in the same agreement
for protein-carbohydrate kinetics/affinity. The affinity constant
for ABG is 1.39 x 107 M-1, while those for the other two
glycopolymers were in the range of ~105 M-1 as calculated by PL
. Little fluctuation in results of affinity constants calculated
by two different methods may occur and it should be emphasized
that the PL method measures solution-solution interactions,
which are different from the solution-solid interactions measured by SPR . The analyte-ligand interactions in our case are
uniquely determined, and the kinetic affinity (KA) for synthetic
glycopolymers is concentration-dependent.
The SPR method has also been shown to be useful for evaluating
the interaction of carbohydrates with proteins in a nano-molar
range. In summary, carbohydrate-protein interactions are key
steps for many physiological and pathological events. Hence, the
development of new carbohydrate conjugates and microarrays
is important for detecting these activities by using biophysical
methods. Such studies are now concerned to the synthesis of
more complex and relevant structures and the study of their
All SPR measurements were carried out on a BIAcore X
100 instrument (GE Healthcare) by using a CM5 sensor chip at
25 °C at a flow rate of 30 μL/ min. HEPES buffer of pH 7.4 used
for all measurements consisted of 10 mM N-(2-hydroxyethyl)
piperazine- N’-(2-ethanesulfonic acid) (HEPES), 500 mM NaCl,
and 0.02% Tween 20 (p20) detergent. To enhance the lectincarbohydrate
binding, 5.0 mM CaCl2  was also added to
the running buffer. SPR experiments were carried out with
immobilized WGA using different glycoconjugates as analytes
to determine the binding constants. The analytes used in this
study were divergent-type ABG glycocluster and linear-type
glycopolymers 1 and 2. The stock of ABG complex (1.028
μM) was prepared in phosphate buffer saline (10 mM PBS, pH
7.4), and 150 μM stock solution of glycopolymer 1 and 100 μM
stock solution of glycopolymer 2 were prepared in MilliQ. Then
subsequent 3-fold dilutions of these polymers were prepared in
10 mM HBS-P, pH 7.4 buffer in a total amount of 200 μL. An aliquot
of the solutions (200 μL) was then injected over the immobilized
chip at a flow rate of 30 μL/ min with contact time of 120 s and
dissociation time of 180 s. These parameters were the same for
all glycopolymers. The chip was regenerated by injection of 30
μL/ min of HBS-EP+ containing 200 mM of EDTA, pH 7.4. The
binding assay was performed by a multi-cycle kinetic (MCK)
approach. The binding assay also included three startup cycles
using running buffer to equilibrate the surface as well as a zero
concentration (30% running buffer plus 70% milliQ) cycle of the
analyte in order to have a blank response usable for reference
subtraction. Each sensorgram was obtained by subtracting the
reference cell: buffer only injection and glycopolymer injection
without ligand immobilization were performed.
Data analysis was conducted with the software BIAcore X 100
evaluation (version: 2.0.1).
The authors would like to thank Dr. Hidenao Arai of Graduate
School of Science & Engineering of Saitama University for his
valuable advice regarding SPR and BIAcore experiments. The
authors are grateful to Rotary Yoneyama Memorial Foundation Japan for continuous support and encouragement. This work
was partly supported by a grant-in-aid from Saitama Prefecture
(K.M.) (Saitama Leading Edge Project).
A.K., T.K., K.H. and K.M. conceived and designed the
experiments; A.K. performed the experiments and analyzed the
data; A.K., T.K., K.H. and K.M. contributed to preparation of the
paper; and A.K. mainly wrote the paper. This study is a PhD work
of A.K. under the supervision of K.M.
Conflicts of Interest
The authors declare no conflict of interest.
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