Modification of bovine serum albumin with aminophenylboronic acid as glycan sensor based on surface plasmon resonance and isothermal titration calorimetry

Article abstract of DOI:10.1515/hc-2017-0049

Aminophenylboronic acid (ABA) modified bovine serum albumin (BSA) was prepared as neolectin and its interactions with oligosaccharides and glycopolymer were studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). The conjuga

Full text of DOI:10.1515/hc-2017-0049

Heterocycl. Commun. 2017; aop
a
a
De-Min Wang , Xin Meng , Xiao-Bin Li, Hao-Jie He, Teng-Fei Zhao, Tian-Wei Jia, Yun He,
Yang Yang* and Peng Yu*
Modification of bovine serum albumin
with aminophenylboronic acid as glycan
sensor based on surface plasmon resonance
and isothermal titration calorimetry
DOI 10.1515/hc-2017-0049
Keywords: aminophenylboronic acid; bovine serum
albumin; glycopolymer; isothermal titration calorimetry;
surface plasmon resonance.
Received March 15, 2017; accepted May 30, 2017
Abstract: Aminophenylboronic acid (ABA) modified
bovine serum albumin (BSA) was prepared as neolectin
and its interactions with oligosaccharides and glycopoly-
mer were studied by surface plasmon resonance (SPR) Introduction
and isothermal titration calorimetry (ITC). The conjuga-
tion between the primary amine group of the ABA mole- Glycans, in the form of oligosaccharides, polysaccha-
cule and lysine residues on BSA was performed with an rides and other glycoconjugates are critical constituents
adipate-based strategy to afford the synthetic neoprotein. of all living organisms [1–4]. It has been found that the
The number of ABA molecules loaded to BSA surface was mammalian cell-surface is decorated with a dense and
determined by matrix-assisted laser desorption/ioniza- complex array of glycans [5]. Due to their abundance, vast
tion – time of flight (MALDI-TOF) mass spectrometry. In structural heterogeneity and the localization at the outer
the BSA-ABA and sugar interaction study, no signal was membrane of cell surface, the carbohydrates as specific
observed for both the SPR and ITC sensor platform using recognition sites for various binders, especially proteins,
monosaccharides as the analyte, indicating a weak bind- play vital roles in many biological and physiological pro-
ing affnity, while the galactose modified polymer showed cesses [6] such as intracellular trafficking, cell signaling,
an enhanced response. The binding affinities of the galac- proliferation and differentiation, cancer progression, viral
tosyl-polymer to BSA-ABA from SPR and ITC data were in and bacterial infection, and immune response [7–11]. Pro-
the micromolar range.
found understanding of carbohydrate-protein interactions
is crucial for the discovery of medicinal, imaging, diag-
nostic and therapeutic agents [12]. Lectins are a family of
carbohydrate-binding proteins that are found in all plants
and animals and are one of the major tools widely used
in the research for carbohydrate-protein interactions [13,
a
De-Min Wang and Xin Meng: These authors contributed equally to
this work.
*
Corresponding authors: Yang Yang and Peng Yu, Key Laboratory
of Industrial Fermentation Microbiology of Ministry of Education,
Tianjin Key Laboratory of Industry Microbiology, China International
Science and Technology Cooperation Base of Food Nutrition/Safety
and Medicinal Chemistry, Sino-French Joint Lab of Food Nutrition/
Safety and Medicinal Chemistry, College of Biotechnology, Tianjin
University of Science and Technology, Tianjin 300457, China, e-mail:
yyang@tust.edu.cn (Y. Yang), yupeng@tust.edu.cn (P. Yu)
De-Min Wang, Xin Meng, Xiao-Bin Li, Hao-Jie He, Teng-Fei Zhao
and Tian-Wei Jia: Key Laboratory of Industrial Fermentation
Microbiology of Ministry of Education, Tianjin Key Laboratory of
Industry Microbiology, China International Science and Technology
Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry,
Sino-French Joint Lab of Food Nutrition/Safety and Medicinal
Chemistry, College of Biotechnology, Tianjin University of Science
and Technology, Tianjin 300457, China
1
4]. However, due to the insufficient resources, poor sta-
bility, complicated purification and high cost of these
commercially available proteins, there is an urgent need
to develop alternatives for the carbohydrate ‘‘binders’’ for
both pharmaceutical and biomaterial applications [15].
Recently, synthetic small molecules have emerged
as carbohydrate-binding lectin mimics [16] for the study
of carbohydrate-protein recognition and interaction.
Boronic acid (BA) can bind with diols of oligosaccharides
under physiological conditions to form boronic acid cyclic
diesters in aqueous solution [17]. Thus, polymeric BA tai-
lored macromolecules including silica beads [18], nano-
particles [19–22], magnetic beads [23] and polymers [24,
25], which mimic the multivalent display of lectins on cell
Yun He: Angstrom Biotechnologies Company, 3350 Scott Blvd.,
Bldg. 9, Santa Clara, CA 95054, USA
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ꢀꢀꢀꢀꢁꢀD.-M. Wang et al.: Modification of BSA with aminophenylboronic acid as glycan sensor
surface, can be used as artificial sugar receptors to inves- Finally, the number of the small organic molecule on the
tigate carbohydrate-protein recognition. However, the BSA surface can be easily determined by matrix-assisted
lack of control over BA presentation on macromolecular laser desorption/ionization – time of flight (MALDI-TOF)
backbones makes it difficult to characterize the surface mass spectrometry [30]. We also used the BSA-ABA conju-
density and spatial distribution of BA after the scaffold gates together with surface plasmon resonance (SPR) and
modification which limits their further application espe- isothermal titration calorimetry (ITC) technology to study
cially in quantitative analysis of carbohydrate-neolectin the dynamic and thermodynamic details of the carbohy-
interaction.
Herein, we directly attached aminophenylboronic acid
ABA)onthesurfaceofbovineserumalbumin(BSA)accord-
drate-(BSA-ABA) interaction at molecular levels.
(
ing to the literature [26] with a different coupling strategy Results and discussion
to prepare neolectin (BSA-ABA) for carbohydrate sensor
applications, because of the biocompatibility and natural BSA-ABA conjugates were prepared via covalent coupling
3
D mimetic property of BSA. The multivalent display of between the primary amine of 3-aminophenylboronic acid
ABA can mimic multivalent interactions at carbohydrate- and lysine residues on BSA by using an adipate NHS ester
protein interfaces at which avidity is crucial [27–29]. method [31, 32] (Scheme 1). The adipate linker can make the
O
O
N
O
O
HO
OH
O
N
O
B
O
HO
OH
HO
OH
B
B
O
O
N
O
O
H
2
O
N
BSA
SH
N
O
H
N
O
n
a
b
NH2
H
O
3
BSA-ABA4, n = 4
BSA-ABA9, n = 9
1
BSA-ABA14
,
n = 14
NH2
O
O
O
c
O
O
O
n
NH OH
m
O
O
O
O
O
n
OH OH
O
NH2
Alkynyl-polymer
O
HO
Poly (methyl vinyl ether-alt-maleic anhydride)
OH
O
MW~130 000
4
O
c
OH OH
O
O
O
O
x
O
y
NH OH
O
O
HO
OH
Not detectable
Detectable
HO OH
HO
OH
HO OH
HO
OH
O
O OH
B
O
Gal-polymer x = 172
B
MW~178 255
O
HN
O
NH
OH
O
HO
HO
OH
O
O
HO
HO
OH
OH
OH
OH
HO
OH
Glucose, Glc
Mannose, Man
O
O
OH OH
O
OH OH
NH
OH
HN
OH
O
O
HO
O
OH
OH
HO
OH
OH
OH
HO
OH
OH
Galactose, Gal
Lactose, Lac
OH
HO
OH
S
S
Oligosaccharide
SPR sensor chip
Scheme 1ꢀPreparation of BSA-ABA conjugates and their immobilization onto a SPR chip for biosensor applications. Reagents and condi-
tions: (a) DMSO, Et N, 70%; (b) PBS, pH 7.4; (c) DMF, room temperature.
3
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D.-M. Wang et al.: Modification of BSA with aminophenylboronic acid as glycan sensorꢂꢁꢀꢀꢀꢂ3
ABA molecule more flexible on the surface of the protein relative low density of the ABA on the BSA surface in our
compared with no linker used [26]. It has been reported study, the binding affinity was too weak to be measured
that cell-surface carbohydrate receptor contains multiple compared to the high density of BA on BA-MIPs. All the
carbohydrate-binding sites and their density regulates the results showed that more sensitive technology should be
biological events [33]. To mimic the phenomenon of the applied to study such interactions.
natural glycan-receptor, various ABA modified BSA con-
Next, another direct-binding measurement on
jugates were prepared by changing the ratio of ABA 3 to a SPR instrument was established to investigate the
BSA. After dialysis, purification on Sephadex G-75 and lyo- dynamic interactions between BSA-ABA and oligosaccha-
philization, the BSA-ABA conjugates were characterized rides (Scheme 1). Together with ITC, SPR is also widely
by MALDI-TOF mass spectrometry (see Supplementary accepted as a sensitive and label-free technique for the
Material). Mass analysis of BSA-ABA conjugates showed study of molecular interactions in biological systems [37].
that an average number of 4, 9 or 14 ABA was attached per First, the BSA-ABA conjugate was immobilized on the
one molecule of BSA (Table 1).
commercial gold chip via gold-thiol covalent coupling in
With the synthetic neolectin in hand, isothermal a PBS buffer (pH 7.0) [38] at a flow rate of 5 μL/min for
titration calorimetry (ITC) [34] was first chosen for exam- channel 2 with the flow of BSA in channel 1 serving as ref-
ining the binding properties of the BSA-ABA conjugates erence. After several minutes for the saturation, different
to oligosaccharides, because this technique does not sugars in PBS solution (1 mꢀ) at a flow rate of 30 μL/min
require labeling and immobilization step. Moreover, for 5 min (180 s association phase) were injected over the
ITC can determine the enthalpy and the entropy of BSA-ABA coated sensor chip. We observed similar results
binding, the association constant (K) and the binding in the ITC experiments as all sugars exhibited no response
stoichiometry (N) from a single measurement [35]. signal (data not shown), verifying the low binding affin-
In the ITC experiment, different sugar (glucose, Glc; ity of BSA-ABA and its monosaccharide ligands. Similarly,
mannose, Man; galactose, Gal; lactose, Lac) solutions with increasing the concentration of the oligosaccharides
in a PBS buffer was loaded into the syringe and titrated to those of the three BSA-ABA conjugates with different
into the BSA-ABA solution in the calorimeter cell. The ABA densities immobilized on the sensor chip did not
heat released at each injection of the PBS buffer to the result in detectable signals and the dissociation con-
BSA-ABA was used as a control to correct the titration stants could not be determined (data not shown). Similar
measurement. To our dismay, trials of thermodynamic BSA-ABA conjugates were prepared previously by amida-
evaluation for the interactions between oligosaccharides tion of carboxylic acid groups in BSA with aminophenyl
and BSA-ABA were unsuccessful even when BSA-ABA14 boronic acid and their sugar binding capacity was also
the highest ABA density coated on BSA was used, due to investigated by SPR technique. The affinity constants KD
the low values of heat in these binding interactions (data were determined to be around a nanomolar range indi-
not shown). Further increase in the concentration of cating a strong binding between BSA-ABA conjugates and
sugar or BSA-ABA did not significantly enhance the ITC monosaccharides [26]. It can therefore be concluded that
signal and the raw data could not be fitted correctly, indi- higher density of ABA on BSA (maximum 26 ABA per 1
cating that the interaction between BSA-ABA and mono- BSA in their system vs 14 ABA per 1 BSA in our study)
saccharide was very weak. It has been reported that the results in stronger interactions.
K value between the boronic acid-functionalized molec-
Although SPR has received wide acknowledgement
D
ularly imprinted polymers (BA-MIPs) and corresponding for applications for investigation of carbohydrate-protein
monosaccharide is typically around a mꢀ-range meas- interaction, the major disadvantage is the difficulty of
ured by ITC experiments [36]. We assumed that due to the measuring low molecular mass analytes because of the
weak signals, especially when monolayer of proteins is
immobilized directly on the flat gold chip sensor surface
Table 1ꢀThe conjugations of ABA with BSA.
[39]. Signal enhancement can be achieved successfully
using larger molecules, such as nanoparticles [40, 41]
and latex particles [42] as backbones for the analytes.
These macromolecular scaffolds enhance the signal not
only through their high molecular weight but also by
the cluster effect [29] for multivalent interactions. It was
reported that when polymeric ABA was immobilized
Entry
Molar ratio
Molecular weight
ABA per BSA
BSA
ꢃ
ꢅꢃ/ꢅ
ꢉꢃ/ꢅ
ꢊꢃ/ꢅ
66 ꢄꢅ8a
6ꢇ 6ꢃꢃ
6ꢈ ꢃꢃꢈ
ꢇꢃ ꢉ8ꢄ
ꢃ
ꢆ
ꢈ
BSA-ABAꢆ
BSA-ABAꢈ
BSA-ABAꢅꢆ
ꢅꢆ
aMolecular weight was determined by MALDI-TOF mass spectrometry. on the gold film of the SPR sensor chip, a selective and
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ꢀꢀꢀꢀꢁꢀD.-M. Wang et al.: Modification of BSA with aminophenylboronic acid as glycan sensor
A
B
Time (min)
20
1000
0
10
30
40
7.5 µM
0
.10
8
6
4
2
00
00
00
00
0
10 µM
0.00
1
2
3
4
5 µM
–0.10
0 µM
0 µM
0 µM
–0.20
–0.30
–0.40
–
50
50
150
250
350
–
200
400
–
0.50
–
Time (s)
–0.60
150.0
–
2
1
1
00
50
00
–200.0
–
–
–
–
250.0
300.0
350.0
400.0
5
0
0
–450.0
0
0.00001 0.00002 0.00003 0.00004 0.00005
Concentration of Gal-polymer (M)
0.00
0.01
0.02
0.03
0.04
Molar ratio
Figure 1ꢀInteraction studies between Gal-polymer and BSA-ABA . (A) SPR sensorgrams and state steady analysis of the binding between
1
4
Gal-polymer and BSA-ABA . (B) ITC titration curves obtained at 29ꢋ K for the titration of BSA-ABA with Gal-polymer. The top panel shows
1
4
14
the raw calorimetric data. Binding parameters were auto-generated after curve fitting using Microcal Origin.
enhanced response to saccharides was observed com- BSA-ABA was observed as shown in Figure 1, while a neg-
1
4
pared with the self-assembled monolayer presentation ligible binding was observed when alkynyl-polymer was
of ABA [43]. These results agree with the ITC strategy injected over BSA-ABA . The dissociation constant k and
14
d
described above [36]. Inspired by this principle, carbo- association constant k were determined by the BI-3000
a
hydrate tailored polymer was chosen in our platform as evaluation software (Table 2). Although the thermody-
the analyte to enhance the binding affinity to BSA-ABA namics constants (entropy and enthalpy) from ITC experi-
coated on the sensor surface.
ments seemed unreliable, similar binding constants K in
D
The synthesis of the galactosyl monomer 4 with an this and the SPR measurement were found. These results
amine group started from peracetylated galactopyrano- can be attributed to the polyvalent interactions between
side, which was obtained using a reported procedure [44] the ABA residues on BSA and the diols of the galactosyl
(
Scheme 1). Glycopolymer (Gal-polymer) was prepared moiety on the Gal-polymer, which monosaccharide does
from [poly(methyl vinyl ether-alt-maleic anhydride), MW ~ not have.
30 000] by treatment with 4. The molecular weight of Gal-
polymer determined by size-exclusion chromatography
SEC) using pullulan as the standard was 178 255 which
1
Table 2ꢀBinding kinetics of alkynyl-polymer and Gal-polymer to
(
BSA-ABA in SPR and ITC assay.
1
4
equals to 172 molecules of galactose on the polymer
surface. Alkynyl-polymer prepared from polyanhydride
by reaction with propargylamine was used as control [45].
The BSA-ABA was used as the lectin mimic due to its high
SPR
ITC
−
ꢂ
χꢃ
K′ (ꢁ)c
K (ꢁ )
K (ꢁ)
A
D
D
14
density of the ABA on the protein surface to investigate the Alkynyl-polymer NBa
NB
ꢆ.ꢈꢅꢁ×ꢁꢅꢃ
NB
ꢉꢆ.ꢅ
NB
ꢆ.6ꢅꢁ×ꢁꢅꢃ
ꢆ
−ꢄ
b
−6
carbohydrate involved interaction (Scheme 1).
Gal-polymer
ꢉ.ꢃꢆꢁ×ꢁꢅꢃ
All bindings were studied again by SPR and ITC
followed by the procedure described above. In the
SPR experiment, Gal-polymer binding to immobilized
a
NB: no binding.
b
c
χ ꢁ=ꢁ0.00015RU 2ꢁ=ꢁ0.1 RU 2.
2
max
max
K′ was calculated as follows: ΔGꢁ=ꢁ–RTlnKꢁ=ꢁΔHꢁ−ꢁTΔS; K′ ꢁ=ꢁ1/K.
D
D
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D.-M. Wang et al.: Modification of BSA with aminophenylboronic acid as glycan sensorꢂꢁꢀꢀꢀꢂ5
containing BSA in sodium phosphate buffer (10 mL, 0.1 ꢀ, pH 7.4).
The mixture was purified on Sephadex G-75 and then freeze-dried.
The resulting BSA-ABA conjugate was characterized by MALDI-TOF
Conclusions
An efficient synthetic strategy was developed for the mass spectrometry and stored at 4°C until use.
synthesis of ABA modified BSA as neolectin from low-
cost, readily available materials. This convenient method
enabled the preparation of a multivalent glycan binder Polymer preparation
as a promising probe for biological studies. The binding
study using SPR and ITC methods indicated that this Amine (100 mg) was dissolved in DMF (10 mL), then poly(methyl
vinyl ether-alt-maleic anhydride) (50 mg) was added and the solution
lectin mimics can be used for the detection of glycopoly-
was slowly stirred at room temperature for 24 h. Then the mixture
mer. Further studies on the polysaccharides binding and
was dialyzed against deionized water and freeze-dried to yield the
detection applications of different densities of BSA-ABA
Gal-polymer or alkynyl-polymer.
are in progress.
Characterization of glycoconjugates with MALDI-TOF
Experimental
The characterization of the BSA-ABA was carried out by matrix
assisted laser desorption ionization–time of flight (MALDI-TOF) mass
The boronic acid-protein conjugates were purified using Sepha-
spectrometry in positive linear ion mode using 2,5-dihydroxybenzoic
dex™ G-75 (GE Healthcare). NMR spectra were recorded on a
acid (DHB) as matrix. BSA-ABA solutions were diluted with the matrix
Bruker AVANCE III (400 MHz) instruments. The molecular weights
(
5 mg/mL matrix in 50% acetonitrile containing 0.1% trifluoroacetic
of the aminophenylboronic acid-protein conjugates were con-
firmed by using a Bruker UltrafleXtreme MALDI-TOF/TOF mass
spectrometer. Molecular weights of the polymers were determined
by size-exclusion chromatography (SEC) using an Aglient 1200 liq-
uid chromatograph with refractive index detector. All surface plas-
acid) at a ratio of 1:5 by volume and deposited onto a MALDI target
plate and dried in air. FlexAnalysis was used for analysis of the data.
mon resonance experiments were carried out on a BIAcore 3000 Surface plasmon resonance analysis
instrument using commercial SPR gold sensor chips (AU 1113 P3,
Xantec Bioanalytics GmbH, Germany) at 25°C. Microcalorimetric
The interactions between BSA-ABA and Gal-polymer were ana-
experiments were performed with an isothermal titration calorim-
eter (ITC200). PBS (0.01 ꢀ, pHꢁ=ꢁ7.4, without calcium and magne-
sium) was the running buffer. Solutions of the analytes in buffer
were filtered through a 0.22 μꢀ micro-membrane and degassed
before being injected.
lyzed at 25°C on a BIAcore 3000 instrument. The BSA and BSA-
ABA (1 mꢀ) as immobilized ligands were injected at a flow rate
of 5 μL/min on channel 1 and 2, respectively, until saturation was
obtained. Gal-polymer analytes were injected over the immobilized
BSA-ABA and BSA at flow rate of 30 μL/min for 5 min. The asso-
ciation and dissociation constants of the glycopolymer binding to
immobilized BSA-ABA were determined by standard BI 3000 evalu-
ation software.
Di-N-succinimidyl adipate (2)
N-hydroxysuccinimide (5 g, 43 mmol) and N-ethyl-N-isopropylpro-
pan-2-amine (7.2 mL, 60.5 mmol) were dissolved in THF (100 mL) at Isothermal titration calorimetry analysis
0
°C and then the solution was slowly treated with adipoyl chloride
(
3 mL, 21.5 mmol). The mixture was stirred for 30 min and the result-
ITC experiments were performed using an ITC200 microcalorimeter
in PBS buffer. The concentration of Gal-polymer was 10 μꢀ, and that
of BSA-ABA14 was 50 μꢀ. In each individual experiment, ~38 μL of
Gal-polymer was injected through the computer-controlled 40 μL
microsyringe at an interval of 2 min into the BSA-ABA solution in the
same buffer (cell volumeꢁ=ꢁ200 μL) while stirring at 750 rpm. A stand-
ard one-site model was fitted to a theoretical titration curve using the
software supplied by MicroCal.
ant precipitate was washed with hot isopropanol and dried under
1
reduced pressure to furnish the title product 2 (5.9 g, 81%): H NMR
(
400 MHz, DMSO-d ): δ 2.79 (s, 8H), 2.72 (m, 4H), 1.70 (m, 4H).
6
General procedure for BSA-ABA conjugates preparation
Di-N-succinimidyl adipate (2, 248 mg, 730 μmol) was dissolved in
1
.9 mL DMSO and trimethylamine (100 μL) was added. Aminophe- Acknowledgements: This work was supported by the Nat-
nylboronic acid (1, 10 mg, 73 μmol) dissolved in 600 μL DMSO was
pipetted slowly under stirring over 30 min to the linker solution.
After an additional 2 h of stirring, sodium phosphate buffer (0.1 ꢀ,
pH 7.4) was added. The unreacted linker and DMSO were extracted
twice with chloroform. The aqueous phase containing 3 was centri-
ural Science Foundation of China (21402140, 21502139),
the International Science and Technology Cooperation
Program of China (2013DFA31160), Key Laboratory of
Industrial Fermentation Microbiology of Ministry of Edu-
fuged for 1 min to remove residual CHCl and then added to a solution cation and Tianjin Key Lab of Industrial Microbiology
3
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