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Measurement of Glycan-Based Interactions by Frontal Affinity Chromatography and Surface Plasmon Resonance
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Measurement of Glycan-Based Interactions by Frontal Affinity Chromatography and Surface Plasmon Resonance Chihiro Sato, Nao Yamakawa, and Ken Kitajima Contents 1. Introduction 2. Frontal Affinity Chromatography (FAC) as a Tool for the Measurement of Glycan-Based Interactions 2.1. FAC principle 2.2. Materials and equipment 2.3. Preparation of affinity adsorbents 2.4. Preparation of neurotransmitters 2.5. Operation of frontal affinity chromatography 2.6. Interaction between polySia and neurotransmitter 3. Surface Plasmon Resonance (SPR)-Based Biosensors as a Tool for the Measurement of Glycan-Based Interactions 3.1. Materials and equipment 3.2. Preparation of biotinylated glycans 3.3. Immobilization of biotinylated glycans on an Au sensor chip 3.4. Immobilization of BDNF on a CM5 sensor chip 3.5. Biacore analysis 4. Conclusions Acknowledgments References
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Abstract Proteins and lipids are often modified with glycan chains, which due to their large hydration effect and structural heterogeneity, significantly alter the surface physicochemical properties of proteins and biomembranes. This ‘‘glycoatmosphere’’ also serves as a field for interactions with various molecules, Bioscience and Biotechnology Center, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Methods in Enzymology, Volume 478 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)78010-1
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2010 Elsevier Inc. All rights reserved.
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including other glycans, lipids, peptides, proteins, and small molecules such as neurotransmitters and drugs as well as lectins. Therefore, sensitive techniques for measuring these glycan-based interactions are becoming more and more necessary, with the appropriate method largely depending on the interacting molecules. In this chapter, we focus on frontal affinity chromatography (FAC) and surface plasmon resonance (SPR) for examining polysialic acid-involved interactions with neurotransmitters and neurotrophins. FAC is characterized by its applicability to analyze weak interactions that are difficult to measure using conventional methods, and by the ease of principle and experimental procedures. SPR is advantageous due to the availability of suitable surface materials and for real-time monitoring with nonlabeled analytes.
1. Introduction Glycosylation is one of the major modifications of proteins and lipids, and a wide variety of glycosylations have been reported. However, the phenomenon of glycosylation remains somewhat mysterious due to a lack of appropriate methodologies for determining glycan structures as well as their biological functions. Recent methods of genetic perturbation for glycan-related enzymes have greatly impacted the understanding of the biological significance of glycans, even if the results are severe or benign. The underlying mechanisms which link gene expression to the resultant phenotypes, however, remain unknown. In this respect, it is important to understand glycan-based interactions with cellular components containing not only proteins, but also glycans, lipids, and other natural substances using appropriate analytical methods. In this chapter, we focus on the measurement of new polysialic acid (polySia)-based interactions using frontal affinity chromatography (FAC) and a surface plasmon resonance (SPR)-based Biacore instrument. PolySia is a polymerized structure of sialic acid present on neural cell adhesion molecules (NCAMs) as a posttranslational modification (Sato, 2004, 2010; Troy, 1996). PolySia-modified NCAM has been well studied in the development of the nervous system since the modification is spatiotemporally regulated (Bonfanti, 2006; Rutishauser, 2008). PolySia is expressed in embryonic brains during neural differentiation and mostly disappears in adult brains, although NCAM expression levels remain unchanged. Based on its bulky polyanionic nature, polySia is thought to function as an antiadhesive molecule against cell–cell and extracellular matrix interactions (Angata et al., 2006; Bonfanti, 2006; Rutishauser, 2008). However, we recently demonstrated by native PAGE analysis that an important neurotrophin in the brain, brain-derived neurotrophic factor (BDNF), interacts with polySia directly to form a large complex (Kanato et al., 2008). Therefore, polySia likely serves an important role as a
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neurogenetic regulator similar to glycosaminoglycans that have been shown to bind growth factors and signaling molecules, such as Wnt (Schwartz and Domowicz, 2004). We thus proposed the hypothesis that, as a novel functional role, polySia may serve as a reservoir of neuroactive molecules, such as neurotrophins, growth factors, and neurotransmitters (Kanato et al., 2008, 2009). To gain insight into this potential novel function of polySia, we applied FAC and SPR for measuring polySia–neurotransmitter and polySia–neurotrophin interactions, respectively.
2. Frontal Affinity Chromatography (FAC) as a Tool for the Measurement of Glycan-Based Interactions Affinity chromatography is a commonly used and powerful tool for the purification of molecules that specifically interact with a target counterpart molecule. In the 1970s, Kasai and Ishii were the first to demonstrate that affinity chromatography is applicable to quantitative analysis for the estimation of dissociation constants for protein–ligand interactions (Kasai and Ishii, 1973). Kasai and his colleagues also applied FAC for the measurement of glycan–lectin interactions (Arata et al., 1997; Oda et al., 1981), which represented the first estimation of Kd. Hindsgaul et al. improved this method through the use of microcolumns and MS as a detector (Ng et al., 2005), allowing materials to be conserved and shortening the time required for analysis. This LC–MS system is particularly applicable for high-throughput screening. At the same time, Hirabayashi also developed an enhanced, high-throughput lectin–glycan interaction-analysis system between immobilized lectins and soluble fluorescent (PA)-labeled glycans (Kuno et al., 2005). Owing to improvements of these FAC-systems, FAC is a widely accepted technique for the analysis of lectin–glycan interactions. The FAC system has been well established and a number of wellwritten reviews are available (Hirabayashi et al., 2003; Kasai et al., 1986; Tateno et al., 2007). In this book, Kamiya and Kato also describe a lectin– glycan interaction revealed using a FAC system; in this chapter, we therefore focus on the general principle and methods for analyzing glycan-polymer and small molecule interactions.
2.1. FAC principle In frontal analysis, the elution front of an analyte and its concentration at a plateau level are measured when the analyte solution at various concentrations is eluted. For the analysis, the volume of the analyte solution should exceed the column volume. Under the assay conditions, as the free analyte
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concentration is always fixed and equal to the initial concentration of analyte, dynamic equilibrium is achieved in the plateau region. Prior to the analysis, the column is packed with a resin on which the ligand of interest (termed B) is immobilized. The column is then isocratically eluted with an excess column volume of analyte (termed A) and the resulting elution curve of A is monitored. Based on the starting point of analyte elution (the elution front), the interaction between B and A can be detected. The retardation of elution (see line 2 in Fig. 10.1) indicates that analyte A interacts with B (Fig. 10.1). In contrast, an analyte which does not interact with B is eluted in the void volume of the column (see line 1 in Fig. 10.1). The generation of a retard volume (VV0) means a volume of an A–B complex formed as a result of specific interaction. Therefore, the area, [A]0(V V0), represents the amount of the A–B complex. Provided the column volume is u, the equation [A]0(VV0) ¼ u [AB] is realized. The dissociation constant (Kd) can be determined from Eq. (10.1), which is based on the equation: A þ B , AB. Kd ¼ ½A½B=½AB ¼ ½A0 ½B0 ½AB =½AB ¼ u½B0 =ðV V0 Þ ½A0 ð10:1Þ
Concentration of analyte
As [B]0 is the concentration of the immobilized ligand, and the effective ligand content can be obtained with the equation Bt ¼ u [B]0. Therefore, Eq. (10.1) can be followed by Eq. (10.2).
1 (no interaction with B) 2 (interaction with B)
[A]0
[A]0(V−V0)
0
V0
V
Elution volume
Figure 10.1 Schematic elution profiles of FAC. The immobilized ligand (B) is packed into a column and a volume in excess of the total column volume of analyte (A) is then eluted. Line 1 is the curve for the analyte that does not interact with B. The elution volume represents the void volume (V0) of the column. Line 2 is the curve for the analyte that interacts with B. The retard (VV0) indicates a specific interaction between A and B. The shaded area, [A]0(VV0), is the amount of the complex [AB].
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Kd ¼ Bt=ðV V0 Þ ½A0
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ð10:2Þ
The value for Bt is obtained from analyte concentration-dependent experiments using either a Lineweaver–Burk type plot, that is, 1/[A]0 versus 1/(V V0)[A]0, or a Woolf–Hofstee type plot, that is, (V V0) versus (V V0) [A]0. Equation (10.2) can be simplified to Eq. (10.3) where [A]0 (10 8 M) is negligible for the Kd value (e.g., >10 6 M). Kd ¼ Bt=ðV V0 Þ;
if ½A0 Kd
ð10:3Þ
The elution volume of an analyte (V ) is determined graphically as the volume corresponding to the elution point, which occurs at the half value of the plateau of the elution curve. V0 is determined as the V of the appropriate control sample without affinity for the immobilized ligand. The values of Bt and Kd are determined from the intercept of the axis and the slope of the fitted curves (Woolf–Hofstee-type plots, (V V0) vs. (V V0)[A]0) (Fig. 10.2), respectively, and Eq. (10.3) can be changed to Eq. (10.4). ½A0 ðV V0 Þ ¼ KdðV V0 Þ þ Bt
ð10:4Þ
Bt [A]0(V−V0)
Amount of complex
It should be noted that strong interactions cannot be measured with this system, as this method is based only on the retardation of elution and not the
Slope = −Kd
0
(V–V0) Retardation of analyte
Figure 10.2 The Woolf–Hofstee plot for the determination of Bt and Kd. Based on Eq. (10.4), [A]0 (V V0) ¼ Kd (V V0) þ Bt, the obtained data can be plotted. The x- and y-axes represent the retarded elution of analyte A (V V0) and the amount of A–B complex ([A]0 (V V0)), respectively. Therefore, from the intercept of y-axis, the Bt (effective ligand content) is calculated. The Kd value is obtained from the slope (¼ Kd).
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complete loss of the analyte from the eluate. Therefore, this method is suited for the measurement of relatively weak interactions that are occasionally encountered, as in the case of glycan-based interactions.
2.2. Materials and equipment Polysialic acid (colominic acid) (Wako) Affigel 102 or Affigel Hz (Bio-rad) 1-ethyl-3-(3-diethylaminopropyl)-carbodiimide (Bio-rad) Phosphate-buffered saline (PBS) (pH 7.2; 10 mM; 137 mM NaCl; 2.7 mM KCl) 5. Neurotransmitters (epinephrine and dopamine) 6. Column (4.0 mm 10 mm, 126 ml, GL Science) 7. HPLC system (Rheodyne injector equipped with a 2 ml-PEEK sample loop and a UV-detector connected to an integrator) 1. 2. 3. 4.
2.3. Preparation of affinity adsorbents For the FAC analysis, two types of polySia (colominic acid)-immobilized resins are used which differed on whether polySia is immobilized through the reducing (Fig. 10.3A) or nonreducing (Fig. 10.3B) terminal end. To prepare the resin with reducing end-immobilized polySia, 5 ml (packed volume) of Affigel 102 in 25 mM Na2HPO4 (pH 6.0) are added to 2 ml of 50 mg/ml colominic acid, and the pH of the resulting solution is adjusted to 5.0 with 1 N HCl. After the addition of 8 mg 1-ethyl-3-(3-diethylaminopropyl)-carbodiimide, the reaction mixture is kept at 4 C for 4 h with gentle mixing by rotation. After washing with PBS, the gel is blocked with acetic anhydrite at room temperature for 30 min and washed with PBS and 1 M NaCl. Based on the amount of unbound polySia measured by the resorcinol method, the extent of immobilization of polySia is estimated to be 0.8 mmol/ml. To prepare the resin to which polySia is immobilized through the nonreducing terminal end (Fig. 10.3B), 5 ml (packed volume) of Affigel Hz in 50 mM sodium acetate buffer (pH 5.5) are added to 2 ml of 48 mg/ml periodate-oxidized colominic acid (prepared by incubation with 25 mM sodium periodate in 100 mM sodium acetate (pH 5.5) followed by desalting), and incubated at 25 C overnight with gentle mixing. After washing the gel with PBS, unbound colominic acid is measured by the resorcinol method to estimate the amount of immobilized colominic acid. The immobilization extent of colominic acid is estimated to be 0.7 mmol/ ml. The two types of resins are each packed into an empty column (4.0 mm 10 mm, 126 ml, GL Science) using a syringe. The prepared columns can be stored at 4 C until use.
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A
H3C
O
H
C
N
O OH
COO− H3C
OH
O
OH
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O H3C
C
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H
C
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H N
O CH
O
H
OH
C
N N
OH
OH
O
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OH
n
N
NH NH
O
COO−
H3C
O
H
C
N
O
O
OH
OH
COO−
OH
O
OH
n
NH
Figure 10.3 PolySia-immobilized beads used for FAC analysis. (A) polySia-affigel 102. (B) Affigel Hz-polySia.
2.4. Preparation of neurotransmitters A variety of neurotransmitters are commercially available. In this study, we selected epinephrine and dopamine for FAC analyses with the immobilized polySia ligand. For the analysis, each neurotransmitter is dissolved in PBS or an appropriate buffer at concentrations ranging from 0 to 30 nM.
2.5. Operation of frontal affinity chromatography A FAC system consists of a pump ( JASCO PU-980i), an injector with a sample loop, a column, and a UV detector ( JASCO 875-UV) connected to a chromato-PRO integrator (Run Time Corporation, Kanagawa, Japan). The 2 ml-sample loop (PEEK) and the column are either kept in an oven (CTO-6A, Shimadzu) or water bath at 25 or 37 C. Prior to analysis, the column is equilibrated with PBS at a flow rate of 0.125 ml/min until a flat baseline of absorbance was achieved. The sample injector is turned to the load position, and 10–20 ml of air is injected using a syringe to empty the sample loop completely. The analyte solution dissolved in PBS is then injected to fill the 2 ml-sample loop. Note that 50–100 ml of excess analyte
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solution is required to completely fill the 2 ml-sample loop. The injector is then turned to the inject position to flow the analyte into the column at a rate of 0.125 ml/min. The analyte eluted from the column is monitored with a UV-detector and the elution curve is recorded for 8 min. After importing the recorded data into Microsoft Excel, the elution volume of the analyte (V) can be calculated, which represents the volume at which half of the plateau concentration is attained. To calculate V0, the elution data from the noninteracting control material (acetylcholine) is used.
2.6. Interaction between polySia and neurotransmitter Using either polySia-affigel 102 or affigel Hz-polySia, the interaction between polySia and the catecholamine neurotransmitters, i.e., epinephrine and dopamine could be observed. Typical elution profiles for epinephrine with polySia-affigel 102 at 37 C are shown in Fig. 10.4A. For the analyses, acetylcholine was used as a noninteracting neurotransmitter with polySia for the determination of V0. The VV0 values were measured for analyte concentrations ranging from 10 to 30 nM. Based on Eq. (10.4), the Kd value determined for epinephrine was 3.1 10 5 (M). Using the FAC, the interaction between two molecules can be examined under different conditions. For example, the effect of pH on the dopamine–polySia interaction was examined by varying the pH of the equilibration buffer and the analyte solution. The Kd value was affected by pH (Fig. 10.4B), indicating that the microenvironmental pH of the cell surface is important for the interaction between dopamine and polySia.
30 nM Acetylcholine
100
20 nM 10 nM
B 3.0
80
Kd(10−5)
% of plateau
A
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20 0
0
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0.4 0.6 V (ml)
0.8
1.0
2.0 5.5
6.0
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7.0
7.5
8.0
8.5
pH
Figure 10.4 The interaction between polySia and catecholamine neurotransmitters as analyzed by FAC. (A) Typical elution profiles for epinephrine. The neurotransmitter was dissolved in PBS at a concentration of 10, 20, and 30 nM and 2 ml of each solution was applied to the column (126 ml) through the 2 ml-sample loop at a flow rate of 0.125 ml/min at 37 C. Each elution curve for epinephrine was superimposed on that of acetylcholine. The observed retardation of elution was dependent upon the concentration of epinephrine. (B) The Kd values for the interaction of dopamine–polySia at different pHs were calculated by FAC analysis. The Kd value for this interaction was dependent upon the pH of the solution.
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3. Surface Plasmon Resonance (SPR)-Based Biosensors as a Tool for the Measurement of Glycan-Based Interactions An SPR-based biosensor was first reported in 1983 (Liedberg et al., 1983) and led to the development of the Biacore instrument. Although Biacore is the most commonly used, several other SPR-based instruments exist for analyzing molecular interactions. The principle of SPR is well documented in other reviews, including a recent one (Willander and Al-Hilli, 2009); thus, in this chapter, we focus on experimental procedures using a Biacore instrument to examine interactions between polySia and a neurotrophin. In order to reliably detect interactions by SPR, we recommend examining several sensor chips with respect to immobilization of the target ligand and the specificity of interaction. Biacore provides numerous types of sensor chips. For example, the CM5 chip is coated with carboxymethyldextran (100 nm thickness of dextran layer), and is the most commonly used because it effectively immobilizes ligands and low nonspecific binding can be achieved. Two other dextran-coated chips, CM4 and CM3, are available and contain a low amount of carboxyl groups and short chain-length dextran (30 nm thickness of dextran), respectively. The carboxyl groups in dextran are activated with either N-ethyl-N0 -[3-(dimethylamino)propyl] carbodiimide) (EDC) or N-hydroxysuccinimide (NHS) to allow conjugation with ligands through their -NH2, -SH, -COOH, and -CHO groups after the addition of appropriate reagents. Other special sensor chips are also available for the immobilization of tagged molecules. These include the SA chip, which is coated with a streptavidin-conjugated dextran for the immobilization of biotinylated ligands, and the NTA chip, which is coated with a NTA-conjugated dextran for immobilization of His-tagged ligands. Although CM5 is widely used, as the carboxymethyldextran coating on this chip contains many anions, it is important to confirm that observed interactions are specific and not due to nonspecific electrostatic forces. When nonspecific interactions are suspected, an Au chip without any coating should be used. In this chapter, we describe the use of the CM5 and Au chips for the immobilization of proteins and glycans, respectively.
3.1. Materials and equipment 1. 2. 3. 4.
Polysialic acid (polySia) (colominic acid) (Wako) Heparan sulfate (HS) (Seikagaku Co.) Tri-N-acetyl-chitotriose (!1GlcNAcb4!)3 (Seikagaku co.) Sensor Chip CM5 or Au (GE Healthcare)
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Biotin-LC-hydrazide (Pierce) Sephadex G-25 (GE Healthcare) 4,4-thio-dibutylic acid (DBA, Aldrich) N-Ethyl-N0 -[3-(dimethylamino)propyl]carbodiimide) (EDC) N-Hydroxysuccinimide (NHS) Streptavidin HBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate 20 (v/v)), pH 7.4) 12. Brain-derived neurotrophic factor (BDNF, Almone) 13. Biacore 2000 (GE Healthcare) 5. 6. 7. 8. 9. 10. 11.
3.2. Preparation of biotinylated glycans To prepare biotinylated glycans, chitotriose (GlcNAc)3 (2 mg/ml), polySia (10 mg/ml), or heparan sulfate (HS) (10 mg/ml) in 50 mM sodium acetate buffer (pH 5.5) are mixed with biotin-LC-hydrazide (final concentration, 5 mM) dissolved in DMSO. After incubation at 50 C for 2 h, NaBH3CN in methanol (22.4 mM final concentration) is added to the reaction mixture. The biotinylated glycans are then applied to a Sephadex G-25 column (1.2 cm 60 cm) and eluted with water to remove free biotin.
3.3. Immobilization of biotinylated glycans on an Au sensor chip The Au sensor surface is washed once with acetone and after drying, the chip is immersed in 10 mM DBA in ethanol to form a self-assembly membrane (SAM) on the Au surface. After gently shaking for 30 min at room temperature, the sensor surface is washed with ethanol three times and allowed to dry. The chip is then placed in a solution of EDC and NHS (a 1:9 mixture of 130 mM EDC in water and 144 mM NHS in 1,4-dioxane) at room temperature for 30 min with gentle shaking to activate the SAM on the Au surface. After adding water, the surface is incubated for 5 min, and then washed the Au surface. The Au chip containing surface-activated SAM is placed on the sensor chip support using the sensor chip assembly unit, and is set in a Biacore 2000 instrument. After priming with water for 7 min, a 0.1 mg/ml streptavidin solution is loaded twice, each time for 7 min at a flow rate of 10 ml/min. The immobilized streptavidin is monitored by the resonance unit (RU) value and typically reaches 490–580 RU. To destroy the excess activated groups, 1 mM ethanolamine is injected into the system for 7 min. After washing with HBS-EP, the target biotinylated glycan (0.1 mg/ml in 500 mM HBS-EP) is injected to allow immobilization on the Au surface (Fig. 10.5). The captured glycans can be monitored and reach around 30 RU for (GlcNAc)3, 120 RU for polySia, and 120 RU for HS.
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O H
O H H3 C
C N
OH OH OH
O
H COO− C 3
C N
O OH OH
O n
O
H
C
N
OH
O N H
H N
Bio tin Streptavidin
O
OH
H N
O C DBA*
S S Au sensor chip
Figure 10.5 PolySia-immobilized Au sensor chip using 40 -dithiodibutyric acid (DBA). DBA was incubated on an Au surface to make self-assembly membrane (SAM). The SAM was activated with NHS and EDC, and then streptoavidin was conjugated. By flowing biotinylated polySia, polySia-immobilized Au surface was applicable to measure interaction.
3.4. Immobilization of BDNF on a CM5 sensor chip To immobilize the neurotrophin BDNF, a research grade CM5 chip is set in a Biacore 2000. After washing with 40% glycerol, activation of the sensor chip surface is performed with a mixture of 400 mM EDC and 100 mM NHS for 7 min at a flow rate of 10 ml/min. Immediately after activation, a BDNF solution (5 ng/ml) in sodium acetate buffer (pH 5.0) is added. After the RU reaches an appropriate value, 1 mM ethanolamine is flowed for 7 min to destroy activated residues (Kanato et al., 2009).
3.5. Biacore analysis The interactions between BDNF and several glycans can be measured using a Biacore 2000 instrument. For the interaction of immobilized glycans with BDNF, varying concentrations of BDNF (0–220 nM) in HBS-EP are injected over the glycan-immobilized sensor chips at a flow rate of 20 ml/min. For the analysis of the interactions between immobilized BDNF and glycans, varying concentrations of polySia (0–80 mM) and HS (0–36 mM) in HBS-EP are injected over the BDNF-immobilized sensor chip at a flow rate of 20 ml/min. After 120 s, HBS-EP is flowed over the sensor surface to monitor the dissociation phase. Following 180 s of dissociation, the sensor surface is fully regenerated by the injection of 10 ml of 3 M NaCl. Using a range of polySia concentrations (0–80 mM) as the analyte and the BDNF-immobilized CM5 sensor chip, several sensorgrams can be obtained (Fig. 10.6). The polySia is flowed for 120 s at 20 ml/min for the association phase, and HBS-EP is then flowed for 120–300 s to monitor the dissociation phase. The sensorgrams allow not only the Kd value, but also the ka (M 1s 1) and kd (s 1) values to be calculated. The Kd value of polySia toward BDNF is 9.1 10 6 (M), whereas that of HS toward BDNF is 1.5 10 9 (M) (Kanato et al., 2009). Interestingly, HS displays nearly the identical affinity to BDNF and polySia as determined by gel-shift assays
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Analyte
Buffer
200 150 80 mM
RU
100
40 mM 20 mM 10 mM 5 mM
50 0
0
50
100 150 200 250 300 Time (s)
Figure 10.6 SPR sensorgrams for the interaction between polySia and BDNF. BDNF was immobilized on a CM5 sensor chip. Several concentrations of polySia (5–80 mM) were flowed over the chip, and the sensorgrams were monitored. BDNF in HBS-EP (analyte) flowed for 0–120 s; HBS-EP (buffer) flowed for 120–300 s.
using native PAGE (Kanato et al., 2008, 2009). It is clearly indicated that polySia requires amine-groups of BDNF for binding and that HS does not require amine group of BDNF for binding. The binding modes of BDNF toward polySia and HS are different. The reverse mode of interaction, that is, immobilized glycans and flowing BDNF, can be also measured. We usually adopt the Au sensor chip for immobilization of polySia to exclude the relatively high affinity of BDNF for the dextran matrix on the CM5 chip. For both the polySiaand HS-immobilized Au sensor chips, BDNF (0–220 mM) is flowed at 20 ml/min for 120 s, followed by elution with HBS-EP. Based on the sensorgram of poySia or HS subtracted with that of (GlcNAc)3, the Kd values of polySia and HS were calculated to be 6.4 10 9 (M) and 2.5 10 9 (M), respectively. Notably, the dissociation constants of polySia obtained using glycan-immobilized and BDNF-immobilized chips are 1000 times different in magnitude, while those of HS are much the same. These results suggest that the binding mechanism is different between the BDNF-polySia and the BDNF-HS complexes.
4. Conclusions FAC has been widely used for analyzing lectin–carbohydrate interactions. The principle and the system of FAC are very simple and it has a merit for measurements of relatively low-affinity interactions that are often the case with
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glycan-based interactions. This chapter features a new application of FAC to a measurement of interaction between glycans and small molecules. Indeed, the polySia–neurotransmitter interaction is demonstrated by this method. Thus, FAC can be applied to non-protein-based interactions that remain unnoticed and low-affinity interactions that are difficult to measure by other methods. With the FAC system, in addition to Kd value, its subtle changes depending on environmental conditions can be easily determined. In FAC, interactions are measured in the flow of analytes, and thus can mimic those in natural flow system such as bloodstream by changing the flow rate using appropriate HPLC pump. This chapter also features an improved protocol in Biacore to measure glycan-based interactions. Biacore has been widely used for glycan-based interactions using glycans immobilized onto the surface of CM-coated chip or flowing glycans as analytes. However, it is a problem that analytes sometimes bind to CM surface nonspecifically. In the improved protocol, we adopted a noncoated Au surface instead of the CM surface. Indeed, with this method, a novel specific interaction between polySia and BDNF together with the Kd value is demonstrated. In the fields of glycomics, researchers are searching for new glycan-based interactions with microarrays and other methods described in this book. Therefore, methodologies have become more and more important to understand quantitatively how specific and how strong the interaction occurs. Furthermore, not only binding but also releasing processes that may be regulated by the microenvironment of cell surface are crucial for functional regulation of the interacting molecule. To understand precise conditions of functioning of the glycan-based interactions, high-throughput and quantitative methods that can be applied under various conditions (flow or static, pH, salt, cations, and so on) would be important.
ACKNOWLEDGMENTS This research was supported in part by Grants-in-Aid for Scientific Research (C) (20570107) (to C. S.) from the Ministry of Education, Science, Sports and Culture and Grants-in-Aid for the Global COE Program: Advanced Systems Biology (to K. K. and N. Y.). We also thank Mr. Ryo Isomura and Miss Sayaka Ono for the results presented in this chapter.
REFERENCES Angata, K., Lee, W., Mitoma, J., Marth, J., and Fukuda, M. (2006). Cellular and molecular analysis of neural development of glycosyltransferase gene knockout mice. Methods Enzymol. 417, 25–37. Arata, Y., Hirabayashi, J., and Kasai, K. (1997). The two lectin domains of the tandemrepeat 32-kDa galectin of the nematode Caenorhabditis elegans have different binding properties. Studies with recombinant protein. J. Biochem. 121, 1002–1009.
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Bonfanti, L. (2006). PSA-NCAM in mammalian structural plasticity and neurogenesis. Prog. Neurobiol. 80, 129–164. Hirabayashi, J., Arata, Y., and Kasai, K. (2003). Frontal affinity chromatography as a tool for elucidation of sugar recognition properties of lectins. Methods Enzymol. 362, 353–368. Kanato, Y., Kitajima, K., and Sato, C. (2008). Direct binding of polysialic acid to a brainderived neurotrophic factor depends on the degree of polymerization. Glycobiology 18, 1044–1053. Kanato, Y., Ono, S., Kitajima, K., and Sato, C. (2009). Complex formation of a brainderived neurotrophic factor and glycosaminoglycans. Biosci. Biotechnol. Biochem. 73, 2735–2741. Kasai, K., and Ishii, S. (1973). Unimportance of histidine and serine residues of trypsin in the substrate binding function proved by affinity chromatography. J. Biochem. 74, 631–633. Kasai, K., Oda, Y., Nishikata, M., and Ishii, S. (1986). Frontal affinity chromatography: Theory for its application to studies on specific interactions of biomolecules. J. Chromatogr. 376, 33–47. Kuno, A., Uchiyama, N., Koseki-Kuno, S., Ebe, Y., Takashima, S., Yamada, M., and Hirabayashi, J. (2005). Evanescent-field fluorescence-assisted lectin microarray: A new strategy for glycan profiling. Nat. Methods 2, 851–856. Liedberg, B., Nylander, C., and Lundstro¨m, I. (1983). Surface plasmon resonance for gas detection and biosensing. Sens. Actuators 4, 299–304. Ng, E., Yang, F., Kameyama, A., Palcic, M., Hindsgaul, O., and Schriemer, D. (2005). High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry. Anal. Chem. 77, 6125–6133. Oda, Y., Kasai, K., and Ishii, S. (1981). Studies on the specific interaction of concanavalin A and saccharides by affinity chromatography. Application of quantitative affinity chromatography to a multivalent system. J. Biochem. 89, 285–296. Rutishauser, U. (2008). Polysialic acid in the plasticity of the developing and adult vertebrate nervous system. Nat. Rev. Neurosci. 9, 26–35. Sato, C. (2004). Chain length diversity of sialic acids and its biological significance. Trends Glycosci. Glycotech. 14, 331–344. Sato, C. (2010). Polysialic Acid. Bentham Science, UAE. (in press). Schwartz, N., and Domowicz, M. (2004). Proteoglycans in brain development. Glycoconj. J. 21, 329–341. Tateno, H., Nakamura-Tsuruta, S., and Hirabayashi, J. (2007). Frontal affinity chromatography: Sugar–protein interactions. Nat. Protoc. 2, 2529–2537. Troy, F. A. II. (1996). Sialobiology and the polysialic acid glycotope. In ‘‘Biology of the Sialic acid,’’ (Rosenerg, ed.). pp.95–144. Plenum press, New York. Willander, M., and Al-Hilli, S. (2009). Analysis of biomolecules using surface plasmons. Methods Mol. Biol. 544, 201–229.
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Measurement of Glycan-Based Interactions by Frontal Affinity Chromatography and Surface Plasmon Resonance Chihiro Sato, Nao Yamakawa, and Ken Kitajima Contents 1. Introduction 2. Frontal Affinity Chromatography (FAC) as a Tool for the Measurement of Glycan-Based Interactions 2.1. FAC principle 2.2. Materials and equipment 2.3. Preparation of affinity adsorbents 2.4. Preparation of neurotransmitters 2.5. Operation of frontal affinity chromatography 2.6. Interaction between polySia and neurotransmitter 3. Surface Plasmon Resonance (SPR)-Based Biosensors as a Tool for the Measurement of Glycan-Based Interactions 3.1. Materials and equipment 3.2. Preparation of biotinylated glycans 3.3. Immobilization of biotinylated glycans on an Au sensor chip 3.4. Immobilization of BDNF on a CM5 sensor chip 3.5. Biacore analysis 4. Conclusions Acknowledgments References
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Abstract Proteins and lipids are often modified with glycan chains, which due to their large hydration effect and structural heterogeneity, significantly alter the surface physicochemical properties of proteins and biomembranes. This ‘‘glycoatmosphere’’ also serves as a field for interactions with various molecules, Bioscience and Biotechnology Center, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Methods in Enzymology, Volume 478 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)78010-1
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including other glycans, lipids, peptides, proteins, and small molecules such as neurotransmitters and drugs as well as lectins. Therefore, sensitive techniques for measuring these glycan-based interactions are becoming more and more necessary, with the appropriate method largely depending on the interacting molecules. In this chapter, we focus on frontal affinity chromatography (FAC) and surface plasmon resonance (SPR) for examining polysialic acid-involved interactions with neurotransmitters and neurotrophins. FAC is characterized by its applicability to analyze weak interactions that are difficult to measure using conventional methods, and by the ease of principle and experimental procedures. SPR is advantageous due to the availability of suitable surface materials and for real-time monitoring with nonlabeled analytes.
1. Introduction Glycosylation is one of the major modifications of proteins and lipids, and a wide variety of glycosylations have been reported. However, the phenomenon of glycosylation remains somewhat mysterious due to a lack of appropriate methodologies for determining glycan structures as well as their biological functions. Recent methods of genetic perturbation for glycan-related enzymes have greatly impacted the understanding of the biological significance of glycans, even if the results are severe or benign. The underlying mechanisms which link gene expression to the resultant phenotypes, however, remain unknown. In this respect, it is important to understand glycan-based interactions with cellular components containing not only proteins, but also glycans, lipids, and other natural substances using appropriate analytical methods. In this chapter, we focus on the measurement of new polysialic acid (polySia)-based interactions using frontal affinity chromatography (FAC) and a surface plasmon resonance (SPR)-based Biacore instrument. PolySia is a polymerized structure of sialic acid present on neural cell adhesion molecules (NCAMs) as a posttranslational modification (Sato, 2004, 2010; Troy, 1996). PolySia-modified NCAM has been well studied in the development of the nervous system since the modification is spatiotemporally regulated (Bonfanti, 2006; Rutishauser, 2008). PolySia is expressed in embryonic brains during neural differentiation and mostly disappears in adult brains, although NCAM expression levels remain unchanged. Based on its bulky polyanionic nature, polySia is thought to function as an antiadhesive molecule against cell–cell and extracellular matrix interactions (Angata et al., 2006; Bonfanti, 2006; Rutishauser, 2008). However, we recently demonstrated by native PAGE analysis that an important neurotrophin in the brain, brain-derived neurotrophic factor (BDNF), interacts with polySia directly to form a large complex (Kanato et al., 2008). Therefore, polySia likely serves an important role as a
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neurogenetic regulator similar to glycosaminoglycans that have been shown to bind growth factors and signaling molecules, such as Wnt (Schwartz and Domowicz, 2004). We thus proposed the hypothesis that, as a novel functional role, polySia may serve as a reservoir of neuroactive molecules, such as neurotrophins, growth factors, and neurotransmitters (Kanato et al., 2008, 2009). To gain insight into this potential novel function of polySia, we applied FAC and SPR for measuring polySia–neurotransmitter and polySia–neurotrophin interactions, respectively.
2. Frontal Affinity Chromatography (FAC) as a Tool for the Measurement of Glycan-Based Interactions Affinity chromatography is a commonly used and powerful tool for the purification of molecules that specifically interact with a target counterpart molecule. In the 1970s, Kasai and Ishii were the first to demonstrate that affinity chromatography is applicable to quantitative analysis for the estimation of dissociation constants for protein–ligand interactions (Kasai and Ishii, 1973). Kasai and his colleagues also applied FAC for the measurement of glycan–lectin interactions (Arata et al., 1997; Oda et al., 1981), which represented the first estimation of Kd. Hindsgaul et al. improved this method through the use of microcolumns and MS as a detector (Ng et al., 2005), allowing materials to be conserved and shortening the time required for analysis. This LC–MS system is particularly applicable for high-throughput screening. At the same time, Hirabayashi also developed an enhanced, high-throughput lectin–glycan interaction-analysis system between immobilized lectins and soluble fluorescent (PA)-labeled glycans (Kuno et al., 2005). Owing to improvements of these FAC-systems, FAC is a widely accepted technique for the analysis of lectin–glycan interactions. The FAC system has been well established and a number of wellwritten reviews are available (Hirabayashi et al., 2003; Kasai et al., 1986; Tateno et al., 2007). In this book, Kamiya and Kato also describe a lectin– glycan interaction revealed using a FAC system; in this chapter, we therefore focus on the general principle and methods for analyzing glycan-polymer and small molecule interactions.
2.1. FAC principle In frontal analysis, the elution front of an analyte and its concentration at a plateau level are measured when the analyte solution at various concentrations is eluted. For the analysis, the volume of the analyte solution should exceed the column volume. Under the assay conditions, as the free analyte
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concentration is always fixed and equal to the initial concentration of analyte, dynamic equilibrium is achieved in the plateau region. Prior to the analysis, the column is packed with a resin on which the ligand of interest (termed B) is immobilized. The column is then isocratically eluted with an excess column volume of analyte (termed A) and the resulting elution curve of A is monitored. Based on the starting point of analyte elution (the elution front), the interaction between B and A can be detected. The retardation of elution (see line 2 in Fig. 10.1) indicates that analyte A interacts with B (Fig. 10.1). In contrast, an analyte which does not interact with B is eluted in the void volume of the column (see line 1 in Fig. 10.1). The generation of a retard volume (VV0) means a volume of an A–B complex formed as a result of specific interaction. Therefore, the area, [A]0(V V0), represents the amount of the A–B complex. Provided the column volume is u, the equation [A]0(VV0) ¼ u [AB] is realized. The dissociation constant (Kd) can be determined from Eq. (10.1), which is based on the equation: A þ B , AB. Kd ¼ ½A½B=½AB ¼ ½A0 ½B0 ½AB =½AB ¼ u½B0 =ðV V0 Þ ½A0 ð10:1Þ
Concentration of analyte
As [B]0 is the concentration of the immobilized ligand, and the effective ligand content can be obtained with the equation Bt ¼ u [B]0. Therefore, Eq. (10.1) can be followed by Eq. (10.2).
1 (no interaction with B) 2 (interaction with B)
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Figure 10.1 Schematic elution profiles of FAC. The immobilized ligand (B) is packed into a column and a volume in excess of the total column volume of analyte (A) is then eluted. Line 1 is the curve for the analyte that does not interact with B. The elution volume represents the void volume (V0) of the column. Line 2 is the curve for the analyte that interacts with B. The retard (VV0) indicates a specific interaction between A and B. The shaded area, [A]0(VV0), is the amount of the complex [AB].
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Kd ¼ Bt=ðV V0 Þ ½A0
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ð10:2Þ
The value for Bt is obtained from analyte concentration-dependent experiments using either a Lineweaver–Burk type plot, that is, 1/[A]0 versus 1/(V V0)[A]0, or a Woolf–Hofstee type plot, that is, (V V0) versus (V V0) [A]0. Equation (10.2) can be simplified to Eq. (10.3) where [A]0 (10 8 M) is negligible for the Kd value (e.g., >10 6 M). Kd ¼ Bt=ðV V0 Þ;
if ½A0 Kd
ð10:3Þ
The elution volume of an analyte (V ) is determined graphically as the volume corresponding to the elution point, which occurs at the half value of the plateau of the elution curve. V0 is determined as the V of the appropriate control sample without affinity for the immobilized ligand. The values of Bt and Kd are determined from the intercept of the axis and the slope of the fitted curves (Woolf–Hofstee-type plots, (V V0) vs. (V V0)[A]0) (Fig. 10.2), respectively, and Eq. (10.3) can be changed to Eq. (10.4). ½A0 ðV V0 Þ ¼ KdðV V0 Þ þ Bt
ð10:4Þ
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It should be noted that strong interactions cannot be measured with this system, as this method is based only on the retardation of elution and not the
Slope = −Kd
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Figure 10.2 The Woolf–Hofstee plot for the determination of Bt and Kd. Based on Eq. (10.4), [A]0 (V V0) ¼ Kd (V V0) þ Bt, the obtained data can be plotted. The x- and y-axes represent the retarded elution of analyte A (V V0) and the amount of A–B complex ([A]0 (V V0)), respectively. Therefore, from the intercept of y-axis, the Bt (effective ligand content) is calculated. The Kd value is obtained from the slope (¼ Kd).
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complete loss of the analyte from the eluate. Therefore, this method is suited for the measurement of relatively weak interactions that are occasionally encountered, as in the case of glycan-based interactions.
2.2. Materials and equipment Polysialic acid (colominic acid) (Wako) Affigel 102 or Affigel Hz (Bio-rad) 1-ethyl-3-(3-diethylaminopropyl)-carbodiimide (Bio-rad) Phosphate-buffered saline (PBS) (pH 7.2; 10 mM; 137 mM NaCl; 2.7 mM KCl) 5. Neurotransmitters (epinephrine and dopamine) 6. Column (4.0 mm 10 mm, 126 ml, GL Science) 7. HPLC system (Rheodyne injector equipped with a 2 ml-PEEK sample loop and a UV-detector connected to an integrator) 1. 2. 3. 4.
2.3. Preparation of affinity adsorbents For the FAC analysis, two types of polySia (colominic acid)-immobilized resins are used which differed on whether polySia is immobilized through the reducing (Fig. 10.3A) or nonreducing (Fig. 10.3B) terminal end. To prepare the resin with reducing end-immobilized polySia, 5 ml (packed volume) of Affigel 102 in 25 mM Na2HPO4 (pH 6.0) are added to 2 ml of 50 mg/ml colominic acid, and the pH of the resulting solution is adjusted to 5.0 with 1 N HCl. After the addition of 8 mg 1-ethyl-3-(3-diethylaminopropyl)-carbodiimide, the reaction mixture is kept at 4 C for 4 h with gentle mixing by rotation. After washing with PBS, the gel is blocked with acetic anhydrite at room temperature for 30 min and washed with PBS and 1 M NaCl. Based on the amount of unbound polySia measured by the resorcinol method, the extent of immobilization of polySia is estimated to be 0.8 mmol/ml. To prepare the resin to which polySia is immobilized through the nonreducing terminal end (Fig. 10.3B), 5 ml (packed volume) of Affigel Hz in 50 mM sodium acetate buffer (pH 5.5) are added to 2 ml of 48 mg/ml periodate-oxidized colominic acid (prepared by incubation with 25 mM sodium periodate in 100 mM sodium acetate (pH 5.5) followed by desalting), and incubated at 25 C overnight with gentle mixing. After washing the gel with PBS, unbound colominic acid is measured by the resorcinol method to estimate the amount of immobilized colominic acid. The immobilization extent of colominic acid is estimated to be 0.7 mmol/ ml. The two types of resins are each packed into an empty column (4.0 mm 10 mm, 126 ml, GL Science) using a syringe. The prepared columns can be stored at 4 C until use.
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Figure 10.3 PolySia-immobilized beads used for FAC analysis. (A) polySia-affigel 102. (B) Affigel Hz-polySia.
2.4. Preparation of neurotransmitters A variety of neurotransmitters are commercially available. In this study, we selected epinephrine and dopamine for FAC analyses with the immobilized polySia ligand. For the analysis, each neurotransmitter is dissolved in PBS or an appropriate buffer at concentrations ranging from 0 to 30 nM.
2.5. Operation of frontal affinity chromatography A FAC system consists of a pump ( JASCO PU-980i), an injector with a sample loop, a column, and a UV detector ( JASCO 875-UV) connected to a chromato-PRO integrator (Run Time Corporation, Kanagawa, Japan). The 2 ml-sample loop (PEEK) and the column are either kept in an oven (CTO-6A, Shimadzu) or water bath at 25 or 37 C. Prior to analysis, the column is equilibrated with PBS at a flow rate of 0.125 ml/min until a flat baseline of absorbance was achieved. The sample injector is turned to the load position, and 10–20 ml of air is injected using a syringe to empty the sample loop completely. The analyte solution dissolved in PBS is then injected to fill the 2 ml-sample loop. Note that 50–100 ml of excess analyte
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solution is required to completely fill the 2 ml-sample loop. The injector is then turned to the inject position to flow the analyte into the column at a rate of 0.125 ml/min. The analyte eluted from the column is monitored with a UV-detector and the elution curve is recorded for 8 min. After importing the recorded data into Microsoft Excel, the elution volume of the analyte (V) can be calculated, which represents the volume at which half of the plateau concentration is attained. To calculate V0, the elution data from the noninteracting control material (acetylcholine) is used.
2.6. Interaction between polySia and neurotransmitter Using either polySia-affigel 102 or affigel Hz-polySia, the interaction between polySia and the catecholamine neurotransmitters, i.e., epinephrine and dopamine could be observed. Typical elution profiles for epinephrine with polySia-affigel 102 at 37 C are shown in Fig. 10.4A. For the analyses, acetylcholine was used as a noninteracting neurotransmitter with polySia for the determination of V0. The VV0 values were measured for analyte concentrations ranging from 10 to 30 nM. Based on Eq. (10.4), the Kd value determined for epinephrine was 3.1 10 5 (M). Using the FAC, the interaction between two molecules can be examined under different conditions. For example, the effect of pH on the dopamine–polySia interaction was examined by varying the pH of the equilibration buffer and the analyte solution. The Kd value was affected by pH (Fig. 10.4B), indicating that the microenvironmental pH of the cell surface is important for the interaction between dopamine and polySia.
30 nM Acetylcholine
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Figure 10.4 The interaction between polySia and catecholamine neurotransmitters as analyzed by FAC. (A) Typical elution profiles for epinephrine. The neurotransmitter was dissolved in PBS at a concentration of 10, 20, and 30 nM and 2 ml of each solution was applied to the column (126 ml) through the 2 ml-sample loop at a flow rate of 0.125 ml/min at 37 C. Each elution curve for epinephrine was superimposed on that of acetylcholine. The observed retardation of elution was dependent upon the concentration of epinephrine. (B) The Kd values for the interaction of dopamine–polySia at different pHs were calculated by FAC analysis. The Kd value for this interaction was dependent upon the pH of the solution.
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3. Surface Plasmon Resonance (SPR)-Based Biosensors as a Tool for the Measurement of Glycan-Based Interactions An SPR-based biosensor was first reported in 1983 (Liedberg et al., 1983) and led to the development of the Biacore instrument. Although Biacore is the most commonly used, several other SPR-based instruments exist for analyzing molecular interactions. The principle of SPR is well documented in other reviews, including a recent one (Willander and Al-Hilli, 2009); thus, in this chapter, we focus on experimental procedures using a Biacore instrument to examine interactions between polySia and a neurotrophin. In order to reliably detect interactions by SPR, we recommend examining several sensor chips with respect to immobilization of the target ligand and the specificity of interaction. Biacore provides numerous types of sensor chips. For example, the CM5 chip is coated with carboxymethyldextran (100 nm thickness of dextran layer), and is the most commonly used because it effectively immobilizes ligands and low nonspecific binding can be achieved. Two other dextran-coated chips, CM4 and CM3, are available and contain a low amount of carboxyl groups and short chain-length dextran (30 nm thickness of dextran), respectively. The carboxyl groups in dextran are activated with either N-ethyl-N0 -[3-(dimethylamino)propyl] carbodiimide) (EDC) or N-hydroxysuccinimide (NHS) to allow conjugation with ligands through their -NH2, -SH, -COOH, and -CHO groups after the addition of appropriate reagents. Other special sensor chips are also available for the immobilization of tagged molecules. These include the SA chip, which is coated with a streptavidin-conjugated dextran for the immobilization of biotinylated ligands, and the NTA chip, which is coated with a NTA-conjugated dextran for immobilization of His-tagged ligands. Although CM5 is widely used, as the carboxymethyldextran coating on this chip contains many anions, it is important to confirm that observed interactions are specific and not due to nonspecific electrostatic forces. When nonspecific interactions are suspected, an Au chip without any coating should be used. In this chapter, we describe the use of the CM5 and Au chips for the immobilization of proteins and glycans, respectively.
3.1. Materials and equipment 1. 2. 3. 4.
Polysialic acid (polySia) (colominic acid) (Wako) Heparan sulfate (HS) (Seikagaku Co.) Tri-N-acetyl-chitotriose (!1GlcNAcb4!)3 (Seikagaku co.) Sensor Chip CM5 or Au (GE Healthcare)
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Biotin-LC-hydrazide (Pierce) Sephadex G-25 (GE Healthcare) 4,4-thio-dibutylic acid (DBA, Aldrich) N-Ethyl-N0 -[3-(dimethylamino)propyl]carbodiimide) (EDC) N-Hydroxysuccinimide (NHS) Streptavidin HBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate 20 (v/v)), pH 7.4) 12. Brain-derived neurotrophic factor (BDNF, Almone) 13. Biacore 2000 (GE Healthcare) 5. 6. 7. 8. 9. 10. 11.
3.2. Preparation of biotinylated glycans To prepare biotinylated glycans, chitotriose (GlcNAc)3 (2 mg/ml), polySia (10 mg/ml), or heparan sulfate (HS) (10 mg/ml) in 50 mM sodium acetate buffer (pH 5.5) are mixed with biotin-LC-hydrazide (final concentration, 5 mM) dissolved in DMSO. After incubation at 50 C for 2 h, NaBH3CN in methanol (22.4 mM final concentration) is added to the reaction mixture. The biotinylated glycans are then applied to a Sephadex G-25 column (1.2 cm 60 cm) and eluted with water to remove free biotin.
3.3. Immobilization of biotinylated glycans on an Au sensor chip The Au sensor surface is washed once with acetone and after drying, the chip is immersed in 10 mM DBA in ethanol to form a self-assembly membrane (SAM) on the Au surface. After gently shaking for 30 min at room temperature, the sensor surface is washed with ethanol three times and allowed to dry. The chip is then placed in a solution of EDC and NHS (a 1:9 mixture of 130 mM EDC in water and 144 mM NHS in 1,4-dioxane) at room temperature for 30 min with gentle shaking to activate the SAM on the Au surface. After adding water, the surface is incubated for 5 min, and then washed the Au surface. The Au chip containing surface-activated SAM is placed on the sensor chip support using the sensor chip assembly unit, and is set in a Biacore 2000 instrument. After priming with water for 7 min, a 0.1 mg/ml streptavidin solution is loaded twice, each time for 7 min at a flow rate of 10 ml/min. The immobilized streptavidin is monitored by the resonance unit (RU) value and typically reaches 490–580 RU. To destroy the excess activated groups, 1 mM ethanolamine is injected into the system for 7 min. After washing with HBS-EP, the target biotinylated glycan (0.1 mg/ml in 500 mM HBS-EP) is injected to allow immobilization on the Au surface (Fig. 10.5). The captured glycans can be monitored and reach around 30 RU for (GlcNAc)3, 120 RU for polySia, and 120 RU for HS.
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O H
O H H3 C
C N
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C N
O OH OH
O n
O
H
C
N
OH
O N H
H N
Bio tin Streptavidin
O
OH
H N
O C DBA*
S S Au sensor chip
Figure 10.5 PolySia-immobilized Au sensor chip using 40 -dithiodibutyric acid (DBA). DBA was incubated on an Au surface to make self-assembly membrane (SAM). The SAM was activated with NHS and EDC, and then streptoavidin was conjugated. By flowing biotinylated polySia, polySia-immobilized Au surface was applicable to measure interaction.
3.4. Immobilization of BDNF on a CM5 sensor chip To immobilize the neurotrophin BDNF, a research grade CM5 chip is set in a Biacore 2000. After washing with 40% glycerol, activation of the sensor chip surface is performed with a mixture of 400 mM EDC and 100 mM NHS for 7 min at a flow rate of 10 ml/min. Immediately after activation, a BDNF solution (5 ng/ml) in sodium acetate buffer (pH 5.0) is added. After the RU reaches an appropriate value, 1 mM ethanolamine is flowed for 7 min to destroy activated residues (Kanato et al., 2009).
3.5. Biacore analysis The interactions between BDNF and several glycans can be measured using a Biacore 2000 instrument. For the interaction of immobilized glycans with BDNF, varying concentrations of BDNF (0–220 nM) in HBS-EP are injected over the glycan-immobilized sensor chips at a flow rate of 20 ml/min. For the analysis of the interactions between immobilized BDNF and glycans, varying concentrations of polySia (0–80 mM) and HS (0–36 mM) in HBS-EP are injected over the BDNF-immobilized sensor chip at a flow rate of 20 ml/min. After 120 s, HBS-EP is flowed over the sensor surface to monitor the dissociation phase. Following 180 s of dissociation, the sensor surface is fully regenerated by the injection of 10 ml of 3 M NaCl. Using a range of polySia concentrations (0–80 mM) as the analyte and the BDNF-immobilized CM5 sensor chip, several sensorgrams can be obtained (Fig. 10.6). The polySia is flowed for 120 s at 20 ml/min for the association phase, and HBS-EP is then flowed for 120–300 s to monitor the dissociation phase. The sensorgrams allow not only the Kd value, but also the ka (M 1s 1) and kd (s 1) values to be calculated. The Kd value of polySia toward BDNF is 9.1 10 6 (M), whereas that of HS toward BDNF is 1.5 10 9 (M) (Kanato et al., 2009). Interestingly, HS displays nearly the identical affinity to BDNF and polySia as determined by gel-shift assays
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Analyte
Buffer
200 150 80 mM
RU
100
40 mM 20 mM 10 mM 5 mM
50 0
0
50
100 150 200 250 300 Time (s)
Figure 10.6 SPR sensorgrams for the interaction between polySia and BDNF. BDNF was immobilized on a CM5 sensor chip. Several concentrations of polySia (5–80 mM) were flowed over the chip, and the sensorgrams were monitored. BDNF in HBS-EP (analyte) flowed for 0–120 s; HBS-EP (buffer) flowed for 120–300 s.
using native PAGE (Kanato et al., 2008, 2009). It is clearly indicated that polySia requires amine-groups of BDNF for binding and that HS does not require amine group of BDNF for binding. The binding modes of BDNF toward polySia and HS are different. The reverse mode of interaction, that is, immobilized glycans and flowing BDNF, can be also measured. We usually adopt the Au sensor chip for immobilization of polySia to exclude the relatively high affinity of BDNF for the dextran matrix on the CM5 chip. For both the polySiaand HS-immobilized Au sensor chips, BDNF (0–220 mM) is flowed at 20 ml/min for 120 s, followed by elution with HBS-EP. Based on the sensorgram of poySia or HS subtracted with that of (GlcNAc)3, the Kd values of polySia and HS were calculated to be 6.4 10 9 (M) and 2.5 10 9 (M), respectively. Notably, the dissociation constants of polySia obtained using glycan-immobilized and BDNF-immobilized chips are 1000 times different in magnitude, while those of HS are much the same. These results suggest that the binding mechanism is different between the BDNF-polySia and the BDNF-HS complexes.
4. Conclusions FAC has been widely used for analyzing lectin–carbohydrate interactions. The principle and the system of FAC are very simple and it has a merit for measurements of relatively low-affinity interactions that are often the case with
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glycan-based interactions. This chapter features a new application of FAC to a measurement of interaction between glycans and small molecules. Indeed, the polySia–neurotransmitter interaction is demonstrated by this method. Thus, FAC can be applied to non-protein-based interactions that remain unnoticed and low-affinity interactions that are difficult to measure by other methods. With the FAC system, in addition to Kd value, its subtle changes depending on environmental conditions can be easily determined. In FAC, interactions are measured in the flow of analytes, and thus can mimic those in natural flow system such as bloodstream by changing the flow rate using appropriate HPLC pump. This chapter also features an improved protocol in Biacore to measure glycan-based interactions. Biacore has been widely used for glycan-based interactions using glycans immobilized onto the surface of CM-coated chip or flowing glycans as analytes. However, it is a problem that analytes sometimes bind to CM surface nonspecifically. In the improved protocol, we adopted a noncoated Au surface instead of the CM surface. Indeed, with this method, a novel specific interaction between polySia and BDNF together with the Kd value is demonstrated. In the fields of glycomics, researchers are searching for new glycan-based interactions with microarrays and other methods described in this book. Therefore, methodologies have become more and more important to understand quantitatively how specific and how strong the interaction occurs. Furthermore, not only binding but also releasing processes that may be regulated by the microenvironment of cell surface are crucial for functional regulation of the interacting molecule. To understand precise conditions of functioning of the glycan-based interactions, high-throughput and quantitative methods that can be applied under various conditions (flow or static, pH, salt, cations, and so on) would be important.
ACKNOWLEDGMENTS This research was supported in part by Grants-in-Aid for Scientific Research (C) (20570107) (to C. S.) from the Ministry of Education, Science, Sports and Culture and Grants-in-Aid for the Global COE Program: Advanced Systems Biology (to K. K. and N. Y.). We also thank Mr. Ryo Isomura and Miss Sayaka Ono for the results presented in this chapter.
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