2CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation, 2-3, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan; 3Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062; and 4Department of Gynecologic Oncology, Roswell Park Memorial Cancer Institute, Buffalo, New York, USA
Received on November 29, 2001; revised on December 25, 2001; accepted on December 28, 2001.
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Abstract |
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Key words: colon/core 1/galectin-4/sulfated glycan/surface plasmon resonance
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Introduction |
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Galectin-4 was initially discovered as a soluble 17-kDa lectin in rat intestinal extracts by Leffler et al. (1989); later the cDNA cloning of galectin-4 revealed that it is a 36-kDa protein (Oda et al., 1993
). Galectin-4 has been found to be localized in the epithelium of the alimentary tract, including oral mucosa, esophagus, and intestinal mucosa (Chiu et al., 1994
; Danielsen and Deurs, 1997
). Chiu et al. (1994)
found that galectin-4 in porcine oral and upper esophageal epithelium is water-insoluble as a component of adherens junction complexes. Danielsen and Deurs (1997)
reported that galectin-4 forms detergent-insoluble complexes with apically sorting brush-border enzymes in porcine small intestine, when the isolation was performed at low temperature. On the contrary, Huflejt et al. (1997)
showed the localization of galectin-4 on the basal membrane of human colon adenocarcinoma T84 cells in a confluent and polarized condition, whereas galectin-3 tends to be concentrated in granular inclusions mostly localized on the apical side. They also showed that galectin-4 was concentrated at the leading edges of lamellipodia in semiconfluent T84 cells. These results suggest that galectin-4 is located in a different area of cells dependently on tissues and culture conditions and that it plays a role in cell adhesion or cell migration. Furthermore, the down-regulation of expression levels of the mRNA for human galectin-4 in colorectal cancer (Rechreche et al., 1997
) and the up-regulation in human hepatocellular carcinoma (Kondoh et al., 1999
) and high metastatic gastric cancer cells (Hippo et al., 2001
) have been reported. These phenomena suggest that galectin-4 may be a good tumor-marker against the latter two cancers and contributes certain characteristics of those cancer cells.
To elucidate physiological functions of galectin-4, it is indispensable to determine the natural ligands and the carbohydrate binding specificity of galectin-4. However, these characters of galectin-4 have not been elucidated. We determined in this study the carbohydrate binding specificity of recombinant human galectin-4 (rhgalectin-4) in comparison with that of recombinant human galectin-3 (rhgalectin-3) by rhgalectin-4 or -3-immobilized column chromatography, surface plasmon resonance method, and inhibitory assay method. As the results, rhgalectin-4 preferentially recognizes Galß13GalNAc (core 1), and 3'-O-sulfation of core 1 dramatically enhanced the binding to rhgalectin-4. The dissociation constant of the binding between SO3
3core 1-O-Bn and rhgalectin-4 was calculated as 3.4 x 106 M at 25°C.
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Results |
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Behaviors of various glycoproteins on rhgalectin-3- or -4-Sepharose column chromatography
In preliminary experiments to elucidate the carbohydrate binding specificity of rhgalectin-4, various glycoproteins that have different type of oligosaccharides were applied on a rhgalectin-3- or -4-immobilized Sepharose column. RNase B bearing one high-mannose type glycan (Liang et al., 1980) (Figure 1A and D) and transferrin bearing two
2
6sialylated biantennary glycans (Spik et al., 1975
) (Figure 1B and E, solid line) flowed through both the columns, whereas asialo-transferrin bound to the rhgalectin-3 column (Figure 1B, dotted line), but not to the rhgalectin-4 column (Figure 1E, dotted line), indicating that rhgalectins-3 and -4 do not recognize high-mannose type glycans and Sia
2
6 substituted ß-galactosyl residues, and that rhgalectin-3 recognizes N-acetyllactosamine residue but rhgalectin-4 does not. Asialofetuin bound to both rhgalectins-3 and -4-Sepharose columns (Figure 1C and F, solid line). Because asialofetuin carries three N-linked glycans and three O-linked glycans (Spiro and Bhoyroo, 1974
), we examined which type of glycan is critical for binding to rhgalectin-3 or -4. The ß-eliminated asialofetuin did not bind to the rhgalectin-4 column (Figure 1F, dotted line), and the ß-eliminated asialofetuin still bound to the rhgalectin-3 column (Figure 1C, dotted line). These results suggested that rhgalectin-3 recognizes type 2 (type 1) glycans and that rhgalectin-4 recognizes some O-linked type glycans. Furthermore, asialofetuin and porcine colorectal mucin bound to rhgalectin-4 immobilized on the sensor chip of BIAcore 2000 but did not bind after ß-elimination (data not shown). These results also suggest that rhgalectin-4 recognizes some O-linked type glycans.
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Although core 1, core 1-O-p-nitrophenyl (pNP), and core 1-O-Bn were weakly retarded to the column (Figure 2D), core 1 derivatives bearing SO33Gal residue bound to the column (Figure 2A and B), and
2
3sialylation of core 1 ([3H]Neu5Ac
2
3core 1) slightly increased the retardation to the column (Figure 2E). These results indicate that galectin-3 recognizes type 1, type 2, and core 1 derivatives that carry Fuc
1
2Gal, Fuc
1
3(4)GlcNAc, GalNAc
1
3(Fuc
1
2)Gal, Neu5Ac
2
3Gal, and SO3
3Gal residues except Neu5Ac
2
6Gal residue.
Behaviors of oligosaccharides on rhgalectin-4-Sepharose column chromatography
On the other hand, most of the oligosaccharides bearing type 1 or type 2 listed in Table I flowed through the rhgalectin-4-Sepharose column (Figure 3A), except SO33LNTOT, which was weakly retarded (Figure 3B). However, 3H-core 1 pyranoside was weakly retarded (Figure 3B) and the reduced core 1, Galß1
3GalNAcOT, flowed through the column (Figure 3A), indicating that rhgalectin-4 preferentially recognizes core 1 disaccharide. Interestingly, 3'-O-sulfation of core 1 increased the binding ability to the column (Figure 3C), whereas a core 1 derivative carrying [3H]Neu5Ac
2
3Gal did not bind to the column (Figure 3A). Substitution at the reducing terminus of 35SO3
3core 1 with pNP or Bn, or substitution of the sulfated core 1 with GlcNAcß1
6GalNAc slightly decreased the affinity to the column (Figures 3D, D, and B, respectively). These results suggested that rhgalectin-4 specifically recognizes SO3
3 core 1 pyranoside.
Inhibitory effects of carbohydrates on binding of rhgalectins-3 and -4 to immobilized asialofetuin measured by surface plasmon resonance method
To determine the relative binding abilities of oligosaccharides to rhgalectins-3 and -4, the inhibitory effects of oligosaccharides on binding of rhgalectins-3 and -4 to immobilized asialofetuin were measured by the inhibition assay based on the surface plasmon resonance. The inhibitory concentration curves and the half inhibition concentrations were summarized in Figure 4 and Table II, respectively.
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The inhibitory effects of oligosaccharides on binding of rhgalectin-4 to immobilized asialofetuin were also investigated. As summarized in Figure 4B and Table II, core 1 showed stronger inhibitory ability than lactose, type 1 and type 2, in contrast to the cases of rhgalectin-3. Substitution on Gal residue of lactose with SO33, Fuc
1
2, or GalNAc
1
3(Fuc
1
2) increased the inhibitory ability. Especially, the addition of sulfate to C-3 position of the Gal residue in core 1-O-Bn increased the inhibitory ability 13.5 times in comparison with core 1-O-Bn. However, in contrast to rhgalectin-3, the addition of sialic acid to C-3 position of the Gal in lactose, 3'SL, decreased the inhibitory ability. Such a unique carbohydrate binding specificity of galectin-4 in the galectin family is the first demonstration.
Kinetic constants for the binding of oligosaccharides to rhgalectin-4 measured by surface plasmon resonance method
To measure the carbohydrate binding ability of rhgalectin-4 directly, the dissociation constants (Kds) of the binding between rhgalectin-4 and oligosaccharides were measured on the basis of rhgalectin-4-immobilized assay system using a BIAcore 2000 instrument. The typical sensorgrams are shown in Figure 5. Injection of oligosaccharides showing weak affinity for rhgalectin-4 resulted in a rapid increase in relative response, followed by a rapid decrease to base line after the injection was completed (Figure 5A and C). The equilibrium binding constants were calculated by the BIA evaluation software. As shown in Table III, lactose, type 1, LNT, type 2, LNnT, and core 1 showed rather high Kds between 2 x 104 M and 8 x 104 M. Lactitol, 3FL, 3'SL, 6'SL, and Gal1
4Galß1
4Glc did not show enough relative responses to calculate the Kd values even at 1 mM. In contrast, the substitution on the Gal residue of lactose with SO3
3, Fuc
1
2, and GalNAc
1
3(Fuc
1
2) decreased the Kd values. Because the aglycon moieties of core 1 influenced the binding abilities to rhgalectin-4 as shown in Tables I and II, we compared the Kds for core 1-O-Bn derivatives. Figure 5A, B, and C are the sensorgrams of core 1-O-Bn, SO3
3core 1-O-Bn, and SO3
3Galß1
3(Galß1
4GlcNAcß1
6) GalNAc
1-O-Bn at 25°C, respectively. SO3
3core 1-O-Bn was very slowly dissociated from rhgalectin-4 on the sensor chip after the injection was completed (Figure 5B). The Kds between SO3
3core 1-O-Bn and rhgalectin-4 were calculated as 3.4 x 106 M at 25°C and 1.2 x 106 M at 4°C. The Kds between other oligosaccharides and the immobilized rhgalectin-4 were also lower at 4°C than those at 25°C. The Kd value for 3'-sulfated core 1-O-Bn was over 94 times smaller than that for core 1-O-Bn itself. However, the Kds for SO3
3 core 1-O-Bn derivatives bearing GlcNAcß1
6 or Galß1
4GlcNAcß1
6 were close to that for core 1-O-Bn. These results indicate that rhgalectin-4 specifically recognizes SO3
3core 1 pyranoside.
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Discussion |
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On the other hand, galectin-4 requires SO33 core 1 sequence for high-affinity binding. Type 1, type 2, and their fucosylated, sialylated, sulfated, or N-acetylgalactosaminylated derivatives at C-2' or C-3' position show the relatively high Kd values (between 5 x 105 M and 8 x 104 M) and flow through rhgalectin-4-Sepharose column as shown in Table I. Although core 1 was weakly retarded to rhgalectin-4-Sepharose column, asialofetuin carrying three core 1 residues per one molecule bound to the column and was eluted with 0.1 M lactose, suggesting that the clustered core 1 in asialofetuin has higher affinity for rhgalectin-4 than core 1 oligosaccharide. The similar effects of clustered O-glycans were reported about the cancer-related monoclonal antibody recognizing the clusters of O-type glycans (Nakada et al., 1993
) and human macrophage C-type lectin recognizing the O-glycans immobilized at high density (Iida et al., 1999
).
The substitution with sulfate at C-3 position of ß-galactosyl residues greatly increased the binding to rhgalectin-4. Because the similar character was also observed in not only galectin-3 but also galectin-1 (Allen et al., 1998), the enhancement of the binding ability to galectins by 3-O-sulfation of ß-galactose residues may be a common character of galectin family.
This is the first report that described the carbohydrate-binding specificity of human galectin-4. Only the carbohydrate-binding specificities of the N- and C-terminal domains of rat galectin-4 were reported (Oda et al., 1993), and these authors reported that each of the two domains in rat galectin-4 has a distinct sugar binding specificity; the N-terminal domain has a similar affinity for lactose, A-tetra, type 1, and core 1, in contrast the C-terminal domain prefers A-tetra to lactose, type 1, type 2, and core 1. More recently, Wasano and Hirakawa (1999) showed that the two domains of rat galectin-4 bind to distinct sites at the intercellular border of colorectal epithelia by separately preparing the N- and C-terminal carbohydrate domains of galectin-4 as recombinant glutathione S-transferase-fused proteins. These results suggested that the specificities of the two domains of human galectin-4 in this study are also different from each other, so which domain has a specific binding ability to 3'-sulfated core 1 has to be clarified at the next step.
The carbohydrate binding specificities of galectins had been determined by measuring the concentrations of carbohydrates that inhibit the binding of the radiolabeled lectins to asialofetuin (or lactose)-Sepharose (Sparrow et al., 1987). However, labeling of lectins sometimes abolishes the binding ability to carbohydrates, and the modification of the reducing termini of oligosaccharides by fluorescence dye or radioactive compounds is not suitable for the study of O-type glycan-specific lectins, such as galectin-4. Furthermore, the inhibition assay system is often influenced by the glycan structures of immobilized glycoproteins and the concentrations of lectins. In this study, we directly measured the dissociation constants between various nonlabeled carbohydrates and rhgalectin-4 by using surface plasmon resonance assay. Although 12 members have been designated as galectins among large numbers of homologs in published databases (Cooper and Barondes, 1999
), studies about their precise carbohydrate binding specificities have been so far very limited. The combined analytical methods used herein will be useful to study the carbohydrate binding specificities of other galectins and galectin homologs.
SO33Galß1
3GalNAc structure in O-linked glycans that can bind to galectin-4 has been found in respiratory mucus glycoproteins from patients with cystic fibrosis (Chance and Mawhinney, 1996
). Information on the occurrence of the sulfated glycan is rather limited to date, but it is possible that SO3
3Galß1
3GalNAc-linked glycoproteins may be distributed in various tissues, because two sulfotransferases, Gal3ST-2 and Gal3ST-4, which recognize Galß1
3GalNAc in glycoproteins as good acceptor are expressed in many tissues (Honke et al., 2001
; Seko et al., 2001
).
The different tissue expression patterns of galectins-3 (Yang et al., 2001) and -4 (Oda et al., 1993
; Rechreche et al., 1997
; Gitt et al., 1998
) shown by northern blot analysis or reverse transcription polymerase chain reaction (PCR) (Guittaut et al., 2001
) have been reported. Human galectin-3 is expressed in heart, skin, kidney, lung, testis, and peripheral blood leukocyte (Guittaut et al., 2001
), whereas human galectin-4 is expressed in small intestine, colon, and rectum (Rechreche et al., 1997
). The marked distribution of galectin-4 in digestive tissues may be related to the carbohydrate binding specificity of galectin-4, and galectin-4 might be involved in the mucous defense mechanism.
It had been reported that O-glycosylation is necessary for apical delivery of human neutropin receptor in caco-2 cells (Monlauzeur et al., 1998) and for the sorting of some intestinal brush border enzymes to the apical side (Naim et al., 1999
). Because porcine intestine galectin-4 forms the clusters with the brush border enzymes and is localized in membranous structures and microvillar actin filament rootlets (Danielsen and Deurs, 1997
), galectin-4 might be participated in the intracellular transport process of the glycoproteins carrying O-type glycans.
The decrease of sulfomucin in the course of human colonic carcinogensis (Felipe, 1969; Yamori et al., 1987
) and the down-regulation of the expression levels of human galectin-4 mRNA in colorectal cancer have been reported (Rechreche et al., 1997
). It seems that SO3
3Galß1
3GalNAc
1
structure is present in sulfomucin, because Gal3ST-2, which recognizes type 1, type 2, and core 1 oligosaccharides as good acceptors, is highly expressed in human colonic normal mucosa (Honke et al., 2001
). The relation of the down-regulation between galectin-4 and sulfomucin has to be clarified in the near future. The natural ligands for galectin-4 should be also determined in relation to the functional roles in the next step.
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Materials and methods |
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Preparation of 35S-labeled and 3H-labeled oligosaccharides
The structures of radiolabeled oligosaccharides used in this study are summarized in Table I. The 3H-labeled oligosaccharide alcohols were prepared by NaB[3H]4-reduction from human milk oligosaccharides according to the methods reported previously (Takasaki and Kobata, 1978). [14C]GalNAc
1
3(Fuc
1
2)Galß1
3GlcNAcß1
3Galß1
4GlcOT (GalNAc
1
3LNF-IOT) was prepared according to the methods reported previously (Yamashita et al., 1976
). The Gal-3-O-[35S]sulfated oligosaccharides were prepared using a Gal-3-O-sulfotransferase partially purified from porcine colonic mucosa (Seko and Yamashita, unpublished data). Briefly, 20 µl of the reaction mixture consisting of 0.1 M sodium cacodylate (pH 6.3), 10 mM MnCl2, 0.1 % (v/v) Triton X-100, 0.1 M NaF, 2 mM ATP-Na2, 6.5 µM [35S]PAPS (2.8 x 105 dpm), 1 mM oligosaccharides, and the enzyme fraction appropriately diluted was incubated at 37°C for 1 h. The 35S-labeled products were purified by paper electrophoresis (pyridine/acetic acid/water = 3:1:387, pH 5.4). The positions of [35S]sulfate residues were determined by the Smith degradation method as described previously (Seko et al., 2001
). The 35SO3
3core 2-O-pNP was prepared from core2-O-pNP using human Gal 3-O-sulfotransferase as described elsewhere (Seko et al., 2001
). [3H]galactose-labeled core 1, core 1-O-pNP, and core 1-O-Bn were prepared using ß1
3galactosyltransferase activity from porcine colonic mucosa as described previously (Seko et al., 2001
). [3H]Neu5Ac
2
3core 1 was prepared using
2
3sialyltransferase from porcine colonic mucosa as follows: 20 µl of reaction mixture consisting of 0.1 M sodium cacodylateHCl buffer (pH 7.0), 10 mM MnCl2, 0.5 % (v/v) Triton CF-54, 150 µM CMP-Neu5Ac, 2 µM CMP-[3H]Neu5Ac (3.6 x 105 dpm), 10 mM core 1, and crude membrane appropriately diluted was incubated 37°C for 1 h. The 3H-labeled product was purified by paper electrophoresis. The linkage position of [3H]Neu5Ac was determined by digestion with Salmonella typhimurium LT2
2
3-sialidase (Takara Shuzo, Kyoto, Japan).
Preparation of rhgalectin-4
A cDNA that contained the entire open reading frame for galectin-4 was obtained using human T-84 colonic epithelial cDNA library by PCR performed by 30 cycles of 95°C for 0.5 min, 50°C for 1 min, and 72°C for 2 min. The primers used were 5'-gctgtcgacATGGCCTATGTCCCCGCA-3' as sense primer and 5'-cctaagcttTCTGGACATAGGACAAGG-3' as antisense primer. After the sequence of the amplified fragment was confirmed using ABI PRISM® 310 Genetic Analyzer (PE Biosystems), the fragment was inserted into the pQE-9 plasmid between the Sal I and the Hind III sites (G4-pQE-9). G4-pQE-9 was transformed with E. coli M15 strain. One and a half L of 2YT-G with 100 µg/ml ampicillin and 25 µg/ml kanamycin was inoculated with 17 ml of overnight culture of the transformants E. coli and then shaken at 30°C until the absorbance at 600 nm reaches 1.0. After addition of IPTG to the final concentration of 0.5 mM, the bacteria were cultured for 1.5 h and harvested by centrifugation at 5000 x g for 5 min. The pellet was frozen and resuspended in 40 ml of PBS-2ME (PBS containing 4 mM 2-mercaptoethanol), and the suspension was incubated on ice for 30 min with 10 mg lysozyme. After centrifugation at 12,000 x g for 25 min, the supernatant was applied to 5 ml of Ni-NTA resin equilibrated with PBS-2ME and was incubated at 4°C. The Ni-NTA resin was washed with PBS-2ME, and then rhgalectin-4 was eluted with PBS-2ME containing 0.25 M imidazole. The eluate was concentrated and dialyzed with PBS-2ME and was applied on an asialofetuin-Sepharose column (5 mg/ml, 1 x 16 cm). After washing the column with PBS-2ME, galectin-4 was eluted with 0.3 M lactose in PBS-2ME. The purified rhgalectin-4 was concentrated by filtration with an Amicon PM10 diaflo membrane, and was frozen at 20°C until use. Protein concentration was determined by a Bio-Rad Protein Assay dye reagent using bovine serum albumin as a standard.
Preparation of rhgalectin-3
A cDNA that contains the entire open reading frame for galectin-3 was obtained using human T-84 colonic epithelial cDNA library by PCR, which was performed by 30 cycles of 95°C for 1 min, 47°C for 1 min, and 70°C for 2 min. The primers used were 5'-agccatATGGCAGACAATTTTTCG-3' as sense primer and 5'-tttggatccTTATATCATGGTATATGA-3' as antisense primer. After the sequence of the amplified fragment was confirmed using ABI PRISM 310 Genetic Analyzer, the fragment was inserted into the plasmid pET3a between the NdeI and the BamHI sites (G3-pET3a). G3-pET3a was transformed with E. coli strain BL21(DE3). The transformants were inoculated and shaken at 37°C overnight in Luria broth (LB) containing 100 µg/ml of ampicillin. One and a half L of LB with 100 µg/ml of ampicillin was inoculated with the overnight culture of the galectin-3-expressing E. coli. When the absorbance at 600 nm reaches 0.9, IPTG was added to the final concentration of 0.5 mM and allowed to stand for 2.5 h, the bacteria were harvested by centrifugation at 5000 x g for 5 min. The pellet was resuspended in 40 ml of PBS-2ME and after centrifugation at 12,000 x g for 25 min, the supernatant was applied on an asialofetuin-Sepharose column(5 mg/ml, 1 x 16 cm). After washing the column with PBS-2ME, rhgalectin-3 was eluted with 0.3 M lactose in PBS-2ME. The purified rhgalectin-3 was concentrated by filtration with an Amicon PM10 diaflo membrane and was frozen at 20°C until use.
Preparation of rhgalectin-immobilized Sepharose 4B
rhGalectin-3 or -4-immobilized Sepharose 4B was prepared according to the manufacturers instructions. The amounts bound to 1 ml Sepharose resin were 2.8 mg for rhgalectin-4 and 2.5 mg for rhgalectin-3.
Fractionation of glycoproteins and oligosaccharides on rhgalectins-immobilized Sepharose 4B columns
Glycoproteins (100 µg each) and 3H- or 35S-labeled oligosaccharides were applied on rhgalectins-immobilized Sepharose 4B columns (0.7 x 2.6 cm), which were equilibrated with PBS-2ME at 4°C. After standing for 15 min, each column was washed with five column volumes of PBS-2ME at 4°C, and the bound glycoprotein or the oligosaccharide was eluted with PBS-2ME containing 0.1 M lactose at room temperature.
ß-Elimination of glycoproteins
Glycoproteins were dissolved in 0.2 N NaOH and were incubated at 45°C for 4 h to release O-linked glycans. The samples were neutralized with 2 N HCl and were dialyzed against PBS.
Inhibition assay based on surface plasmon resonance
The inhibition effects of various carbohydrates on the binding of rhgalectins-3 and -4 to immobilized asialofetuin were measured using a BIAcore 2000 instrument (BIAcore, Uppsala, Sweden). Asialofetuin was covalently immobilized on the CM5 sensor surface by amine coupling at pH 4 according to the manufacturers instructions. The amount of immobilized asialofetuin was 10,000 RU. Thirty µg/ml of rhgalectin-3 and various concentrations of carbohydrates were mixed in HBS-EP buffer (0.01 M HEPES-NaOH [pH 7.4], 0.15 M NaCl, 3 mM ethylenediamine tetra-acetic acid, 0.005% [v/v] polysorbate 20) and introduced onto the asialofetuin-immobilized surface at a flow rate of 20 µl/min at 25°C. In the case of rhgalectin-4, the concentration introduced was 2.5 µg/ml. For removing the bound rhgalectins on the surface, 0.1 M lactose in HBS-EP buffer was used. The interaction was monitored as the change in the surface plasmon resonance response.
Estimation of kinetic constants based on surface plasmon resonance
The dissociation constants between rhgalectin-4 and carbohydrates were measured using rhgalectin-4-immobilized sensor in a BIAcore 2000 instrument. The purified rhgalectin-4 was immobilized on the CM5 sensor surface at pH 6 according to the manufacturers instructions. The amount of rhgalectin-4 immobilized by amine coupling was approximately 10,000 RU. Various carbohydrates in HBS-EP buffer were introduced onto the surface at a flow rate of 20 µl/min. The interaction was monitored at 25°C or 4°C as the change in the surface plasmon resonance response. For removing the bound carbohydrates on the surface, 0.1 M lactose in HBS-EP buffer was used. The dissociation constants were calculated using BIA evaluation 3.0 software.
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Abbreviations |
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Footnotes |
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References |
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