Glycobiology Advance Access originally published online on August 12, 2008
Glycobiology 2008 18(11):882-890; doi:10.1093/glycob/cwn077
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Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6
2 Department of Biological Chemistry, School of Pharmaceutical Science, Teikyo University, 1091-1 Suarashi, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan
3 Lectin Application and Analysis Team, Research Center for Medical Glycoscience, AIST, Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan
1 To whom correspondence should be addressed: Tel: +81-42-685-3741; Fax: +81-42-685-3742; e-mail: t-take{at}pharm.teikyo-u.ac.jp
Received on July 9, 2008; revised on August 4, 2008; accepted on August 5, 2008
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Abstract |
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We report a detailed structural analysis of the N-glycans of Caenorhabditis elegans recognized by C. elegans galectin LEC-6. Glycoproteins of C. elegans captured by an immobilized LEC-6 affinity adsorbent were isolated. The N-glycans of these glycoproteins were then liberated by hydrazinolysis and labeled with the fluorophore 2-aminopyridine (PA). The derived pyridylaminated (PA)-sugars were further fractionated by rechromatography on immobilized LEC-6 adsorbent and by reversed-phase high-performance liquid chromatography (HPLC). The structures of the PA-sugars thus obtained were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We confirmed that all PA-sugars having affinity for LEC-6 contain a Gal-Fuc disaccharide unit, and that this unit is bound to the innermost GlcNAc residue of the N-glycan chain. The dissociation constants of LEC-6 for these glycans were measured by frontal affinity chromatography. LEC-6 exhibited higher affinity for oligosaccharides having a Gal-Fuc unit linked to position 6 of the innermost GlcNAc residue than for those having Galβ1-4GlcNAc units. Affinity for the former disappeared, however, following treatment with β-galactosidase. If the glycan contained a Hex-Fuc disaccharide linked to the penultimate GlcNAc residue, the affinity would be diminished. We propose, therefore, that the galectins of C. elegans utilize the Gal-Fuc disaccharide unit for recognition instead of the Gal-GlcNAc unit that is common in vertebrates.
Key words: Caenorhabditis elegans / core chitobiose modifications / frontal affinity chromatography / galectin / N-glycan
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Introduction |
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The nematode Caenorhabditis elegans is considered to be a good target animal to study the functions of glycans and their receptor lectins at the whole body level. In recent years, notable advances have been made in the study of the structure and function of C. elegans glycans (Cipollo et al. 2005
; Hanneman et al. 2006; Shi et al. 2006; Gutternigg et al. 2007; Paschinger et al. 2008); these have been found to be somewhat different from those of vertebrates. In contrast, research on C. elegans lectins, decipherers of the glycocodes carried by these glycans, has proceeded comparatively slowly.
Galectins are a family of animal lectins that have conserved carbohydrate recognition domains (CRDs) and recognize β-galactosides (Kasai and Hirabayashi 1996). The galectins mediate cell–cell and cell–extracellular matrix interactions and are involved in a variety of phenomena such as signal transduction, cell differentiation, immunity, and tumorigenesis. In the C. elegans genome, at least 11 putative galectin genes have been identified. The products of two of these genes, LEC-1 and LEC-6, have been purified from the worm body by using affinity chromatography on immobilized asialofetuin (Hirabayashi, Satoh, Kasai 1992; Hirabayashi, Satoh, Ohyama, et al. 1992; Hirabayashi et al. 1996). LEC-6 is one of the best-characterized lectins in C. elegans (Ahmed et al. 2002; Hirabayashi, Hayama, et al. 2002; Kaji et al. 2003, 2007). The sugar-binding specificity of LEC-6 has previously been analyzed in detail by frontal affinity chromatography (FAC) by using fluorescence-labeled sugars (PA-sugars) and found to be similar to that of vertebrate galectins: LEC-6 recognizes sugars containing Galβ1-4GlcNAc (lactosamine) units (Hirabayashi, Hashidate, et al. 2002). The sugars used by Hirabayashi et al. are mainly of vertebrate origin. To date, however, there have been no reports that have confirmed the existence of glycans containing lactosamine units in C. elegans. This difference between C. elegans and vertebrates with respect to the glycan structure urged us to isolate and characterize the C. elegans glycans that interact with LEC-6.
In this study, we isolated the N-glycans of C. elegans recognized by LEC-6. Their structures were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase treatment. We identified a series of N-glycans whose chitobiose cores are extensively modified and found that all of these contained a Gal-Fuc disaccharide unit linked to position 6 of the innermost GlcNAc residue. The dissociation constants of the LEC-6 for these glycans were measured by FAC; these measurements revealed that LEC-6 exhibits higher affinity for oligosaccharides having a Gal-Fuc unit than for those having Galβ1-4GlcNAc units. Affinity for the former disappeared, however, following treatment with β-galactosidase that leads to the loss of one galactose residue. In addition, a Hex-Fuc disaccharide attached to the penultimate GlcNAc residue was found to reduce affinity. On the basis of these results, we propose that endogenous ligands of LEC-6 should have a Gal residue linked to Fuc. C. elegans appears to utilize the Gal-Fuc disaccharide unit instead of the Gal-GlcNAc unit for sugar recognition.
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Results |
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Isolation of C. elegans N-glycans recognized by LEC-6
Since a variety of N-linked glycoproteins have been found to bind to the immobilized LEC-6 adsorbent (Hirabayashi, Hayama, et al. 2002
; Kaji et al. 2003, 2007), we focused on the endogenous ligands composed of N-glycans. In order to analyze the structures of the C. elegans N-glycans recognized by LEC-6, we isolated glycoproteins bound to immobilized LEC-6. An extract from mixed-stage C. elegans was applied to an immobilized LEC-6 column. Following extensive washing of the column, bound materials were eluted with lactose. Each fraction was analyzed in its protein content and was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Figure 1). From the isolated glycoproteins having affinity for LEC-6 (Figure 1B, compare fraction 31 with 34), N-glycans (fractions 34–36) were liberated by hydrazinolysis and labeled with the fluorescent reagent 2-aminopyridine. After the removal of excess 2-aminopyridine, derived PA-sugars were used for subsequent analyses.
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Since the glycoproteins obtained might have contained multiple glycan chains, the prepared PA-sugars were subjected to a further affinity separation by using an immobilized LEC-6 column (Figure 2A). The PA-sugars were separated into the following three fractions: pass-through fraction (fraction T), delayed fraction (fraction D), and adsorbed-eluted fraction (fraction E). The PA-sugars in each fraction were subsequently subjected to MALDI-TOF MS analysis (Figure 2B and Table I). The MS spectrum of each fraction is clearly different. It should be noted, however, that in fractions D and E, we found no PA-sugar having more than two HexNAc residues; that is, all of these sugars possessed only two HexNAc residues. This observation is consistent with our previous results (Hirabayashi, Hayama, et al. 2002
) and suggests that the endogenous glycans recognized by LEC-6 lack the Galβ1-4GlcNAc unit, although they are expected to have at least one β-galactoside residue.
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LEC-6 recognizes a β-galactoside residue of C. elegans N-glycans
In order to confirm the above assumption, we examined the binding ability of LEC-6 for β-galactosidase-treated glycans. First, we further separated LEC-6-binding glycans by reversed-phase high-performance liquid chromatography (HPLC) (Figure 3). Both fractions D and E were separated into four major peaks: D1–D4 for fraction D and E1–E4 for fraction E. These peaks were subjected to further purification by normal-phase HPLC (data not shown). We successfully obtained approximately 300 pmol of each glycan. Next, these glycans were treated with β-galactosidase from Streptococcus 6646K, and the reaction products were isolated by reversed-phase HPLC. The glycans thus prepared were also subjected to FAC analysis by using an immobilized LEC-6 column having relatively weak affinity (Figure 4A). Figure 4A shows the elution profiles of each PA-sugar: in these profiles, the extent of retardation of the elution front is proportional to the binding constants of each PA-sugar. As control PA-sugars, we used NA2, NA3, and NA4, containing two, three, and four Galβ1-4GlcNAc (lactosamine) units, respectively. As a result, retardations of the elution fronts of all of the LEC-6-binding glycans were observed, whereas with those treated with β-galactosidase, no retardation was observed (Figure 4A). This suggests that LEC-6-binding glycans should contain at least one β-galactoside residue. The glycans of fraction D were retarded slightly, whereas those of fraction E were retarded markedly, indicating that the affinity of the glycans of fraction D is weaker than that of the glycans of fraction E (Figure 4B and Table II). The Ka values of LEC-6 for E1–E4 were higher than that for NA4, which contains four lactosamine units. The Ka values for D1–D4 were similar to that for NA2.
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Structural analysis of LEC-6-binding N-glycans
In order to examine the structures of LEC-6-binding glycans, each of the purified glycans was subjected to MALDI-TOF MS analysis (Table III). Treatment of these glycans with β-galactosidase resulted in the loss of 162 Da—equal to the mass of a galactose residue. Since this treatment resulted in the disappearance of the affinity for LEC-6, it is assumed that one galactose residue is essential for the binding.
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Next, the structures of the glycans were further analyzed by MALDI-TOF MS/MS (Figure 5 for D3 and E3, and data not shown for the others; see Figure 8 for the deduced structures). A fragment ion corresponding to Fuc2Hex1HexNAc1PA (m/z 774) was observed for D3 and E3, but not for those treated with β-galactosidase. Instead, a fragment ion corresponding to Fuc2HexNAc1PA (m/z 612) was observed. These results indicate that the innermost GlcNAc residue of D3 and E3 was modified with one galactose and two fucose residues, and that the galactose residue is responsible for the interaction with LEC-6. In the MS/MS spectra of D3 and D3 + Gal, a fragment ion corresponding to Fuc1Hex4HexNAc1 (m/z 1019) was observed, implying that the penultimate GlcNAc residues of these sugars are modified with a Hex-Fuc disaccharide unit. On the basis of these observations, we deduced their structures (Figure 8). All of the reducing end GlcNAc residues were modified with a Gal-Fuc unit and the galactose residue was found to be essential for recognition by LEC-6. A comparison of the deduced structures of fraction D with those of fraction E revealed a clear difference: the penultimate GlcNAc residues of fraction D glycans were modified with a Hex-Fuc disaccharide unit, whereas those of fraction E were not. It is considered that the Hex-Fuc disaccharide linked to the penultimate GlcNAc residue may have a suppressive effect on the recognition of glycans due to steric hindrance.
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In order to confirm the linked position of the Gal-Fuc disaccharide, D3, D4, E3, and E4 were treated with
-fucosidase (data not shown). However, no reaction products were obtained. Therefore, each of these fractions was initially treated with β-galactosidase and then subsequently treated with -fucosidase (Figure 6, and data not shown for D3 and D4). After the removal of the galactose residue, treatment with -fucosidase successfully removed the fucose residues. Since -fucosidase from bovine kidney preferentially cleaves 1-6-linked fucose residues (Takahashi et al. 1999), the Gal-Fuc disaccharides are suggested to link to position 6 of the innermost GlcNAc residues. It was found, however, that D3 and D4 were rather resistant to -fucosidase digestion even after β-galactosidase treatment: only small amounts of product were obtained. Their compositions were also confirmed by MALDI-TOF MS. The Hex-Fuc disaccharide linked to the penultimate GlcNAc residue probably interferes with the enzyme due to steric hindrance. The products of glycosidase treatment were further subjected to MALDI-TOF MS/MS analysis (Figure 7). Fragment ions corresponding to Hex4HexNAc1 (m/z 873) for E3 and methylatedFuc1Hex4HexNAc1 (m/z 1033) for E4 were observed. Therefore, we concluded that a Gal-Fuc disaccharide is linked to position 6 of the innermost GlcNAc residue.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterial and methodsConflict of interest statementReferences |
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The ability of galectins to bind to the lactosamine unit appears to have been conserved throughout evolution (Hirabayashi, Hashidate, et al. 2002
). However, although C. elegans galectin LEC-6 also retains affinity for this unit, to date, few reports have appeared that indicate the existence of this unit in the glycans of C. elegans except that of Cipollo et al. (2005) who found some fragments consistent with the lactosamine unit by MS/MS analysis. In the present study, we isolated a series of N-glycans from C. elegans, which serve as endogenous ligands of LEC-6 (Figure 8). Structural analysis and FAC analysis of these glycans revealed that the galactose residue attached to the fucose residue linked to position 6 of the innermost N-acetylglucosamine is responsible for the interaction with LEC-6, and that the Hex-Fuc disaccharide unit linked to the penultimate N-acetylglucosamine interferes with this interaction. Oligosaccharides having only a single Gal-Fuc unit exhibited significantly strong affinity for LEC-6, while those having multiple Gal-GlcNAc units exhibited relatively weak affinity. On the basis of these results, we propose that C. elegans utilizes the Gal-Fuc disaccharide unit instead of the Gal-GlcNAc unit for sugar recognition and subsequent cellular regulation.
C. elegans N-glycans have been classified into six groups: oligomannosidic, truncated complex, phosphorylcholine-rich, paucimannosidic, fucose-rich, and core chitobiose modifications (CCMs) as reviewed by Paschinger et al. (2008). According to this classification, LEC-6-binding glycans are classified as CCMs. The CCM-type glycans of C. elegans were originally identified by Hanneman et al. (2006), and similar structures have been found in octopus, squid, and keyhole limpet (Zhang et al. 1997; Takahashi et al. 2003; Wuhrer et al. 2004). All of these glycans contain a Galβ1-4Fuc disaccharide unit attached to position 6 of the innermost GlcNAc residue. Although we have not yet confirmed the linkage between Gal and Fuc residues owing to sample limitation, we predict that this will also be a β1-4 linkage.
N-Glycans containing the Gal-Fuc disaccharide unit have been found in nematodes, squids, and limpets, all of which belong to the Protostomia. These observations raise the question as to whether recognition of the Gal-Fuc disaccharide unit by galectins is a common feature in the Protostomia. Drosophila melanogaster, one of the other well-studied model organisms belonging to the Protostomia, has been shown to have N-glycans containing the lactosamine unit, but not the Gal-Fuc disaccharide unit (Aoki et al. 2007). However, unlike the N-glycans of vertebrates, those of D. melanogaster were revealed to contain only one lactosamine unit. At least six putative galectin genes have been found in the D. melanogaster genome (Vasta et al. 2004), one of which, Dmgal (Drosophila melanogaster galectin) has been demonstrated to bind to β-lactosyl-Sepharose (Pace et al. 2002). Characterization of the endogenous ligands of Dmgal is of particular interest because it may reveal the presence of the Gal-Fuc unit as the counterparts of Drosophila galectins. The search for galectins in squid and limpet is also of considerable interest.
Several enzymes required for the biosynthesis of CCM-type N-glycans have been identified (Zhang et al. 2003; Paschinger et al. 2004, 2005, 2006; Gutternigg et al. 2007; Nguyen et al. 2007). One of these, UDP-N-acetylglucosamine:-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I), an enzyme essential for the biosynthesis of paucimannose, complex, and CCM-type N-glycans, is of particular interest. In C. elegans, there are three GnT I genes homologous to mammalian GnT I (Chen et al. 1999). Triple null mutant worms with respect to these genes have been obtained and analyzed for their phenotypes. However, they were found to exhibit no distinguishing phenotype under laboratory conditions, except for some altered susceptibilities to bacterial pathogens (Zhu et al. 2004; Shi et al. 2006). In the present study, we demonstrated that LEC-6 recognizes the Gal-Fuc disaccharide; however, its in vivo function is still poorly understood. Although genome-wide analysis of gene functions (i.e., knockdown of lec-6 mRNA) has been carried out, no phenotype has been observed (Gonczy et al. 2000; Kamath et al. 2003; Rual et al. 2004; Fernandez et al. 2005; Sonnichsen et al. 2005). Investigation of the behavior of LEC-6 in the worms with triple null mutant for GnT I genes, and also examination of other mutants lacking genes associated with the recognition or biosynthesis of N-glycans, may be helpful in clarifying the in vivo function of LEC-6.
Using a proteomic approach, it has been demonstrated that approximately 500 C. elegans proteins were adsorbed to an immobilized LEC-6 column (Hirabayashi, Hayama, et al. 2002; Kaji et al. 2003, 2007). One of these, epi-1, which encodes the laminin chain, is of particular interest since the role of galectins in the adhesion of cells to laminin has been demonstrated in vertebrates (Friedrichs et al. 2007). These observations imply that C. elegans LEC-6 play a similar role in epithelial cell adhesion; the interaction between LEC-6 and CCM-type N-glycans may be involved in this phenomenon.
This is the first study to demonstrate that the Gal-Fuc disaccharide unit is recognized by a nematode galectin. Homology modeling of LEC-6 and vertebrate galectins has revealed that LEC-6 has a shorter loop between strands 4 and 5 (Ahmed et al. 2002; Vasta et al. 2004). This loop might facilitate recognition of the Gal-Fuc disaccharide. X-ray analysis of the crystals of recombinant LEC-6 protein binding the Gal-Fuc disaccharide is essential for future investigation of this interaction.
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Material and methods |
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Preparation of immobilized LEC-6 adsorbent
Recombinant LEC-6 was expressed as a fusion protein with β-galactosidase
-peptide (Hirabayashi et al. 1996). In order to construct the recombinant plasmid pET-LEC-6, the cDNA coding for -peptide and the ORF of lec-6 was inserted into the NdeI and BamHI sites of a pET21a vector (Novagen, Darmstadt, Germany). Escherichia coli strain BL21 (DE3) transformed with pET-LEC-6 was cultured overnight at 37°C in a LBA medium (Luria–Bertani medium supplemented with 50 µg/mL of ampicillin), transferred to a 25-fold volume of the LBA medium, and then incubated at 37°C for 3 h. Following chilling in ice-cold water, isopropyl-1-thio-β-D-galactopyranoside was added to the cells to a final concentration of 0.1 mM and the cells were further incubated at 20°C for 18 h. The cells were then harvested and suspended in phosphate-buffered saline-ethylenediaminetetraacetic acid (PBS-EDTA; 8.1 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.68 mM KCl, pH 7.4, supplemented with 2 mM EDTA), and then lysed by sonication. Following centrifugation of the lysate, the recombinant LEC-6 contained in the supernatant was adsorbed on an asialofetuin-Sepharose column prepared as described previously (Arata et al. 1997). The adsorbed protein was eluted with PBS-Lac (PBS containing 0.1 M lactose). The buffer of the eluate was changed to a coupling buffer (0.2 M Na2HCO3, 0.5 M NaCl, 0.1 M lactose, pH 8.3) by ultrafiltration using Amicon® Ultra-15 filter devices (Millipore, Billerica, MA). The resulting solution was then concentrated to an appropriate volume for immobilization. The immobilization of LEC-6 on NHS-activated Sepharose (GE Healthcare) was performed essentially according to the manufacturer's instructions. The immobilized LEC-6 adsorbent thus prepared was packed into a disposable column (BIO-RAD, Hercules, CA). Immobilized LEC-6 columns for HPLC and FAC were prepared by immobilizing recombinant LEC-6 on HiTrap NHS-activated Sepharose according to the manufacturer's instructions, with the exception that we used the above-described coupling buffer. For FAC analysis, the LEC-6 Sepharose thus prepared was packed into a stainless steel column (0.4 x 1.0 cm; GL Sciences, Tokyo, Japan).
Isolation of glycoproteins bound by LEC-6
Mixed stages of C. elegans strain N2 were prepared as described previously (Hirabayashi, Hayama, et al. 2002). The worms (25 g, wet weight) were suspended in ice-cold TBS (20 mM Tris–HCl, pH 7.5, 150 mM NaCl), and then disrupted by sonication. The mixture was centrifuged, and the resulting supernatant was applied to an immobilized LEC-6 column (bed volume, 6 mL; 8 mg LEC-6 protein/mL gel). After extensive washing of the column with TBS, the adsorbed glycoproteins were eluted with TBS containing 0.1 M lactose. These procedures were performed at 4°C. The fraction volume was set to 5 mL throughout the experiment. The protein concentration of each fraction was determined using a BIO-RAD Protein Assay with BSA as a standard. Fractions containing LEC-6-binding glycoproteins were subjected to ultrafiltration using Amicon® Ultra-15 filter devices in order to remove lactose. The resulting solution was subjected to ethanol precipitation, and the precipitate was dried for subsequent use.
Preparation of pyridylaminated-sugars (PA-sugars)
Hydrazinolysis, N-acetylation, and pyridylamination were performed at Masuda Chemical Industries (Kagawa, Japan). In brief, hydrazinolysis was performed for 4 mg of glycoprotein bound to LEC-6. The liberated N-glycans were N-acetylated and pyridylaminated. Excess 2-aminopyridine was removed by phenol–chloroform extraction, chloroform extraction, and gel filtration essentially as described previously (Hirabayashi, Hayama, et al. 2002).
Second affinity separation and high-performance liquid chromatography (HPLC) of PA-sugars
Affinity fractionation was performed on an immobilized LEC-6 column (bed volume, 1 mL; 6 mg LEC-6 protein/mL gel) equilibrated with 10 mM ammonium acetate, pH 6.0, at a flow rate of 0.25 mL/min at 4°C. The adsorbed PA-sugars were eluted with 2 mL of 10 mM ammonium acetate, pH 6.0, containing 20 mM lactose. Reversed-phase HPLC was performed on a Palpak type R column (0.46 x 25 cm; Takara Biomedicals, Shiga, Japan) equilibrated with 0.1 M ammonium acetate, pH 4.0, at a flow rate of 1.0 mL/min at 30°C. Normal-phase HPLC was performed on a Palpak type N column (0.46 x 25 cm; Takara Biomedicals) equilibrated with 22.5 mM ammonium acetate, pH 7.0, containing 55% acetonitrile, at a flow rate of 1.0 mL/min at 30°C. The elution of PA-sugars was detected using a fluorescence spectrophotometer at an excitation wavelength of 310 nm and an emission wavelength of 380 nm. The N-glycan fractions were collected, lyophilized, and then analyzed by MALDI-TOF/MS. Prior to MALDI-TOF/MS analysis, fraction E (adsorbed and eluted fraction) was subjected to gel filtration on an Asahipak GS-310 column (0.78 x 25 cm; Asahi Chemicals, Tokyo, Japan) equilibrated with 10 mM ammonium acetate, pH 6.0, for the removal of lactose.
Glycosidase treatment
In order to eliminate galactose residues, we used β-galactosidase from Streptococcus 6646K (Seikagaku Kogyo, Tokyo, Japan) as described previously (Arata et al. 2006). PA-sugars (20–50 pmol) were digested with 1 mU of β-galactosidase in 25 µL of 40 mM ammonium acetate, pH 5.5, containing 0.02% bovine serum albumin, for 24–48 h at 37°C. In order to eliminate fucose residues, 50 pmol of PA-sugar was digested with 100 mU of -L-fucosidase from bovine kidney (Sigma, St. Louis, MO) in 20 µL of 50 mM ammonium acetate, pH 5.5, for 48 h at 37°C. The reaction products of glycosidase treatment were isolated by reversed-phase HPLC, and then subjected to further analysis.
Frontal affinity chromatography (FAC) analysis
FAC was performed as described previously (Arata et al. 2001; Hirabayashi, Hashidate, et al. 2002). In brief, 2.0 mL of a PA-sugar in a buffer comprising 20 mM Na2HPO4, pH 7.2, 150 mM NaCl, and 1 mM EDTA, at a concentration of 5 nM was applied to an immobilized LEC-6 column (1.35 mg LEC-6 protein/mL gel) prepared as described above, at a flow rate of 0.25 mL/min, at 20°C. The elution of PA-sugars was detected using a fluorescence spectrophotometer at an excitation wavelength of 310 nm and an emission wavelength of 380 nm. The Kd values for the interaction between LEC-6 and each of the PA-sugars were determined according to a basic equation of FAC: Kd = Bt/(Vf – V0) – [A]0. In this equation, Bt is the effective ligand content (expressed in mol), Vf is the volume of the elution front of each PA-sugar (expressed in mL), V0 is the Vf of PA-rhamnose that is not bound by LEC-6, and [A]0 is the initial concentration of the PA-sugar. If [A]0 is negligibly small compared with Kd, the equation described above can be simplified to the equation Kd = Bt/(Vf – V0). The Bt value of an immobilized LEC-6 column was calculated by concentration-dependence analysis using lacto-N-fucopentaose I (LNFP-I) (Dextra Laboratories, Reading, UK) and PA-LNFP-I. The Ka values were calculated according to the equation Ka = 1/Kd. The PA-sugars (NA2, Galβ1-4GlcNAcβ1-2Man1-3(Galβ1-4GlcNAcβ1-2Man1-6) Manβ1-4GlcNAcβ1-4GlcAc-PA; NA3,Galβ1-4 GlcNAcβ1-2(G alβ1-4GlcNAcβ1-4)Man1-3(Galβ1-4GlcNAcβ1-2Man1-6) Manβ1-4GlcNAcβ1-4GlcNAc-PA; NA4,Galβ1-4GlcNAcβ1-2 (Galβ1-4GlcNAcβ1-4)Man1-3(Galβ1-4 GlcNAcβ1-2(Galβ1- 4GlcNAcβ1-6)Man1-6)Manβ1-4GlcNAcβ1-4GlcNAc-PA) and PA-rhamnose were purchased from Takara Biomedicals.
MALDI-TOF MS analysis
The MS and MS/MS spectra of PA-sugars were acquired with an UltraflexTM TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) as described previously (Kamekawa et al. 2006), with the exception that we used a matrix solution comprising 10 mg/mL 2,5-dihydroxybenzoic acid, 10 mM sodium acetate, and 20% EtOH in order to accelerate the generation of [M + Na]+ions.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterial and methodsConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We are grateful to Dr. Hideki Matsuzaki for his assistance with MS experiments. We also thank Drs. Yoichiro Arata and Yoko Nemoto-sasaki for helpful discussions.
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Abbreviations |
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CCMs, core chitobiose modifications; CRDs, carbohydrate recognition domains; HPLC, high-performance liquid chro- matography; FAC, frontal affinity chromatography; MALDI- TOF MS, matrix-assisted laser desorption/ionization time-of- flight mass spectrometry; PA-sugars, pyridylaminated-sugars; PBS-EDTA, phosphate-buffered saline-ethylenediaminetetr- aacetic acid; SDS–PAGE, sodium dodecyl sulfate–polyacry- lamide gel electrophoresis
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References |
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