Glycobiology Advance Access originally published online on July 16, 2008
Glycobiology 2008 18(10):789-798; doi:10.1093/glycob/cwn068
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Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central 2, 1-1-1 Umezono, Ibaraki 305-8568, Japan
1 To whom correspondence should be addressed: Tel: +81-29-861-3124; Fax: +81-29-861-3125; e-mail: jun-hirabayashi{at}aist.go.jp
Received on April 29, 2008; revised on June 10, 2008; accepted on July 8, 2008
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Abstract |
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The extensive involvement of glycan-binding proteins (GBPs) as regulators in diverse biological phenomena provides a fundamental reason to investigate their glycan-binding specificities. Here, we developed a glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of GBPs. Eighty-nine selected multivalent glycoconjugates comprising natural glycoproteins, neo-glycoproteins, and polyacrylamide (PAA)-conjugated glycan epitopes were immobilized on an epoxy-activated glass slide. The GBP binding was monitored by an evanescent-field fluorescence-assisted scanner at equilibrium without washing steps. The detection principle also allows direct application of unpurified GBPs with the aid of specific antibodies. Model experiments using plant lectins (RCA120, ConA, and SNA), galectins (3 and 8), a C-type lectin (DC-SIGN) and a siglec (CD22) provided data consistent with previous work within 4 h using less than 40 ng of GBPs per analysis. As an application, serum profiling of antiglycan antibodies (IgG and IgM) was performed with Cy3-labeled secondary antibodies. Moreover, novel carbohydrate-binding ability was demonstrated for a human IL-18 binding protein. Thus, the developed glycan array is useful for investigation of various types of GBPs, with the added advantage of wash-free analysis.
Key words: evanescent / glycoconjugate / lectin / microarray / specificity
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Glycan-binding proteins (GBPs), or lectins, exist as diverse families of proteins that bind specifically to glycans (Sharon and Goldstein 1998
; Sharon and Lis 2004). Their ubiquitous occurrence in the biosphere has been established in viruses, bacteria, fungi, plants, and animals including humans, while their endogenous functions largely remain to be elucidated. However, there is accumulated evidence that they play regulatory roles in diverse biological processes, such as microbe infection, immune response, cell differentiation, and tumor-cell metastasis through specific binding to their glycan ligands (Robinson et al. 2006; Crocker et al. 2007; van Vliet et al. 2007). Lectins of known specificity have served as useful tools in biochemical research fields such as in cell typing, tissue staining, enrichment and purification of glycoproteins, western blotting, flow cytometry, and glycan profiling (Hirabayashi 2004; Wu et al. 2008). A lectin microarray provides a comprehensive approach to profiling diverse glycan structures displayed at the cell surface and in glycoconjugates (Kuno et al. 2005; Pilobello et al. 2005; Hsu et al. 2006; Tateno, Uchiyama, et al. 2007). The different specificities of lectins for complex glycans render them valuable tools for recognizing a particular type of glycan (epitope), and hence, fuel the search for yet more novel lectins (Goldstein 2002).
From an historical viewpoint, most of the known lectins have been identified through agglutination and binding assays using model cells (e.g., erythrocytes) and glycoproteins (e.g., asialofetuin), respectively (Boyd and Shapleigh 1954; Sharon and Lis 2004). In addition, there is a variety of approaches to the determination of detailed sugar-binding specificity: these include enzyme-linked immunosorbent assay (ELISA) (Blixt et al. 2003; Wu et al. 2008), equilibrium dialysis (Mega and Hase 1991), surface plasmon resonance (Shinohara et al. 1994), isothermal titration calorimetry (Dam and Brewer 2004), frontal affinity chromatography (FAC) (Tateno, Nakamura-Tsuruta, et al. 2007), and glycan microarray (Fukui et al. 2002; Feizi et al. 2003; Blixt et al. 2004; Manimala et al. 2006). Among the analytical systems developed, the glycan microarray may be the most promising approach for high-throughput investigation of GBPs and has now been established as an essential tool for functional glycomics. The glycan arrays developed in the Consortium for Functional Glycomics (CFG) (Blixt et al. 2004) and the UK Glycoarray Consortium (Fukui et al. 2002) have been providing enormous information on the glycan-binding properties of various types of GBPs. The current CFG glycan array (version 3.2) has now 406 of distinct glycan compounds, including a series of N- and O-linked glycans and glycolipid-type glycans. The library size of both of the glycan arrays is still expanding. However, there is still a room to improve the glycan array technology such as detection methods.
An evanescent-field fluorescence-assisted detection principle is one of the most sensitive detection methods, which was successfully applied for detection of sugar–protein interactions in the lectin array (Kuno et al. 2005). The system enables detection of interactions between glycans and immobilized lectins without any washing step. Thus, even weak interactions between sugars and lectins can be detected (Uchiyama et al. 2008). However, there has been no report on the application of the detection system for the glycan microarray.
Here we developed a glycoconjugate microarray with the evanescent-field fluorescence-assisted detection principle. To enhance the avidity of GBPs, glycoproteins and glycopolymers displaying highly multivalent glycan ligands were immobilized on epoxy-activated glass slides. The resulting glycoconjugate array is capable of detection of carbohydrate-binding activities of diverse families of lectins and glycan-binding antibodies, even in crude samples. Thus, the detection system was proved to be versatile for investigation of sugar–protein interactions, in either formats of lectin microarray or glycan microarray.
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Results |
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Strategy for specificity profiling of lectins
In general, lectins show much lower affinity for monovalent oligosaccharides than do antibodies, with Kd values in the µM to mM range, whereas it is well known that they show much higher binding avidity for multivalent glycan ligands by "glycoside cluster effect" (Lee and Lee 2000
; Gestwicki et al. 2002; Collins and Paulson 2004; Dam et al. 2007). Such monovalent interactions, if any, would not withstand the extensive washing steps adopted in general microarray techniques. Therefore, we adopted a strategy to immobilize multivalent rather than monovalent glycans on microarray glass slides: natural glycoproteins, neo-glycoproteins, and glycan epitope-conjugated polyacrylamide (PAA) polymers (Figure 1). In order to detect even weak interactions that should be eliminated by extensive washing steps, we took advantage of an evanescent-field fluorescence-assisted scanner, which was originally developed for the purpose of lectin microarray (Kuno et al. 2005).
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The use of epoxy-activated glass slides allows covalent attachment of (neo)glycoproteins containing primary amines. As an extension of this approach, commercially available glycosides attached to the PAA backbone of 30 kDa, which have often been used as high-affinity probes for lectins (Bovin 1998
; Collins et al. 2006; Tateno, Li, et al. 2007), were found to be efficiently immobilized on epoxy-activated glass slides under the same conditions (see Materials and methods). Varying concentrations of BSA and PAA conjugates of 
-L-Fuc (0–500 µg/mL, Fuc-BSA and Fuc-PAA, respectively) were spotted on glass slides, and ligand coupling efficiency was measured by binding to a Cy3-labeled Aleuria aurantia lectin (AAL). As shown in Figure 2, binding of AAL was observed on the spots of both Fuc-BSA and Fuc-PAA with similar intensities in a concentration-dependent manner, whereas no signal was observed for control BSA and PAA. Uniform spot morphology and a high S/N ratio were obtained. The CV (%) of the signal intensity of the positive spots was low (2.2), demonstrating the reliability and reproducibility of the array. In this study, 61 glycoside-PAA conjugates containing representative terminal glycan structures (epitopes) of glycoproteins and glycolipids were spotted at a concentration of 0.1 mg/mL, whereas 28 (neo)glycoproteins expressing glycan structures of representative N- and O-glycans were spotted at 0.5 mg/mL. All samples were printed in triplicate in each 14-well glass slide (Figure 3 and supplementary Table I).
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Validation study using established plant lectins
Sensitivity is one of the most important factors for developing a glycan array system, since the quantities of natural samples are usually limited. To validate the sensitivity of the developed glycoconjugate array, we first analyzed three representative plant lectins: a Gal-binding lectin (RCA120), a Man-binding lectin (Con A), and an
2-6Sia-binding lectin (SNA). Varying amounts of the Cy3-labeled plant lectins (0–40 ng/well, 0–1000 ng/mL) were applied to the array and incubated at 20°C for 3 h, and binding was directly detected by the evanescent-field fluorescence-assisted scanner without washing steps. As shown in Figure 4A, concentration-dependent signals were obtained, where 4–12 ng of lectins per well (100–300 ng/mL, 40 µL) gave signals (i.e., >10,000 net intensity) strong enough for analysis. The three plant lectins exhibited distinct binding profiles on the array as described in detail below.
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RCA120, an R-type lectin isolated from the seeds of the common castor bean (Ricinus communis), exhibited binding to glycoproteins containing galactosylated N-glycans (23, 24, 36–39), but not to their agalactosylated forms (42–44) (Figure 4B, top). Among synthetic glycans tested, strong binding was observed for β-D-Gal (25), but not for its anomer,
-D-Gal (69), demonstrating that the protein is a βGal-specific lectin. Linkage specificity of RCA120 was also demonstrated on this array. It showed preference for type 2 (Galβ1-4GlcNAc, 31) over type 1 (Galβ1-3GlcNAc, 29) structures. Binding to type 2 LacNAc was almost abolished by modification of βGal either by 1-2Fuc (03), 2-3Sia (17), 1-3Gal (71), or 1-4Gal (72) as well as that of βGlcNAc by 1-3Fuc (09), these results being consistent with a previous finding (Itakura et al. 2007). Interestingly, binding to type 2 LacNAc was strongly enhanced by the addition of sulfate at 6-OH of Gal (33), but not to its position isomer (LacNAc with sulfate at 3-OH of GlcNAc, 32), demonstrating a novel effect of sulfate at 6-OH of Gal on RCA120 affinity enhancement.
Con A, a legume lectin isolated from jack bean (Canavalia ensiformis), showed strong binding to glycoproteins containing high mannose-type N-glycans, such as thyroglobulin (24, 39), ovalbumin (OVA, 46), yeast invertase (INV, 50), and yeast mannan (84, 85) (Figure 4B, middle). The lectin also showed substantial binding to human transferrin (23, 38, 44) having multiple biantennary N-glycans, but to a lesser extent to fetuin (21, 36, 42) having such glycans as minor components. Among the synthetic ligands tested, the lectin showed selective binding to -D-Man (47), and to a lesser extent to -D-Glc (74, 76). All these features are consistent with those of a previous report (Ohyama et al. 1985).
SNA, another R-type lectin isolated from elderberry (Sambucus nigra L.), bound exclusively to glycoproteins containing sialylated N-glycans (21–24) and O-glycans (67, 68), but not to their desialylated forms (36–39, 61, 62) (Figure 4B, bottom). Among the synthetic glycosides, SNA bound to 2-6sialyllactose (20), but not to its linkage isomer, 2-3sialyllactose (16), indicating that the lectin recognizes 2-6Sia-containing glycans, consistent with a previous finding (Shibuya et al. 1987).
Application to purified animal lectins: galectin-3C and 8N
In order to extend applicability of the system for specificity profiling, we analyzed a few members of the galectin family, a major group of animal lectins with β-galactoside specificity and an evolutionally conserved carbohydrate-recognition domain (CRD). As shown in Figure 5, overall binding profiles of the C-terminal domain of galectin-3 (Gal-3C) and the N-terminal domain of galectin-8 (Gal-8N) are different despite the fact that they share basic specificity for β-galactoside. Detailed interpretations are as follows.
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Gal-3 is a chimera-type galectin composed of an N-terminal collagenase-sensitive domain and a C-terminal CRD. Using 4 ng of Gal-3C, a characteristic specificity profile was obtained (Figure 5, top). As described previously (Hirabayashi et al. 2002
; Stowell et al. 2008), Gal-3C exhibited strong affinity to blood group A (5) as well as blood group B antigens (6, 71). Gal-3C also displayed significant binding to both type 1 (Galβ1-3GlcNAc, 29) and core 1 (Galβ1-3GalNAc, 52) structures as well as to their 3'-O-sulfated forms (30 and 59, respectively), while binding to core 2 structure (Galβ1-3[GlcNAcβ1-6]GalNAc, 53) was somewhat reduced. Binding to these O-glycans was confirmed for asialoglycophorin (Asialo-GP, 62). In contrast, Gal-3C showed much weaker avidity to glycoproteins with galactosylated N-glycans that were recognized by RCA120 at the same concentration (4 ng/well) (36–39). These results suggest that Gal-3 might preferentially recognize mucin-type O-glycans rather than branched N-glycans.
Gal-8 is a tandem-repeat-type galectin consisting of N-terminal and C-terminal CRDs. A specificity profile of Gal-8N was obtained using 12 ng (300 ng/mL) of the recombinant protein (Figure 5, bottom). Gal-8N displayed particular preference for LacNAc substituted with Sia (15–17) or sulfate (30) at the 3-OH of Gal, although binding to neither type 1 (29) nor type 2 (31) LacNAc was detectable under this condition. Indeed, Gal-8N bound to 2-3Sia-containing glycoproteins such as fetuin (21) and 1-acid glycoprotein (AGP, 22), but not to their desialylated forms (36, 37). Gal-8N also exhibited binding to O-glycans such as sialyl T (Sia2-3Galβ1-3GalNAc, 65) and 3'-O-sulfo T (3-O-sulfate-Galβ1-3GalNAc, 59), but not detectably to T antigen (Galβ1-3GalNAc, 52), demonstrating the strong effect of substitutions with Sia or sulfate at the 3-OH of Gal on affinity enhancement (Hirabayashi et al. 2002).
Application to crude extracts of recombinant lectins: DC-SIGN and CD22
The above results confirmed that the developed system worked satisfactorily for elucidation of sugar-binding specificities of several established lectins. However, the main purpose of this study is to develop a rapid profiling system of extensive GBPs, and hence its direct applicability to crude samples is a critical factor in determining whether or not the method is of high utility. For this purpose, we carried out two-step experiments by using DC-SIGN-Fc and CD22-Fc fusion proteins, which represent C-type lectins and siglecs, respectively. Firstly, varying concentrations of these fusion proteins were premixed with Cy3-labeled goat antihuman IgG and incubated on the array at 20°C for 3 h, and their binding was directly detected by the scanner without washing steps. As shown in Figure 6A, concentration-dependent binding signals were obtained, where 4–12 ng of the lectins per well (100–300 ng/mL) gave stable signals, comparable to the case using Cy3-labeled lectins directly. Thus, indirect labeling via the fluorescent-labeled anti-Fc secondary antibody could be readily achieved. However, when the DC-SIGN-Fc and CD22-Fc were Cy3-labeled and analyzed by the array, much weaker binding signals could be obtained, indicating that antibody precomplexing also helps to enhance the binding signals of lectins with low affinity to glycans.
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The method was then examined for its applicability to unpurified lectins. Expression vectors encoding DC-SIGN-Fc and CD22-Fc were transfected into CHO cells and HEK293T cells, respectively. The resulting cell culture supernatant was pre-incubated with the above detection probe (Cy3-labeled goat antihuman IgG) and applied to the array, and the binding was detected by the evanescent-field-type scanner without washing. As shown in Figure 6B, the control culture supernatant gave no detectable signals, whereas the culture supernatant (CS) of cells expressing either DC-SIGN-Fc or CD22-Fc gave stable profiles, which were similar to those obtained for their purified forms (Figure 6A). This result clearly indicates that glycan-binding activities of the recombinant lectins expressed in CS could be easily and precisely analyzed without the need for purification. Detailed explanations of sugar-binding specificities of the two recombinant lectins are as follows.
DC-SIGN, a member of the type 2 receptor subgroup of the C-type lectin family, is a dendritic cell-specific immune receptor implicated in viral and pathogen infections and cancer cell immune tolerance. As shown in Figure 6C (top), DC-SIGN-Fc displayed strong binding to both fucosylated (1, 3, 7–10) and mannose-containing glycans (47, 48, 50, 84, 85), consistent with previous reports (Cambi et al. 2003; Guo et al. 2004). CD22, a member of the siglec family, is a negative regulator of B cell signaling and binds selectively to 2-6Sia (Powell and Varki 1994; Blixt et al. 2003). As expected, CD22-Fc exhibited binding to synthetic 2-6sialyllactose (20) as well as to 2-6Sia-containing glycoproteins (22–24), but not to their desialylated forms (37–39) (Figure 6C, bottom). CD22-Fc also bound to sialyl-Tn (Sia2-6GalNAc, 63, 64) only at a high concentration (40 ng/well, 1 µg/mL) (Figure 6A). The binding of CD22 to sialyl-Tn was previously reported by Brinkman-Van der Linden et al. (Brinkman-Van der Linden and Varki 2000).
Challenge for investigating novel glycan-binding activities of GBPs
Having demonstrated that the system is valid for rapid and sensitive profiling of established groups of lectins, we finally investigated the glycan-binding activities of unidentified groups of GBPs, such as cytokine-binding proteins and antibodies. Among cytokine-binding proteins, poxvirus IL-18 binding proteins (IL-18BPs), which bind IL-18 and inhibit IL-18-mediated immune responses, were reported to have glycosaminoglycan (GAG)-binding activity (Xiang and Moss 2003; Esteban et al. 2004). However, there is no report on the glycan-binding activity of its human homolog, human IL-18BP. As shown in Figure 7 (top panel), human IL-18BP exhibited binding activity toward a series of GAGs (78–82), similar to that of poxvirus IL-18BPs. Interestingly, it also showed clear preference for a sulfated disaccharide, i.e., LacNAc with sulfate at the 6-OH of Gal (33), but not for its position isomer (LacNAc with sulfate at 3-OH of GlcNAc, 32) nor nonsulfated LacNAc (31). To our surprise, human IL-18BP also bound to -L-rhamnose (-L-Rha, 83) that is often seen in the outermost cell surface components of bacteria, but not in eukaryotes. Although the actual ligand(s) of human IL-18BP has not been identified, the present finding implies that human IL-18BP has sugar-binding function, which is related to glycosaminoglycans or bacterial polysaccharides.
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The final attempt was to examine whether or not the developed microarray system was useful for rapid glycan-epitope profiling of human serum immunoglobulin. Human type H serum was diluted at 1000 times with a probing buffer and applied to the array. In order to detect IgM and IgG classes independently, the array was probed with either Cy3-labeled goat antihuman IgM or goat antihuman IgG antibodies, respectively. As shown in Figure 7 (middle and bottom panels), IgG and IgM gave distinct profiles from each other. Antiglycan IgM were found to be generated mostly against glycan epitopes representing microorganisms, such as β-D-Man (48), yeast invertase (50), β-D-Glc (75),
-L-Rha (83), and mannan from S. cerevisiae (84) and C. albicans (85) except for A and B blood group antigens (5, 6) and Gal1-3Gal, a xenotransplantation epitope (70, 71). In contrast, antiglycan IgG exhibited more selective binding to particular structures, such as Tn (-D-GalNAc, 51), melibiose (Gal1-6Glc, 73), and mannan from C. albicans (85) but not that from S. cerevisiae (84). Binding to the A and B blood group antigens (5, 6) and yeast invertase (50) is common to both antiglycan IgM and IgG. |
Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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In conventional hemagglutination assay, the approximate sugar-binding specificity of lectins is determined by hapten inhibition using a panel of free saccharides. Although the hemagglutination assay is the most common and convenient method for the detection of lectin activity, it has limitations: (i) it is applicable only for lectins with affinity to abundant glycans expressed at the cell surface, (ii) it is applicable only for multivalent lectins to cross-link glycans expressed at the surface of different cells, and (iii) relatively large amounts of lectins and saccharides are required, which are often not available (Wu et al. 2008
). The assay has proved useful for the detection of conventional soluble lectins, represented by plant lectins and galectins, but not for membrane proteins, such as siglecs and type 2 C-type lectins. Alternatively, binding ability toward appropriate cells and glycoconjugates has been assessed for glycan-binding activity (Crocker and Gordon 1989; Powell and Varki 1994). For the need of throughput and sensitivity, a glycan microarray was recently introduced into glycomics research. After the development of a glycan array, the studies of GBPs were significantly accelerated. In this report, we have described a glycoconugate microarray based on the evanescent-field fluorescence-assisted detection principle for the purpose of GBP assessment and discovery. The main novelty of the developed system over previously described glycan arrays is the use of evanescent-field fluorescence-assisted detection, which allows "wash-free analysis," by simply applying fluorescence-labeled lectins, even in crude samples, to the array without washing steps. Such merit would further increase the throughput of glycan array analysis.
Admittedly, the present system has several limitations: (i) the current version of the array has only a limited number of structurally defined glycans. For example, among the lectins analyzed so far, siglec-8 gave no detectable signals since its preferred glycan ligand, 6'-sulfo-sialyl Lewis x (Neu5Ac2-3[6-SO4]Galβ1-4[Fuc1-3]GlcNAc), is not employed on the present array (Bochner et al. 2005). (ii) The method is semiquantitative, and therefore, is not suitable for determining accurate and reliable binding constants (Ka or Kd). For more quantitative interaction analysis, frontal affinity chromatography (FAC) may be a better measuring system for determination of accurate binding constants of lectins. For this purpose, more than a hundred structure-defined oligosaccharides are commercially available (Tateno et al. 2007).
Approximately a hundred of lectins have been reported so far, while a much larger number of potential lectins have been nominated in the human genome (Drickamer 1999). Moreover, it is likely that there exist a significant number of unidentified GBPs belonging to other protein families that have never been nominated. In this respect, the developed system is likely to be involved in a discovery phase of novel lectins and novel specificities using extensive cDNA and protein libraries. Indeed, we could find novel glycan-binding activities for IL-18BP in this study. It might also have potential for diagnostic and therapeutic applications, as the specificities of antiglycan antibodies using a series of human sera can be analyzed in a rapid and systematic manner.
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Materials and methods |
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Materials
A panel of glycoside-polyacryamide (PAA) conjugates synthesized by coupling spacered oligosaccharides and spacered biotin to poly(4-nitrophenyl acrylate) were purchased from Glycotech (MD) (Bovin et al. 1993
). Fetuin from fetal bovine serum, 1-acid glycoprotein from bovine serum, transferrin from human serum, porcine thyroglobulin, mucin from bovine submaxillary glands, glycophorin from human blood type MN and its desialylated form, ovoalbumin from chicken egg white, ovomucoid from chicken egg white, invertase from S. cerevisiae, and mannan from S. cerevisiae were purchased from Sigma (MO). Mannan from C. albicans was purchased from Takara (Shiga, Japan). Hyaluronic acid, chondroitin sulfate (CSA), dermatan sulfate (CSB), heparin sulfate (HS), keratin sulfate (KS), RCA120, and ConA were purchased from Seikagaku Co. (Tokyo, Japan). Sambucus nigra agglutinin (SNA) was obtained from Vector Laboratories (CA). Goat antihuman IgG (Fc
-specific) and goat antihuman IgM (Fc5µ-specific) were purchased from Jackson ImmunoResearch Laboratories, Inc. (PA). The recombinant human IL-18 binding protein was purchased from R&D Systems (MN). BSA-glycosaminoglycan conjugates (GAG-BSA) were prepared using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as previously described (Nishioka et al. 2007). Asialo-glycoproteins were prepared by incubating in 0.1 N HCl at 80°C for 1 h. Agalactosylated glycoproteins were prepared by Streptococcus 6646K β-galactosidase (Seikagaku) treatment.
Preparation of CD22-Fc and DC-SIGN-Fc chimera
The human CD22-Fc chimeric construct (CD22-Fc) was generated by cloning the N-terminal three Ig domains (1-324 amino acids) into the EK-Fc/pcDNA3.1(–) vector (Angata et al. 2002). The DC-SIGN-Fc fusion construct (DC-SIGN-Fc) was generated by cloning the carbohydrate-recognition domain (CRD, 251–404 amino acids) into the pSecTag/FRT/V5-His vector containing the Fc portion of human IgG1. The CD22-Fc and DC-SIGN-Fc constructs were transfected into HEK293T cells cultured in OPTI-MEM/2.5% fetal calf serum (FCS) and CHO cells in F12/10% FCS media, respectively. Culture supernatants were recovered and CD22-Fc and DC-SIGN-Fc fusion proteins were then purified by Protein A Sepharose. Culture supernatants were also directly analyzed by the glycoconjugate microarray.
Preparation of human galectins
Expression plasmids for the C-terminal domain of Galectin-3 (Gal-3C) and the N-terminal domain of Galectin-8 (Gal-8N) were constructed using pET-27b (Hirabayashi et al. 2002). The plasmids were cloned into Escherichia coli (E. coli) BL21-CodonPlus (DE3)-RIL strain (Stratagene, CA). The recombinant proteins were produced by the addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG) and purified by lactosyl-Sepharose.
Fabrication of the glycoconjugate microarray
Glycoproteins and glycoside-PAA conjugates were dissolved in a spotting solution (Matsunami Glass Ind., Ltd, Osaka, Japan) at a final concentration of 0.5 and 0.1 mg/mL (25 µL for 12 glass slides, 168 wells), respectively. After filtration using 0.22 µm poresize filter to remove insoluble particles, they were spotted on a microarray-grade epoxy-coated glass slide (Schott AG, Mainz, Germany) attached with a silicone rubber sheet with 14 chambers, using a noncontact microarray printing robot (MicroSys 4000; Genomic Solutions Inc., MI) with a spot diameter size of 220 µm spaced at a 260 µm interval. The glass slide was incubated in a humidity-controlled incubator at 25°C for 3 h to allow immobilization. After incubation, excess amounts of nonimmobilized materials were washed out with the probing buffer (25 mM Tris–HCl, pH 7.4 containing 0.8% NaCl, 1% (v/v) Triton-X, 1 mM MnCl2, 1 mM CaCl2) and blocked with 100 µL of TBS (25 mM Tris–HCl, pH 7.4 containing 0.8% NaCl) containing 1% BSA at 20°C for 1 h.
Binding assay
Cy3-labeled GBPs or GBPs precomplexed with Cy3-labeled antibodies in the probing buffer were applied to each chamber of the glass slides (40 µL/well) and were incubated at 20°C for 3 h, after which fluorescent images were immediately acquired using an evanescent-field activated fluorescence scanner, SC-Profiler (Moritex, Tokyo) under Cy3 mode without washing steps. Throughout experiments, scanning conditions of the cooled charge-coupled device (CCD) camera, i.e., resolution (5 µm), number of times for integration (4), and exposure time (200 msec) were fixed. Data were analyzed with the Array Pro analyzer Ver. 4.5 (Media Cybernetics, Inc., MD). Net intensity value for each spot was determined by signal intensity minus background value. Data are the average ± S.D. of triplicate determinations.
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Supplementary Data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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New Energy and Industrial Technology Development Organization (NEDO), Japan.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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The authors thank Ms. Yoshiko Kubo and Jinko Murakami for help in the preparation of the array and Dr. James C. Paulson for providing full-length cDNA of human CD22.
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Abbreviations |
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CFG, Consortium for Functional Glycomics; CS, culture supernatant; FAC, frontal affinity chromatography; FCS, fetal calf serum; GBP, glycan-binding protein; PAA, polyacrylamide
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References |
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