Printed covalent glycan array for ligand profiling of diverse glycan binding proteins

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   Fig. 1.

   Covalent printing of a diverse glycan library onto an amino-reactive glass surface and image analysis by using standard microarray technology.

   
   
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   Fig. 2.

   Representative glycan structures on the array. Glycan structures detected by GBPs in this article are shown in the symbol nomenclature adopted by the Consortium for Functional Glycomics (www.functionalglycomics.org). A full list of glycans can be found in Fig. 7.

   
   
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   Fig. 3.

   Printing optimization and specificity of selected plant lectins. (A) Optimization of glycan concentration and length of printing time were determined by printing mannose structures and detected by Con A. A representative mannose glycan (136) was printed at various concentrations (4–500μM) in replicates of eight at six different time points. (B) Binding specificities of Con A-FITC and ECA-FITC on the complete array.m

   
   
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   Fig. 4.

   Mammalian GBP specificity. C-type lectin (DC-SIGN): DC-SIGN-Fc chimera (30 μg/ml) detected by secondary goat anti-human-IgG-Alexa 488 antibody (10 μg/ml) bound selectively to α1–2- and/or α1–3/4-fucosylated glycans and to Manα1–2 glycans. Siglec (CD22): CD22-Fc chimera (10 μg/ml) precomplexed with secondary goat anti-human-IgG-Alexa 488 (5 μg/ml) and tertiary rabbit anti-goat-IgG-FITC (2.5 μg/ml) antibodies bound exclusively to Neu5Acα2–6Gal-glycans. Galectin (galectin-4): Human galectin-4-Alexa 488 (10 μg/ml) evaluated with glycans printed at 100 and 10 μM bound preferentially to blood group glycans.m

   
   
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   Fig. 5.

   Anticarbohydrate antibody specificity. (Top) Mouse anti-CD15-FITC mAb (BD Biosciences clone HI98, 100 tests) bound exclusively to Lewis^X glycans. (Middle) 2G12 mAb (30 μg/ml) precomplexed with goat anti-human-IgG-FITC (15 μg/ml) bound to specific Manα1–2 glycans, including the Man8 and Man9 N-glycans. (Bottom) Human serum of 10 healthy individuals (1:25 dilution) was individually bound to glycan arrays and detected by subsequent overlay with monoclonal mouse anti-human-IgG-IgM-IgA-biotin antibody (10 μg/ml) and streptavidin-FITC (10 μg/ml). Results represent the mean and standard deviation for binding in all 10 experiments. Anticarbohydrate antibodies detecting various blood group antigens as well as mannans and bacterial fragments were found.^m

   
   
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   Fig. 6.

   Bacterial and viral GBP specificity. (Top) CVN (30 μg/ml) detected with secondary polyclonal rabbit anti-CVN (10 μg/ml) and tertiary anti-rabbit-IgG-FITC (10 μg/ml) bound various α1–2 mannosides. (Middle) Pure recombinant hemagglutinin (150 μg/ml) derived from duck/Ukraine/1/63 (H3/N7), precomplexed with mouse anti-HisTag-IgG-Alexa 488 (75 μg/ml) and anti-mouse-IgG-Alexa 488 (35 μg/ml), bound exclusively to Neu5Acα2–3Gal-terminating glycans. (Bottom) Intact influenza virus A/Puerto Rico/8/34 (H1N1) was applied at 100 μg/ml in the presence of 10 μM of the neuraminidase inhibitor oseltamivir carboxylate. The virus bound a wide spectrum of sialosides with both NeuAcα2–3Gal and NeuAcα2–6Gal sequences.^m