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

Abstract

Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses.

Fig. 1.

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

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.

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.

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.

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

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