Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions

Abstract

We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are obtained from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis(x) sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis(x) sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-gamma (IFN-gamma) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis(x), and sulfo-Lewis(a), in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.

Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides

“…Binding Assays of Soluble Dectin-1 to Arrays of Glucan-derived NGL Probes-Unless otherwise stated, NGLs (1 l of 50 pmol/l solutions of each, in chloroform/methanol/water (25:25:8 by volume)) were arrayed by jet spray as 2-mm bands onto nitrocellulose membranes or nitrocellulose-coated FAST TM glass slides (Schleicher & Schuell), and the binding of the soluble Dectin-1 was assayed essentially as described (21,27). In brief, nonspecific binding sites on the slides were blocked for 1 h with Pierce Blocker solution; the binding of Fc-Dectin-1 (1 g/ml in Pierce Blocker solution) was detected after 2 h of incubation, using biotinylated anti-IgG (10 g/ml), followed by streptavidin-conjugated horseradish peroxidase (10 g/ml) and color development with FAST-3, 3Ј-diaminobenzidine peroxidase substrate (Sigma).…”

“…not recognized by Dectin-1. Among them are oligosaccharide probes recognized by other mammalian carbohydrate-binding proteins (21,31,42). Here, the ability to generate tailor-made oligosaccharide probes from targeted sources, fungal and bacterial polysaccharides, in conjunction with MS and methylation analysis has been crucial for assigning oligosaccharide ligands for Dectin-1.…”

“…Here we investigate the oligosaccharide ligands for Dectin-1 using the neoglycolipid (NGL)-based oligosaccharide microarray technology (21,22), a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. Our approach is to first select ligand-positive and -negative ␤-glucans, to partially fragment these and chemically link the oligosaccharide fragments to a lipid; the NGL probes thus generated are arrayed and used for Dectin-1 binding studies in conjunction with mass spectrometry (MS) and methylation analysis for chain length and sugar linkage assignments.…”

Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen

“…Thus, the microarray provides a more appropriate and more sensitive model system for studying cellular events than traditional solution-phase ELISA analysis. Successful glycoarray systems have followed advancements in carbohydrate synthesis and have implemented both covalent (32)(33)(34)(35)(36)(37)(38)(39)(40)(41) and noncovalent (42)(43)(44)(45)(46) strategies for sugar immobilization.…”

Chemokine-Glycosaminoglycan Binding

“…The characterization of chemokine-GAG interactions has been greatly facilitated by the development of reasonably efficient screening and detection techniques (22)(23)(24)(25)(26). However, there are still many limitations, and the resulting data have been somewhat nebulous.…”