With the development of microarray technologies, glycan microarrays come to predominant for the high throughput study of protein–glycan binding events and screening glycan-binding proteins (GBPs). Glycan microarrays are presentations of multiple glycans or glycoconjugates printed on a single slide for screening with GBPs, which include lectins, antibodies, bacteria, and viruses. Glycans are usually derivatized with functional groups and immobilized onto the activated glass slides at similar concentrations for each glycan. The utility of a glycan microarray is directly related to the number and variety of the glycans available on the printed surface for interrogation by GBPs.
The rationale for developing a glycan array is to interrogate it with GBPs or potential GBPs and quantify the specific fluorescent signals that indicate the glycan structures to which the GBP in question binds. By comparing relative binding of the GBP to related glycan structures, it is possible to define a glycan motif or motifs that the GBP recognizes with higher binding compared to other related structures.
Figure 1. The general strategy for preparing covalent glycan microarrays
There is a general strategy to construct glycan microarrays. Free glycans obtained from natural sources or chemically synthesized to contain reactive amine space groups at the reducing termini, are used for covalent printing. The immobilized glycans are deposited on slides in a spatially discrete pattern and stored in desiccated conditions. The slides may then be hydrated and interrogated or incubated with compounds or cells that may bind glycans, including purified natural or recombinant GBPs, antibodies, toxins, serum, cells, viruses, and bacteria. Binding is detected by direct or indirect fluorescence approaches, which provide an image of fluorescent spots that may be identified to coincide with deposited glycans. The data intensity of fluorescence is measured and graphed versus glycan number on the microarray.
Figure 2. Schematic diagram of the various applications of glycan microarrays
Glycan microarray has become a sensitive and versatile approach for the analysis of glycosylation changes in complex biological samples. It can not only investigate glycan-GBPs binding but also provide kinetic analysis of glycan–protein interactions. Nowadays, this application has rapidly expanded and now achieves successful discrimination of selective interactions between glycans and, not only proteins, but also viruses, bacteria and eukaryotic cells, and most recently even live cell responses to immobilized glycans. Glycan microarray can be used to discover functional glycans, characterize glycan-processing enzymes, and detect pathogens for diagnosis. It is also suitable for monitoring multiple molecular interactions with biomolecules where glycans are involved, offering a large variety of bioassay options.
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