The principle and applications of glycan microarrays
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Glycan microarrays first described in 2002 provide a high-throughput means of profiling the interactions of glycan-binding proteins with their ligands. This method can be used to identify specific binding of glycans by lectins, antibodies, toxins, viruses, etc. For more information:https://www.creative-proteomics.com/services/glycan-related-microarray-assay.htm
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The principle and applications of glycan microarrays
- 1. Creative Proteomics Presentation Glycan Microarrays
- 2. The compounds that consist of a large number of monosaccharides linked glycosidically. Introduction of Glycan Biological functions Structural and modulatory properties of glycans Specific recognition of glycans by other molecules Glycan-binding proteins (GBPs) Intrinsic GBPs Extrinsic GBPs Varki A, Lowe J B. Biological roles of glycans. 2009.
- 3. Introduction Glycan-binding proteins (GBPs) Intrinsic GBPs recognize glycans from the same organism and mediate cell-cell interactions or recognize extracellular molecules. Extrinsic GBPs recognize glycans from different organisms and also mediate symbiotic relationships. pathogenic microbial adhesins agglutinins toxins Varki A, Lowe J B. Biological roles of glycans. 2009.
- 4. The interaction between glycans and proteins is accomplished through the multivalent and intensifying affinity between them, and this force is relatively weak Have a high degree of structural specificity Since synthesis of glycans is not guided by the template, it is formed under the action of various forms of enzymes Characteristics Complexity of glycan Glycan microarray • First described in 2002 • A high-throughput means of profiling the interactions of glycan-binding proteins with their ligands. • Identify specific binding of glycans by lectins, antibodies, toxins, viruses, etc.
- 5. The Principle Cummings R D, Pierce J M. The challenge and promise of glycomics. Chemistry & biology, 2014, 21(1): 1-15. Individual glycans from natural sources or chemical or enzymatic syntheses are modified to allow themselves to covalently or non-covalently be attached to a solid surface. The glycan microarray can be interrogated with GBPs or other reagents or even cells and viruses. They can be visualized by either direct or indirect fluorescent tagging.
- 6. Carbohydrate microarrays are incubated with fluorophore-labeled GBPs, fluorophore-labeled secondary reagents that interact with the GBPs, or a tag (like biotin or His tag) conjugated to the GBPs. A high-resolution fluorescence microarray scanner is applied to quantify the fluorescence intensity of the bound proteins. Surface plasmon resonance (SPR) imaging and mass spectrometry (MS) techniques are employed as label- free detection methods in glycan microarrays. Applications Rapid profiling of glycan binding properties of proteins, including plant and animal lectins, antibodies, and growth factors. Hyun J Y, Pai J, Shin I. The Glycan Microarray Story from Construction to Applications. Accounts of chemical research, 2017, 50(4): 1069-1078.
- 7. Many pathogenic bacteria display the pathogenic properties, which are caused by the binding of pathogens to host cell surface glycans through specific glycan−protein interactions. The researchers incubated a microarray containing glycans with the Escherichia coli strain, which was stained with the fluorescent dye. The microspots containing mannose epitopes exhibited fluorescence because of the mannose-binding protein on pili expressed by the strain. Applications Examine glycan binding properties of whole pathogens and to detect pathogens as part of diagnoses. Hyun J Y, Pai J, Shin I. The Glycan Microarray Story from Construction to Applications. Accounts of chemical research, 2017, 50(4): 1069-1078.
- 8. Researchers applied glycan microarrays to determine concentrations (IC50) of soluble inhibitors to inhibit 50% of protein binding to glycans. A series of pre-incubated mixtures of the fluorophore- labeled lectin and an inhibitor were applied to glycan microarrays. The IC50 values of the inhibitors are then determined by measuring the fluorescence intensities of bound proteins that remain on the microarrays after washing. Applications Determine the quantitative binding affinities of proteins to glycans. Hyun J Y, Pai J, Shin I. The Glycan Microarray Story from Construction to Applications. Accounts of chemical research, 2017, 50(4): 1069-1078.
- 9. Carbohydrate microarrays are constructed by attaching unmodified glycans to the hydrazide-coated surface. Mammalian cells expressing lectins were pretreated with a fluorescent probe, PF1. Microarrays containing glycans were incubated with these mammalian cells. The functions were identified by measuring the fluorescence intensity of each spot. The results show that cells that adhere to glycans on the microarrays generate ROS, as reflected in an increase in fluorescence signals induced by the ROS probe. Applications For rapid screening of functional glycans that elicit cellular responses by interacting with cell-surface lectins. PFI can selectively detect reactive oxygen species (ROS). The binding of glycan ligands to lectins on cell surfaces can trigger a cellular response that results in the generation of ROS. Therefore, the resulting fluorescence signal emanating from PF1 is directly related to glycan binding to cell-surface lectins. Hyun J Y, Pai J, Shin I. The Glycan Microarray Story from Construction to Applications. Accounts of chemical research, 2017, 50(4): 1069-1078.
Notas del editor
- Hello, welcome to watch Creative Proteomics’Video. Today, we are going to learn some basic knowledge about Glycan Microarrays
- Glycans are defined as the compounds that consist of a large number of monosaccharides linked glycosidically. Glycans have diverse biological functions and it can be classified into two broad categories. One is the structural and modulatory properties of glycans and the other is the specific recognition of glycans by other molecules, most commonly, glycan-binding proteins (GBPs). The GBPs can be divided into two types, intrinsic GBPs and extrinsic GBPs.
- The intrinsic GBPs recognize glycans from the same organism and mediate cell-cell interactions or recognize extracellular molecules. The extrinsic GBPs which contain pathogenic microbial adhesins, agglutinins, or toxins, recognize glycans from different organisms and some also mediate symbiotic relationships.
- Due to the complexity of glycan molecules, glycomics has its own characteristics. First, glycans have a high degree of structural specificity. Second, since the synthesis of glycans is not guided by the template, it is formed under the action of various forms of enzymes, and even in the same glycosylation site of the same kind of molecule, the structure of the carbohydrate chain is also different. In addition, the interaction between glycans and proteins is accomplished through the multivalent and intensifying affinity between them, and this force is relatively weak. These characteristics place very high demands on analytical techniques. Therefore, in order to carry out research on the structure and function of glycans, researchers have established a new research method - glycan microarray. Glycan microarrays was first described in 2002 provide a high-throughput means of profiling the interactions of glycan-binding proteins with their ligands. This method can be used to identify specific binding of glycans by lectins, antibodies, toxins, viruses, etc.
- In glycan microarrays, individual glycans which are from natural sources or chemo or enzymatic syntheses are modified to allow themselves covalently or non-covalently to attach to a solid surface. The glycan microarray can be interrogated with GBPs or other reagents or even cells and viruses to identify those glycan ‘‘spots’’ that are recognized, which can be visualized by either direct or indirect fluorescent tagging. As shown in the Figure 2, the results indicate that the GBPs bound strongly to one glycan, less strongly to another glycan, and did not bind appreciably to any other glycan. Such microarrays can also be prepared from glycolipids, glycopeptides, whole glycoproteins, or polysaccharides.
- Rapid profiling of glycan binding properties of proteins, including plant and animal lectins, antibodies, and growth factors. To achieve the goals, carbohydrate microarrays are incubated with fluorophore-labeled GBPs, fluorophore-labeled secondary reagents that interact with the GBPs, or a tag (like biotin or His tag) conjugated to the GBPs. And then a high-resolution fluorescence microarray scanner is applied to quantify the fluorescence intensity of the bound proteins. In addition, surface plasmon resonance (SPR) imaging and mass spectrometry (MS) techniques are employed as label-free detection methods in glycan microarrays.
- Examine glycan binding properties of whole pathogens and to detect pathogens as part of diagnoses. Many pathogenic bacteria display the pathogenic properties are caused by the binding of pathogens to host cell surface glycans through specific glycan−protein interactions. The researchers incubated a microarray containing glycans with the Escherichia coli strain, which was stained with the fluorescent dye. The microspots containing mannose epitopes exhibited fluorescence because of the mannose-binding protein on pili expressed by the strain. In order to make this approach become widespread, experimental conditions employed for this purpose should be optimized.
- Determine the quantitative binding affinities of proteins to glycans. Researchers applied glycan microarrays to determine concentrations (IC50) of soluble inhibitors to inhibit 50% of protein binding to glycans. In this assay, a series of pre-incubated mixtures of the fluorophore-labeled lectin and an inhibitor are applied to glycan microarrays. The IC50 values of the inhibitors are then determined by measuring the fluorescence intensities of bound proteins that remain on the microarrays after washing.
- employ the microarray technology for rapid screening of functional glycans that elicit cellular responses by interacting with cell-surface lectins. Carbohydrate microarrays are constructed by attaching unmodified glycans to the hydrazide-coated surface. Mammalian cells expressing lectins are pretreated with a fluorescent probe, PF1. PFI can selectively detect reactive oxygen species (ROS). The binding of glycan ligands to lectins on cell surfaces can trigger a cellular response that results in the generation of ROS. Therefore, the resulting fluorescence signal emanating from PF1 is directly related to glycan binding to cell-surface lectins. And the microarrays containing glycans are incubated with these mammalian cells. The functions are identified by measuring the fluorescence intensity of each spot. The results show that cells that adhere to glycans on the microarrays generate ROS, as reflected in an increase in fluorescence signals induced by the ROS probe.
- As one of the leading companies in the omics field with over years of experience in omics study, Creative Proteomics provides offers a variety types of glycan-related microarray assays, including Lectin Microarray Assay, Glyco-gene Microarray Assay, Glycan Microarray Assay, Microbial Glycan Microarray Assay, Glycopeptide Microarray Assay.
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