Glycan Microarray Service

What is Glycan Microarray?

Glycan microarrays, also known as carbohydrate microarrays, are innovative platforms for studying the interactions between glycans and various biomolecules, including proteins, antibodies, lectins, viruses, and bacteria. These microarrays consist of immobilized glycans arranged in a spatially defined and miniaturized fashion, enabling the simultaneous screening of numerous glycan-protein interactions on a single chip.

Principle of Glycan Microarray

The principle underlying glycan microarray technology revolves around the immobilization of diverse glycans onto solid substrates, followed by the incubation of target biomolecules, such as proteins or antibodies, with the arrayed glycans. Specific binding events between the immobilized glycans and the target molecules are detected using fluorescence-based or label-free methods, providing insights into glycan-binding specificities and affinities.

Glycan Microarray Platform Offered by Creative Proteomics

Carbohydrate-Protein Interactions: Our platform allows you to screen and characterize interactions between glycans and various biomolecules, including proteins, antibodies, lectins, viruses, and cells. By immobilizing diverse glycans on solid substrates, we enable you to precisely investigate carbohydrate recognition events.

Glycan Binding Specificity: We offer you the capability to profile the binding specificities of glycan-binding proteins (GBPs) and other carbohydrate-binding molecules. Through high-throughput screening on our microarray platform, you can elucidate the binding preferences of GBPs towards different glycan structures, providing valuable insights into glycan recognition mechanisms.

Kinetic Assessments: Our platform supports kinetic assessments of glycan-protein interactions, allowing you to measure binding affinities, association rates, and dissociation rates. By employing fluorescence-based detection methods, we enable you to monitor binding kinetics in real-time, facilitating quantitative analyses of molecular interactions.

Pathogen Interactions: You can use our glycan microarray platform to probe the binding of viruses, bacteria, and other pathogens to host cell glycans. This analysis can aid in understanding pathogen-host interactions, vaccine development, and the identification of potential therapeutic targets.

Enzymatic Profiling: Our platform enables you to profile enzyme substrate specificities and activities towards glycan substrates. By incubating glycan microarrays with enzymatic samples, you can identify enzyme substrates and characterize enzymatic reactions, shedding light on glycan biosynthesis and degradation pathways.

Biological Response Profiling: You can utilize our glycan microarray platform to profile the inflammatory immune response and other biological responses mediated by glycans. By incubating microarrays with biological samples, such as serum or cell lysates, you can identify glycan-binding antibodies and other biomolecules involved in immune recognition processes.

Workflow of Glycan Microarray Services

1) Project Consultation:

• Initiate project discussion to understand research objectives and requirements.

2) Sample Preparation:

• Provide samples according to recommended volumes and types.
• Our experts handle purification, labeling, and dilution for optimal analysis.

3) Glycan Microarray Design:

• Customize microarray layout based on research goals and sample characteristics.
• Curate diverse glycan library for comprehensive analysis.

4) Glycan Immobilization:

• Ensure precise immobilization of glycans for accurate binding interactions.

5) Sample Incubation and Binding Assay:

• Incubate samples with microarrays to facilitate specific binding events.
• Utilize fluorescence-based or label-free detection for visualization and quantification.

6) Data Acquisition and Analysis:

• Capture fluorescence signals and process data using advanced algorithms.
• Identify binding patterns, affinities, and specificities.

7) Result Interpretation and Reporting:

• Provide detailed interpretation of findings and implications for research.
• Deliver comprehensive analysis reports for easy understanding and dissemination.

8) Project Conclusion and Follow-Up:

• Summarize experimental procedures, results, and conclusions in final report.
• Offer ongoing support and guidance to address any follow-up questions or concerns.

Advantages of Glycan Microarray

High Throughput: Our glycan microarray platform can analyze hundreds to thousands of glycan samples simultaneously, significantly increasing throughput compared to traditional methods. For example, our system can process up to 1,000 glycan samples in a single run.

High Sensitivity: Our technology can detect glycan components in samples at concentrations as low as picograms per milliliter (pg/mL), demonstrating exceptional sensitivity. This level of sensitivity ensures that even low-abundance glycans are accurately identified and quantified.

Diversity: We cater to a wide range of sample types, including cells, tissues, serum, proteins, and glycoproteins, from various organisms and sources. Our service covers over 100 different glycan structures, ensuring comprehensive analysis across diverse biological contexts.

Customization: We offer customizable services tailored to the specific needs of each client. This includes flexibility in experimental designs, such as sample selection criteria, assay conditions, and data analysis parameters. Our team works closely with clients to ensure that our services meet their exact requirements.

Reliability and Accuracy: With a quality control (QC) process that includes rigorous validation steps, our services consistently deliver accurate and reproducible results. Our QC metrics demonstrate an accuracy rate of over 95% across all glycan microarray experiments.

Sample Requirements for Glycan Microarrays

Applications of Glycan Microarrays

Investigation of Glycan-Protein Interactions: Glycan microarrays facilitate the identification and characterization of glycan-binding proteins, shedding light on cellular recognition mechanisms and disease pathways.

Characterization of Glycosylation Patterns: By profiling the binding specificities of antibodies and lectins to glycans, microarrays aid in deciphering glycosylation patterns and their implications in health and disease.

Pathogen Interactions: Glycan microarrays enable the screening of virus and bacterial binding to host cell glycans, providing insights into pathogen-host interactions and aiding in the development of therapeutics and vaccines.

Enzyme Substrate Specificity: Microarrays are utilized to profile the substrate specificity of glycosyltransferases and glycosidases, facilitating the study of glycan biosynthesis and metabolism.

Biomarker Discovery: By identifying anti-glycan antibodies as potential biomarkers for diseases such as cancer, autoimmune disorders, and infectious diseases, glycan microarrays offer new avenues for diagnostic and therapeutic development.

Title: Role of O-Acetylated Disialogangliosides as Receptors for Coronaviruses

Background

Coronaviruses (CoVs) like OC43 and HKU1 cause respiratory infections in humans, and understanding their receptor preferences is crucial for developing prevention and intervention strategies. Previous studies have highlighted the importance of glycan receptors in viral entry, prompting investigations into the role of O-acetylated disialogangliosides as potential receptors for these viruses.

Sample

HEK293T cells were used in infection assays to assess susceptibility to VSV particles pseudotyped with OC43 and HKU1 spikes. Additionally, human respiratory tract tissues were analyzed to determine the distribution of O-acetylated disialoside receptors.

Technical Methods

Sialylation and Isopropylidene Removal: Diol acceptors were reacted with a sialyl donor in the presence of TMSOTf, followed by quenching and purification steps.

Hydrogenation, Acetyl Migration, and Biotin Conjugation: Starting materials underwent hydrogenation, regioselective acetyl migration using DMSO and caesium fluoride, and subsequent biotin conjugation for downstream applications.

HE-Mediated De-O-acetylation: Glycan precursors were treated with HEs to remove O-acetyl groups, followed by purification using HPLC.

Glycan Microarray Printing and Screening: Biotinylated compounds were printed on streptavidin-coated glass slides, incubated with human-Fc tagged proteins, and analyzed to determine binding affinities.

Immunofluorescence Assay of Cultured Cells: HEK-293T cells were transfected with plasmids, treated with inhibitors or BCoV-LUN HE, and then incubated with S1A-mFc lectins. Confocal fluorescence microscopy was used to visualize binding.

Virolectin Staining of Human Tissues: Paraffin-embedded human tissue sections were incubated with S1A-mFc virolectins, followed by staining and examination using light microscopy.

VSV Pseudotyping and Infectivity Assay: VSV particles pseudotyped with OC43 and HKU1 spikes were produced and used to infect HEK-293T cells. Infection levels were assessed by measuring firefly luciferase expression.

Glycan microarray studies reveal binding patterns associated with host specificities

Results

Overexpression of ST8Sia1 significantly increased infection of HEK293T cells with VSV particles pseudotyped with OC43 and HKU1 spikes. Immunofluorescence assays showed binding of OC43 and HKU1 S1A proteins to ciliated pseudostratified columnar epithelial cells in the human respiratory tract tissues. Additionally, glycan microarray analysis indicated binding of OC43 and HKU1 spikes to gangliosides containing α2,8-disialosyl, such as 9-Ac-GD3, in a 9-O-acetyl-dependent manner. These findings suggest that O-acetylated disialogangliosides may serve as receptors for these coronaviruses, facilitating viral entry into host cells.

Reference

1. Li, Zeshi, et al. "Synthetic O-acetylated sialosides facilitate functional receptor identification for human respiratory viruses." Nature Chemistry 13.5 (2021): 496-503.