Quantitative Analysis of Carbohydrate−Protein Interactions Using Glycan Microarrays:  Determination of Surface and Solution Dissociation Constants

Liang, Pi‐Hui; Wang, Shengkai; Wong, Chi‐Huey

doi:10.1021/ja072931h

Cited by 241 publications

(247 citation statements)

References 38 publications

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“…The glycan microarray is a powerful tool for investigating carbohydrate-protein interactions (15)(16)(17)(18) and provides a new platform for influenza virus subtyping (16)(17)(18). Although powerful, understanding HA-glycan interactions by array analysis has been complicated by two issues.…”

Section: Creating Defined Ha Glycoforms For Quantitative Glycan Micromentioning

confidence: 99%

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Glycans on influenza hemagglutinin affect receptor binding and immune response

Wang

Chen

Tseng

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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283

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Recent cases of avian influenza H5N1 and the swine-origin 2009 H1N1 have caused a great concern that a global disaster like the 1918 influenza pandemic may occur again. Viral transmission begins with a critical interaction between hemagglutinin (HA) glycoprotein, which is on the viral coat of influenza, and sialic acid (SA) containing glycans, which are on the host cell surface. To elucidate the role of HA glycosylation in this important interaction, various defined HA glycoforms were prepared, and their binding affinity and specificity were studied by using a synthetic SA microarray. Truncation of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. The contribution of each monosaccharide and sulfate group within SA ligand structures to HA binding energy was quantitatively dissected. It was found that the sulfate group adds nearly 100-fold (2.04 kcal/mol) in binding energy to fully glycosylated HA, and so does the biantennary glycan to the monoglycosylated HA glycoform. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. Thus, removal of structurally nonessential glycans on viral surface glycoproteins may be a very effective and general approach for vaccine design against influenza and other human viruses.flu vaccine ͉ glycan binding ͉ glycosylation T he highly pathogenic H5N1 and the 2009 swine-origin influenza A (H1N1) viruses have caused global outbreaks and raised a great concern that further changes in the viruses may occur to bring about a deadly pandemic (1, 2). Important contributions to our understanding of influenza infections have come from the studies on hemagglutinin (HA), a viral coat glycoprotein that binds to specific sialylated glycan receptors in the respiratory tract, allowing the virus to enter the cell (3-6). To cross the species barrier and infect the human population, avian HA must change its receptorbinding preference from a terminally sialylated glycan that contains ␣2,3 (avian)-linked to ␣2,6 (human)-linked sialic acid motifs (7), and this switch could occur through only two mutations, as in the 1918 pandemic (8). Understanding the factors that affect influenza binding to glycan receptors is thus critical for developing methods to control any future crossover influenza strains that have pandemic potential.HA is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem region (3). Both regions carry N-linked oligosaccharides (9), which affect the functional properties of HA (10, 11). Among different subtypes of influenza A viruses, there is extensive variation in the glycosylation sites of the head region, whereas the stem oligosaccharides are more conserved and required for fusion activity (11). Glycans near antigenic peptide epitopes interfere with antibody recognition (12), and glycans near the proteolytic ...

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Section: Creating Defined Ha Glycoforms For Quantitative Glycan Micromentioning

confidence: 99%

“…We next prepared a quantitative array to determine surface dissociation constants (17). To avoid any skewing by antibody layering, HA was directly labeled with the fluorescent dye Cy3 (19).…”

Section: Creating Defined Ha Glycoforms For Quantitative Glycan Micromentioning

confidence: 99%

Glycans on influenza hemagglutinin affect receptor binding and immune response

Wang

Chen

Tseng

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

283

256

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“…Both protein and carbohydrate microarrays have been used to investigate the kinetics and thermodynamics of the interactions between biomolecules in solution phase and solid phase (40)(41)(42). To determine the dissociation constants of Globo H and truncated analogs on surface interacting with antibodies in a multivalent manner, we adopted the direct measurement method reported by our group in ref.…”

Section: Determination Of Surface Dissociation Constants (Kdsurf)mentioning

confidence: 99%

“…To determine the dissociation constants of Globo H and truncated analogs on surface interacting with antibodies in a multivalent manner, we adopted the direct measurement method reported by our group in ref. 42, using different concentrations of antibodies and printed sugars. The Langmuir isotherm was then used for analyzing the binding curves to generate the dissociation constants on surface (K D , surf ).…”

Section: Determination Of Surface Dissociation Constants (Kdsurf)mentioning

confidence: 99%

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Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer

Wang

Huang

Ren

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Cancer-associated carbohydrate antigens are often found on the surface of cancer cells. Understanding their roles in cancer progression will lead to the development of new therapeutics and high-sensitivity diagnostics for cancers. Globo H is a member of this family, which is highly expressed on breast cancer cells. Here, we report the development of a glycan microarray of Globo H and its analogs for measurement of the dissociation constants on surface (K D,surf) with three different monoclonal antibodies (VK-9, Mbr1, and anti-SSEA-3), to deduce their binding specificity. The glycan microarray was also used to detect the amount of antibodies present in the plasma of breast cancer patients and normal blood donors. It was shown that the amount of antibodies against Globo H from breast cancer patients were significantly higher than normal blood donors, providing a new tool for possible breast cancer diagnosis. Compared with the traditional ELISA method, this array method required only atto-mole amounts of materials and is more effective and more sensitive (5 orders of magnitude). The glycan microarray thus provides a new platform for use to monitor the immune response to carbohydrate epitopes after vaccine therapy or during the course of cancer progression.array ͉ diagnosis

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Glycochips

Hillringhaus¹,

Seibel²

2010

Encyclopedia of Industrial Biotechnology

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An overview is given about the different carbohydrate‐array types as well as immobilization and read out techniques, which offer a broad spectrum for many applications used in academia and the industries. Also the glycan acquisition is discussed, which is a fundamental task since pure and defined glycans are required to gain sound results. Finally, applications are mentioned, which give an impression of how glycan arrays impact the understanding of glycan‐mediated biological processes and give rise to new medical applications.

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Quantitative Analysis of Carbohydrate−Protein Interactions Using Glycan Microarrays: Determination of Surface and Solution Dissociation Constants

Cited by 241 publications

References 38 publications

Glycans on influenza hemagglutinin affect receptor binding and immune response

Glycans on influenza hemagglutinin affect receptor binding and immune response

Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer

Glycochips

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