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J Mol Biol. 2019 Feb 15;431(4):842-856. doi: 10.1016/j.jmb.2018.12.014. Epub 2018 Dec 29.

Fluorescent Trimeric Hemagglutinins Reveal Multivalent Receptor Binding Properties.

Author information

1
Pathology Division, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, 3584, CL, Utrecht, the Netherlands.
2
Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584, CG, Utrecht, the Netherlands.
3
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
4
Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; Department of Immunology & Microbiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
5
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd, Athens, GA 30602, USA.
6
Department of Pharmaceutical Sciences, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584, CG, Utrecht, the Netherlands; Drug sector, Saudi Food and Drug Authority, Riyadh, Saudi Arabia.
7
Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584, CG, Utrecht, the Netherlands; Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd, Athens, GA 30602, USA.
8
Pathology Division, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, 3584, CL, Utrecht, the Netherlands. Electronic address: m.h.verheije@uu.nl.
9
Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584, CG, Utrecht, the Netherlands. Electronic address: r.vries@uu.nl.

Abstract

Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.

KEYWORDS:

Influenza; attachment protein; bidentate; multivalent; precomplexing

PMID:
30597163
PMCID:
PMC6397626
[Available on 2020-02-15]
DOI:
10.1016/j.jmb.2018.12.014
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