Metabolic Oligosaccharide Engineering with Alkyne Sialic Acids Confers Neuraminidase Resistance and Inhibits Influenza Reproduction

Abstract

Metabolic incorporation of azide- or alkyne-modified sialic acids into the cellular glycosylation pathway enables the study of sialoglycan expression, localization, and trafficking via bioorthogonal chemistry. Herein, we report that such modifications of the sialic acid sugar can have a profound influence on their hydrolysis by neuraminidases (sialidase). Azidoacetyl (Az)-modified sialic acids were prone to neuraminidase cleavage, whereas propargyloxycarbonyl (Poc)-modified sialic acids were largely resistant to cleavage. Because the influenza virus infection cycle depends on the hydrolysis of host-cell-surface sialic acids, influenza cell-to-cell transmission was strongly reduced in Poc sialic acid glycoengineered host cells. The use of Poc sialic acids may disturb biological processes involving neuraminidase cleavage but also provides perspective for use in applications in which sialic acid hydrolysis is not desired, such as antibody modification, viral infection, etc.

Figure 1

Schematic presentation of metabolic labeling with Ac₅SiaNAz or Ac₅SiaNPoc and the resistance of Ac₅SiaNPoc against influenza neuraminidase.

Figure 2

Metabolic labeling of THP-1 cells. (a) The cells were incubated for 3 days with PBS, 100 μM Ac₅SiaNAz, or 100 μM Ac₅SiaNPoc and reacted to biotin–alkyne or biotin–azide, respectively, using CuAAC. Biotin-containing sialoglycans were stained with PE–streptavidin and visualized by confocal microscopy. (b) Fluorescence was quantified by flow cytometry, and the mean fluorescence intensity ± SEM is shown (n = 3).

Figure 3

Enzymatic removal of Az and Poc sialic acids from the cell surface of THP-1 cells. Cells incubated for 3 days with PBS, 100 μM Ac₅SiaNAz, or 100 μM Ac₅SiaNPoc were treated for 1 h with 250mU/mL Clostridium perfringens neuraminidase. Az and Poc sialoglycans were reacted to fluorescent biotin using CuAAC (a), α2,3-sialoglycans were detected with MALII lectin (b), α2,6-sialoglycans were detected with SNA-I lectin (c), and terminal β-galactose was detected with PNA lectin (d). Bar diagrams show mean fluorescence intensity or mean lectin binding normalized to control ± SEM of three independent experiments. MALII: Maackia amurensis lectin; PE: phycoerythrin, PNA: Peanut agglutinin lectin; SEM: standard error of the mean; SNA-I: Sambucus nigra lectin.

Figure 4

Effect of Az and Poc sialic acid on influenza reproduction (333× dilution). A549 cells cultured with either Ac₅SiaNAz or Ac₅SiaNPoc were treated with influenza virus strain A/PR8-GFP/8/34, and the GFP expression was measured as a readout for successful viral infection after 6 h and reinfection after 24 h (a). Az and Poc sialoglycans were detected 24 h post-infection by reaction with fluorescent biotin using the CuAAC (b). Bar diagrams show mean fluorescence intensity ± SEM (n = 4).