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. 2022 Sep 9;8(9):1883-1893.
doi: 10.1021/acsinfecdis.2c00155. Epub 2022 Aug 18.

Sialic Acid and Fucose Residues on the SARS-CoV-2 Receptor-Binding Domain Modulate IgG Antibody Reactivity

Ebba Samuelsson  1 , Ekaterina Mirgorodskaya  2 , Kristina Nyström  1 , Malin Bäckström  3 , Jan-Åke Liljeqvist  1 , Rickard Nordén  1   4
Affiliations

Sialic Acid and Fucose Residues on the SARS-CoV-2 Receptor-Binding Domain Modulate IgG Antibody Reactivity

Ebba Samuelsson et al. ACS Infect Dis. .

Abstract

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of the recombinant RBD produced in different mammalian cells. We found a higher degree of complex-type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated, and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity, while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity, verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it is possible to enhance the immunogenicity of recombinant viral proteins.

Keywords: SARS-CoV-2; antibody reactivity; glycoepitope; glycoproteomics; mass spectrometry; receptor binding domain.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Schematic presentation of glycan distribution at the respective sites of the CHO-S- and HEK293F-produced RBD. The glycan structures are inferred from the obtained mass spectrometry data as well as from previously reported glycan structures and known glycan biosynthesis pathways in mammalian cells. (A) Degree of glycosylation, distribution between the glycan types, degree of sialylation, and degree of fucosylation at site N331 as detected on the CHO-S- and HEK293F-produced RBD. (B) Degree of glycosylation, distribution between the glycan types, degree of sialylation, and degree of fucosylation at site N343 as detected on the CHO-S- and HEK293F-produced RBD. (C) Degree of glycosylation, distribution between glycan compositions, and degree of sialylation at site T323/S325 as detected on the CHO-S- and HEK293F-produced RBD. (D) Degree of glycosylation, distribution between glycan compositions, and degree of sialylation at site T523 as detected on the CHO-S- and HEK293F-produced RBD. (E) Glycosylation of the recombinant RBD produced in CHO-S cells with the most prevalent glycans drawn at the respective site. The yellow circle highlights the glycan hotspot. (F) Glycosylation of the recombinant RBD produced in HEK293F cells with the most prevalent glycans drawn at the respective site. The yellow circle highlights the glycan hotspot.
Figure 2
Figure 2
NT-titre (left y axis, gray bars) and anti-RBD IgG-levels (right y axis, transparent circles) for the 24 characterized serum samples. The sera were divided into three groups based on the neutralizing capability: non-neutralizing (NT-negative, n = 7), weakly neutralizing (NT titre 3–6, n = 7), and highly neutralizing (NT titre 48–96, n = 10). Anti RBD-IgG value ≥50 AU/mL is considered positive.
Figure 3
Figure 3
Reactivity of highly neutralizing sera (NT titre 48–96, n = 10) against the fully glycosylated RBD (mock treated) and against the deglycosylated RBD produced in CHO-S and HEK293F cells. (A) Removal of both N-linked and O-linked glycans, removal of N-linked glycans alone, or removal of O-linked glycans alone from the RBD produced in CHO-S cells. (B) Removal of both N-linked and O-linked glycans, removal of N-linked glycans alone, or removal of O-linked glycans alone from the RBD produced in HEK293F cells. (C) Removal of sialic acids alone from the RBD produced in CHO-S and HEK293F cells. (D) Removal of fucose alone from the RBD produced in CHO-S and HEK293F cells. Data information: dark red color symbolizes a serum with high levels of anti-RBD IgG, and white color indicates anti-RBD IgG-negative serum (<50 AU/mL). Statistical analysis was performed with the Wilcoxon matched-pair signed rank test, ** = p < 0.001.
Figure 4
Figure 4
Antibody reactivity of highly neutralizing sera (NT titre 48–96, n = 10). (A) Reactivity against the fully glycosylated RBD (untreated) expressed in CHO-S, HEK293F, and Lec3.2.8.1 cells. (B) Reactivity against the fully glycosylated RBD (mock treated) produced in Lec-3.2.8.1 cells, or against the Lec3.2.8.1-produced RBD following enzymatic removal of sialic acids and fucose. Data information: dark red color symbolizes a serum with high levels of anti-RBD IgG, and white color indicates anti-RBD IgG-negative serum (<50 AU/mL). Statisitical analysis was performed with the Wilcoxon matched-pair signed rank test, ** = p < 0.001.
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