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. 2021 Dec 7;11(1):23561.
doi: 10.1038/s41598-021-02904-w.

N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry

Dongxia Wang  1 , Bin Zhou  2 , Theodore R Keppel  3 , Maria Solano  3 , Jakub Baudys  3 , Jason Goldstein  4 , M G Finn  5 , Xiaoyu Fan  2 , Asheley P Chapman  5 , Jonathan L Bundy  3 , Adrian R Woolfitt  3 , Sarah H Osman  3 , James L Pirkle  3 , David E Wentworth  2 , John R Barr  6
Affiliations

N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry

Dongxia Wang et al. Sci Rep. .

Abstract

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
SDS gel analysis of the SARS-CoV-2 S proteins interacting with other proteins. (A) Binding of the S to the biotinylated ACE2. lane 1: S-614D; lane 2: S-614D + ACE2; lane 3: S-614G; lane 4: S-614G + ACE2. (B) Binding of the S to monoclonal antibodies. Lane 1–6: S-614D, S-614D + 3G7, S-614D + 3A2, S-614G, S-614G + 3G7, and S-614G + 3A2. The gels were run under reduced condition and visualized with SYPRO Ruby stains.
Figure 2
Figure 2
Comparison of N-linked glycosylation profiles in ectodomain spike protein between S-614D (blue) and S-614G (orange) samples. Glycosylation abundances were calculated from one representative native peptide sequence for glycan sites (A) N17, (B) N61, (C) N74, (D) N122, (E) N165, (F) N234, (G) N282, (H) N331, (I) N343, (J) N603, (K) N616, (L) N657, (M) N709, (N) N717, (O) N801, (P) N1074, (Q) N1098, (R) N1134, (S) N1158, and (T) N1173. Inserted pie charts (upper: 614D; lower: 614G) depict the relative composition of high-mannose (green), hybrid (purple), and complex (gray) types of glycoforms. In the short names of individual glycans, N, H, A, and F symbolize HexNAc, Hex, NeuAc, and Fuc, respectively. X-axis represent individual glycans.
Figure 2
Figure 2
Comparison of N-linked glycosylation profiles in ectodomain spike protein between S-614D (blue) and S-614G (orange) samples. Glycosylation abundances were calculated from one representative native peptide sequence for glycan sites (A) N17, (B) N61, (C) N74, (D) N122, (E) N165, (F) N234, (G) N282, (H) N331, (I) N343, (J) N603, (K) N616, (L) N657, (M) N709, (N) N717, (O) N801, (P) N1074, (Q) N1098, (R) N1134, (S) N1158, and (T) N1173. Inserted pie charts (upper: 614D; lower: 614G) depict the relative composition of high-mannose (green), hybrid (purple), and complex (gray) types of glycoforms. In the short names of individual glycans, N, H, A, and F symbolize HexNAc, Hex, NeuAc, and Fuc, respectively. X-axis represent individual glycans.
Figure 3
Figure 3
(A) Schematic SARS-CoV-2 S protein primary structure. (B) N-glycans depicted on a representative full-length, fully-glycosylated prefusion conformation of the trimetric SARS-CoV-2 spike protein (file 6vsb 1 1 1.pdb from the CHARMM-GUI Archive displayed in PyMOL),. Blue-colored glycans indicate no change in the glycosylation site between the S-614G mutant and the S-614D wild type. Magenta-colored glycans indicate a modification in the glycan distribution and type between the mutant and wild type. The RBD is shown in green. The N149 and N1194 glycans are gold. The glycans depicted do not necessarily match those described in this report, and the O-linked glycans in the model are hidden due to low occupancy.
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References

    1. Huang C, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet (London, England) 2020;395:497–506. doi: 10.1016/S0140-6736(20)30183-5. - DOI - PMC - PubMed
    1. Wrapp D, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367:1260–1263. doi: 10.1126/science.abb2507. - DOI - PMC - PubMed
    1. Yan R, et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020;367:1444–1448. doi: 10.1126/science.abb2762. - DOI - PMC - PubMed
    1. Watanabe Y, Bowden TA, Wilson IA, Crispin M. Exploitation of glycosylation in enveloped virus pathobiology. Biochim. Biophys. Acta. 2019;1863:1480–1497. doi: 10.1016/j.bbagen.2019.05.012. - DOI - PMC - PubMed
    1. Watanabe Y, et al. Vulnerabilities in coronavirus glycan shields despite extensive glycosylation. Nat. Commun. 2020;11:2688. doi: 10.1038/s41467-020-16567-0. - DOI - PMC - PubMed

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