Dataset Information

In Depth Characterization of Protein N-Glycosylation for a COVID-19 Variant-Design Vaccine Spike Protein

ABSTRACT: In this work, we performed comprehensive glycosylation analysis to a candidate vaccine Spike protein. The Spike protein studied here was featured by a set of structural designs such as the six-proline substitution, furin cleavage site replacement, trimeric foldon incorporation as well as “hotspots” of SARS-CoV-2 variants. Besides, it was expressed in Chinese Hamster Ovary (CHO) Cell lines and manufactured at pilot scales, and thus, well represented latest variant-design vaccine of COVID-19. A combination of LC-MS/MS based analytical methods was dedicatedly applied to fully unravel the Spike N-glycosylation. Utilization of different protease was firstly explored and discussed. As a result, RPLC-MS/MS following Trypsin and GluC enzyme plus PNGase F treatment proved an effective and complementary method in analyzing N-glycosites and site-specific glycan. An HILIC-LC-MS based analytical method together with 2-AB labeling was also utilized to analyze N-glycan abundance for the Spike protein.

ORGANISM(S): Cricetulus Griseus

SUBMITTER: Chenliang Zhou

PROVIDER: PXD038220 | iProX | Sat Nov 19 00:00:00 GMT 2022

REPOSITORIES: iProX

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In-depth characterization of protein N-glycosylation for a COVID-19 variant-design vaccine spike protein.

Huang Jiangming J Hou Shouzeng S An Jiao J Zhou Chenliang C

Analytical and bioanalytical chemistry 20230126 8

COVID-19 is caused by SARS-CoV-2 infection and remains one of the biggest pandemics around the world since 2019. Vaccination has proved to be an effective way of preventing SARS-CoV-2 infection and alleviating the hospitalization burden. Among different forms of COVID-19 vaccine design, the spike protein of SARS-CoV-2 virus is widely used as a candidate vaccine antigen. As a surface protein on the virus envelop, the spike was reported to be heavily N-glycosylated and glycosylation had a great im ...[more]

PMID: 36698045

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes an extensively glycosylated spike protein that protrudes from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the predominant recombinant spike protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, recombinant full-size spike protein was produced transiently in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on spike glycans, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on glycans and glycopeptides from spike protein. We determined global and site-specific N-linked glycosylation patterns in soluble SARS-CoV-2 spike using MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. We identified the glycan compositions at 21 and 19 out of the 22 predicted N-glycosylation sites of the SARS-CoV-2 spike proteins produced in CHO and HEK, respectively. The N-glycan site at 1158 position (N1158) and the N-glycan sites at 122 ,282 and 1158 positions (N122, N282 and N1158) were found to be unoccupied on spike secreted from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human spike proteins revealed that CHO-Spike presented more complex and higher sialylation (α2,3-linked) content while HEK-Spike displayed more high-mannose and minor content of α2,3- and α2,6-linked sialic acids. The N74 site represents the most heavily N-glycosylated site on both spike proteins while some high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801 and N1134) on HEK-Spike may differentially shield the virus compared to CHO-spike and provide a different host immune system interaction strategy. Collectively, these data underscore the importance of characterizing site-specific glycosylation on recombinant human spike protein from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in diagnostics and vaccines.

Dataset Information

In Depth Characterization of Protein N-Glycosylation for a COVID-19 Variant-Design Vaccine Spike Protein

Publications

In-depth characterization of protein N-glycosylation for a COVID-19 variant-design vaccine spike protein.

Similar Datasets

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