Dataset Information

In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM

ABSTRACT: The spike protein of the porcine epidemic diarrhea virus from various strains was generated from different mammalian expression systems. The integration of cryo-EM and MS delineated the glycosylation patterns on Spike proteins and also helped understand how individual glycans contribute to biological functions.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Cell Culture

SUBMITTER: Yu-Chun Chien

LAB HEAD: Kay-Hooi Khoo

PROVIDER: PXD035778 | Pride | 2022-10-15

REPOSITORIES: Pride

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Publications

In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM.

Huang Cheng-Yu CY Draczkowski Piotr P Wang Yong-Sheng YS Chang Chia-Yu CY Chien Yu-Chun YC Cheng Yun-Han YH Wu Yi-Min YM Wang Chun-Hsiung CH Chang Yuan-Chih YC Chang Yen-Chen YC Yang Tzu-Jing TJ Tsai Yu-Xi YX Khoo Kay-Hooi KH Chang Hui-Wen HW Hsu Shang-Te Danny SD

Nature communications 20220819 1

Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by porcine epidemic diarrhea virus (PEDV). PED causes enteric disorders with an exceptionally high fatality in neonates, bringing substantial economic losses in the pork industry. The trimeric spike (S) glycoprotein of PEDV is responsible for virus-host recognition, membrane fusion, and is the main target for vaccine development and antigenic analysis. The atomic structures of the recombinant PEDV S proteins of two diffe ...[more]

PMID: 35986008

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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 situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM

Publications

In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM.

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