Dataset Information

Comprehensive glycomic and glycoproteomic analysis of human programmed cell death protein 1 extracellular domain

ABSTRACT: Mass spectrometric analyses of N- and O-glycosylation on human programmed cell death protein 1 extracellular domain (hPD-1 ECD) that expressed by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells

ORGANISM(S): Homo Sapiens

SUBMITTER: Xuechen Li

PROVIDER: PXD048448 | iProX | Mon Jan 22 00:00:00 GMT 2024

REPOSITORIES: iProX

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Publications

Comprehensive Glycomic and Glycoproteomic Analyses of Human Programmed Cell Death Protein 1 Extracellular Domain.

Chen Qiushi Q Tan Zhiwu Z Tang Yang Y Fung Yi Man Eva YME Chen Sheng S Chen Zhiwei Z Li Xuechen X

Journal of proteome research 20240805 9

Human programmed cell death protein 1 (hPD-1) is an essential receptor in the immune checkpoint pathway. It has played an important role in cancer therapy. However, not all patients respond positively to the PD-1 antibody treatment, and the underlying mechanism remains unknown. PD-1 is a transmembrane glycoprotein, and its extracellular domain (ECD) is reported to be responsible for interactions and signal transduction. This domain contains 4 <i>N</i>-glycosylation sites and 25 potential <i>O</i ...[more]

PMID: 39101792

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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

Comprehensive glycomic and glycoproteomic analysis of human programmed cell death protein 1 extracellular domain

Publications

Comprehensive Glycomic and Glycoproteomic Analyses of Human Programmed Cell Death Protein 1 Extracellular Domain.

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