Dataset Information

Glycosylation landscape of CAR-T in AAVS1 and SPPL3 knockout

ABSTRACT: In this study, we delved into deciphering the glycosylation patterns and precise localization of glycosylation sites within CAR-T proteins. Specifically, we focused on samples derived from human T cells that underwent gene knockout of AAVS1 and SPPL3, two critical genes that could potentially influence CAR-T function. To ensure accuracy and precision, we employed optimal tribrid orbitrap mass spectrometry settings for glycosylation landscapes of the CAR-T proteins. By analyzing these data, we aimed to gain a deeper understanding of how AAVS1 and SPPL3 knockout impacted the glycosylation profiles and ultimately the function and efficacy of CAR-T cells in immunotherapy applications.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell

SUBMITTER: Xue Sun

LAB HEAD: Catherine CL Wong

PROVIDER: PXD052748 | Pride | 2025-05-23

REPOSITORIES: Pride

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Source:

			Action	DRS
	AAVS1_KO_CAR1_OT_NO_et.raw	Raw
	AAVS1_KO_CAR2_TP_O_et.raw	Raw
	AAVS1_KO_CAR3_OTO_N_et.raw	Raw
	AAVS1_KO_T1_OT_NO_et.raw	Raw
	AAVS1_KO_T2_TP_O_et.raw	Raw

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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

Glycosylation landscape of CAR-T in AAVS1 and SPPL3 knockout

Dataset's files

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