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An integrated multi-proteomics approach reveals the altered N-glycosylation patterns during Jurkat T cell activation

ABSTRACT: Protein glycosylation plays essential roles in regulating innate and adaptive immune response. Previous studies have typically focused on individual protein-glycan interaction or specific glycoform changes associated with T cell activation, yet a systematic characterization of protein glycosylation alterations remains insufficiently elucidated. To address these limitations, we conducted temporally-resolved quantitative multi-proteomic analysis on activated Jurkat T cells. Through an integrative analysis of glycoforms, site-specific glycans, glycoproteins, and glycosylation enzymes, we portrayed the dynamic landscape of protein glycosylation during Jurkat T cell activation. We found the heterogeneity and number of significantly up-regulated intact glycopeptides increased along with stimulation time. For most glycopeptides, their alteration patterns did not correlate well with the abundance of their glycoprotein substrates. Comparative analysis of global peptides, glycopeptides, and phosphopeptides revealed their distinctive changing patterns along Jurkat T cell activation, and only glycosylation demonstrated a steady increase trend with a large proportion of up-regulated glycopeptides. Collectively, this integrated multi-proteomics characterization of activated Jurkat T cells provided insights for the crucial role of glycosylation in modulating immune response, and would aid the development of novel therapeutic strategy targeting glycosylation.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Mingliang Ye

PROVIDER: PXD058431 | JPOST Repository | Sun Nov 30 00:00:00 GMT 2025

REPOSITORIES: jPOST

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			Action	DRS
	ZCR_QE_20220701_jurkat_pho_15min1_200ug.raw	Raw
	ZCR_QE_20220701_jurkat_pho_15min2_200ug.raw	Raw
	ZCR_QE_20220701_jurkat_pho_15min3_200ug.raw	Raw
	ZCR_QE_20220701_jurkat_pho_18h1_200ug.raw	Raw
	ZCR_QE_20220701_jurkat_pho_18h2_200ug.raw	Raw

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Project description:Our goal is to understand how glycosylation on human B cells is regulated during the differentiation and activation. Accumulating evidences have indicated that functions of immune cells are regulated in a glycan-dependent manner. One of well-known examples is B cell regulation by CD22 through its association with sialylated glycans. While recent studies uncovered that B cell activation leads to loss of high affinity glycan ligands for human CD22 (Neu5Ac-a2,6-Gal-B1,4-GlcNAc(6-sulfo)), the gene expression profile of human B cells before and after activation is unknown. Therefore we would like to perform gene expression analysis of human B cells before and after activation. We already have published the glycan profiling and gene expression of mouse B cells before and after activation done in collaboration with the CFG. This study revealed programmed changes in glycosylation relevant to regulation of B cell signaling by CD22. Since the ligands of human CD22 differ in several respects from that of mouse CD22, these experiments are relevant to the evolution of siglec ligands and their conserved functions in B cell biology. In brief, the experimental protocol is as follows: human peripheral B cells are isolated from human peripheral blood of healthy donors and stimulated for 0, 24, 48 and 72 hrs. Human B cells were isolated from human peripheral blood of healthy donors by the negative selection kit (Miltenyi). For the resting B cell samples, isolated B cells were immediately shock-frozen in the liquid N2 and kept at -80˚C until the RNA extraction. For the activated B cell samples, the cells were activated in the RPMI medium (Invitrogen) supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 1 mM non-essential amino acid, 1 mM sodium pyruvate, and 50 M 2-melcaptoethanol, 10 g/mL anti-human IgA, G, M (Jackson Immunoresearch), and 50 nM CpG ODN 2006 (Invivogen) for 3 days. Total RNA was isolated using the TRIzol reagent (Invitrogen).

Project description:SRD5A3-CDG is a congenital disorder of glycosylation (CDG) resulting from pathogenic variants in SRD5A3 and follows an autosomal recessive inheritance pattern. The enzyme encoded by SRD5A3, polyprenal reductase, plays a crucial role in synthesizing lipid precursors essential for N-linked glycosylation. Despite insights from functional studies into its enzymatic function, there remains a gap in understanding global changes in patient cells. We sought to identify glycoproteomic and proteomic signatures specific to SRD5A3-CDG, potentially aiding in biomarker discovery and advancing our understanding of disease mechanisms. Using tandem mass tag (TMT)-based relative quantitation, we analyzed fibroblasts derived from five patients along with control fibroblasts. Glycoproteomics analysis by liquid chromatographyâ€“tandem mass spectrometry (LC-MS/MS) identified 3,047 glycopeptides with 544 unique glycosylation sites from 276 glycoproteins. Of these, 418 glycopeptides showed statistically significant changes with 379 glycopeptides decreased (p<0.05) in SRD5A3-CDG patient-derived samples. These included high mannose, complex and hybrid glycan-bearing glycopeptides. High mannose glycopeptides from protocadherin Fat 4 and integrin alpha-11 and complex glycopeptides from CD55 were among the most significantly decreased glycopeptides. Proteomics analysis led to the identification of 5,933 proteins, of which 873 proteins showed statistically significant changes. Decreased proteins included cell surface glycoproteins, various mitochondrial protein populations and proteins involved in the N-glycosylation pathway. Lysosomal proteins such as N-acetylglucosamine-6-sulfatase and procathepsin-L also showed reduced levels of phosphorylated mannose-containing glycopeptides. Our findings point to disruptions in glycosylation pathways as well as energy metabolism and lysosomal functions in SRD5A3-CDG, providing clues to improved understanding and management of patients with this disorder.

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An integrated multi-proteomics approach reveals the altered N-glycosylation patterns during Jurkat T cell activation

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