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Pressure Cycling Technology-Based Quantitative Glycoproteomics Reveals Distinctive N‑glycosylation Patterns in Human Kidney Tissues with Minor Glomerular Abnormalities

ABSTRACT: Kidney diseases present substantial clinical challenges, with aberrant glycoprotein emerging as a key pathogenic driver. Minor glomerular abnormalities (MGAs), a category of unclassified glomerular lesions defined by subtle structural changes, are commonly detected in patients with persistent, asymptomatic, isolated proteinuria or microhematuria., yet their site-specific N-glycosylation patterns remain unelucidated. To address this gap, we applied a laboratory-developed pressure cycling technology (PCT)-based quantitative glycoproteomics workflow (GlycoPCT) to compare intact N-glycopeptides (IGPs) between distant non-neoplastic tissues (DNT; n=24) and trace renal biopsy samples from MGA patients (n=27). Integrated with high-sensitivity, high-resolution mass spectrometry (EThcD-sceHCD-MS/MS), GlycoPCT identified 672 upregulated IGPs (FC>1.5, p<0.05) and 573 downregulated IGPs (FC<0.67, p<0.05) in MGA tissues. Total fucosylation type N-glycans were significantly downregulated (p<0.05), while sialylation type N-glycans were markedly downregulated (p<0.001). Additionally, 24 glycoproteins associated with the PI3K-Akt signaling pathway (a well-documented mediator of renal injury) showed broadly N-glycosylation upregulation. Site-specific N-glycosylation analysis further revealed differential patterns in IgG subclasses and complement-related markers that distinguish MGA from DNT, offering new mechanistic insights into MGA pathogenesis. These novel glyco-signatures clarify the role of N-glycosylation in renal disease, highlight TINAGL1, IgG, IgM, and complement-related glycoproteins as potential MGA biomarkers, and validate GlycoPCT as a powerful tool for trace tissue analysis. This study lays the groundwork for translating N-glycosylation findings into clinical applications to improve MGA diagnosis and management.

ORGANISM(S): Homo Sapiens

SUBMITTER: Yong Zhang

PROVIDER: PXD069708 | iProX | Tue Oct 21 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:STT3A and STT3B are the main catalytic subunit of oligosaccharyltransferase (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and posttranslocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomics and glycoproteomics analyses to characterize the protein expression and glycosylation in STT3A-knockout (KO), STT3B-KO, and wild-type (WT) HEK293 cells. In total, 3,993 proteins, 4,020 unique N-linked intact glycopeptides (IGPs) and 723 glycosites, representing 401 glycoproteins were identified in KO and WT cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, mannosylated N-glycans from glycoproteins were reduced, while fucosylated N-glycans were increased in STT3A-KO cells. The glycan changes may be caused by the differential expression of glycosyltransferases and the activation of unfolded protein response (UPR) with further analysis of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells. We verified that the hyperglycosylation of ENPL was caused by the ER stress and UPR, which were induced by the deletion of the STT3A. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.STT3A and STT3B are the main catalytic subunit of oligosaccharyltransferase (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and posttranslocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomics and glycoproteomics analyses to characterize the protein expression and glycosylation in STT3A-knockout (KO), STT3B-KO, and wild-type (WT) HEK293 cells. In total, 3,993 proteins, 4,020 unique N-linked intact glycopeptides (IGPs) and 723 glycosites, representing 401 glycoproteins were identified in KO and WT cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, mannosylated N-glycans from glycoproteins were reduced, while fucosylated N-glycans were increased in STT3A-KO cells. The glycan changes may be caused by the differential expression of glycosyltransferases and the activation of unfolded protein response (UPR) with further analysis of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells. We verified that the hyperglycosylation of ENPL was caused by the ER stress and UPR, which were induced by the deletion of the STT3A. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

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Pressure Cycling Technology-Based Quantitative Glycoproteomics Reveals Distinctive N‑glycosylation Patterns in Human Kidney Tissues with Minor Glomerular Abnormalities

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