Protein glycosylation, a crucial post-translational modification, adds complexity and functionality to proteins. While N-linked glycosylation has been extensively studied, O-linked glycosylation analysis faces challenges due to structural intricacies and methodological limitations. O-linked glycosylation involves attaching glycans to Ser, Thr, or Tyr residues in proteins. O-GalNAc addition is a primary form of O-linked glycosylation, characterized by the addition of N-acetyl-galactosamine. Definitive characterization of O-linked glycoproteins necessitates quantitative analysis of glycosylation sites and their glycans, which lack a consensus motif compared to N-linked glycosylation.
To overcome the scarcity of suitable methodologies for detailed analysis of O-linked glycosylation, a new chemoenzymatic method called EXoO was developed. This method enables the site-specific extraction of O-linked glycopeptides, facilitating the mapping of over 3,000 O-linked glycosylation sites and their glycans on more than 1,000 proteins in human tissues. EXoO revealed conserved motifs and topological orientations of glycosylation sites, indicating its effectiveness in defining the O-linked glycoproteome across different sample types. Moreover, EXoO unveiled significant differences in O-linked glycoproteins between normal and tumor kidney tissues, showcasing its potential for clinical diagnostics and therapeutics.
Various enrichment methodologies have been employed to study O-linked glycoproteins, including lectins, HILIC, and metabolic labeling. However, precise identification of O-linked glycosylation sites and their glycans remains challenging. Mass spectrometry, particularly using ETD for fragmentation, has been pivotal in localizing O-linked glycosylation sites efficiently. The EXoO method streamlines the extraction of site-specific O-linked glycopeptides by utilizing a solid support and the OpeRATOR enzyme, derived from Akkermansia muciniphila, which cleaves glycopeptides at glycan-occupied Ser or Thr residues.
The application of EXoO in human kidney tissues, T cells, and serum samples demonstrated its effectiveness in accessing the O-linked glycoproteome. Motif analysis of O-linked glycosylation sites revealed conserved patterns, with Pro being overrepresented at specific positions. Gene ontology analysis highlighted the cellular components and biological processes associated with O-linked glycoproteins, emphasizing their diverse functionalities. The precise mapping of O-linked glycosylation sites in heavily glycosylated proteins like VCAN and MUC1 underscored the utility of EXoO in revealing novel insights into mucin-type glycoproteins.
Comparative analysis of normal and tumor kidney tissues using EXoO identified aberrant O-linked glycoproteins, with significant changes observed in glycan structures primarily in proteins like VCAN, ACAN, and FBLN2. The remodeling of the extracellular matrix and altered expression of proteins associated with tumor progression were evident, emphasizing the clinical relevance of EXoO in investigating disease-specific glycoprotein signatures. Overall, EXoO emerges as a powerful tool for comprehensive mapping of the O-linked glycoproteome, offering new avenues for studying protein glycosylation in health and disease.
Tags: lyophilization, bioinformatics, immunotherapy, validation, mass spectrometry
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