Glycosylation, an essential process where sugars are added to proteins after they are made, holds significant importance in maintaining normal bodily functions. More than half of the proteins in living organisms undergo glycosylation, indicating its crucial role. This modification comes in two main forms: N-glycosylation and O-glycosylation. Among them, O-GalNAc modification, commonly found in immunoglobulins, is of particular interest due to its association with various physiological processes like inflammation, immune response, viral infections, cell adhesion, metastasis, and apoptosis.
However, due to the complexity of glycosyltransferase enzymes and the intricate structures of O-polysaccharide modifications, determining the exact glycosylation structures at each site poses significant technical challenges. This limitation makes it difficult to comprehensively study O-glycoproteins in the body and establish their structure-function relationships. Therefore, creating a detailed map of site-specific O-glycoproteins in complex tissues or biological fluids under realistic conditions becomes crucial in understanding their role in diseases.
Case 1 Insights into Tissue-Specific O-Glycoprotein Regulation (1)
In a recent study published in the journal PNAS on October 20th, a team led by Lawrence A Tabak from the National Institutes of Health presented their findings titled "Quantitative mapping of the in vivo O-GalNAc glycoproteome in mouse tissues identifies GalNAc-T2 O-glycosites in metabolic disorder." They used advanced techniques in glycoproteomics and proteomics to map the entire landscape of O-GalNAc glycoproteins in mice, establishing a practical method for quantitatively mapping these proteins in complex samples like tissue extracts and biological fluids. By studying mice lacking the GALNT2 gene, they aimed to understand lipid and metabolic imbalances seen in congenital glycosylation disorders.
Select Services
The researchers integrated various methods and software packages to optimize the workflow for quantifying O-glycosylation. They collected samples from different tissues of healthy mice and subjected them to extensive analysis. The results provided insights into 2154 O-glycosylation sites, 2834 glycopeptide sequences, 38 different polysaccharide compositions, and 4020 glycopeptide sequences from 595 glycoproteins. Through meticulous operations, they established a reliable method for precisely locating O-glycosylation sites.
Whole Process of Glycoproteomics Analysis
Further analysis revealed that serine and threonine residues constituted the majority of O-glycosylation sites, with a high correlation between experimentally identified and predicted sites. Additionally, comparison with phosphorylation sites highlighted overlaps between O-glycosylation and phosphorylation sites. The researchers also explored tissue-specific regulation of O-glycosylation and identified potential substrates of GalNAc-T2, shedding light on the mechanisms underlying congenital glycosylation disorders.
Tissue-specific regulation of O-glycosylation revealed by integrating glycoproteomics and proteomics
Quantitative analysis of glycoproteomics and proteomics identifies significant changes in O-glycosylation sites and protein abundance
In conclusion, this study provides a comprehensive understanding of tissue-specific O-glycoprotein regulation through detailed mapping in mouse tissues and blood. By comparing data from congenital glycosylation disorder mouse models, the study confirms the reliability of mapping specific O-glycosylation sites and their role in metabolic disorders. This research not only enriches our databases but also emphasizes the importance of building comprehensive maps of protein modifications for a deeper understanding of biological mechanisms.
Case 2 Proteomic Insights into Hepatoblastoma: Unraveling Phosphorylation and O-Glycosylation Dynamics (2)
Hepatoblastoma (HB) originates from abnormal development of pluripotent stem cells or liver progenitor cells and is a highly malignant embryonic stem cell cancer in children. HB is rare and difficult to diagnose. Despite some progress in treatment, poor prognosis and high risk of early death still exist. Therefore, understanding the pathogenesis and molecular mechanisms of HB is crucial for improving early diagnosis, treatment, and prognosis of HB patients.
Post-translational modifications (PTMs) of proteins are important mechanisms that regulate protein function and influence cellular pathway regulation. In this study, researchers focused on hepatoblastoma and, from the perspective of proteomic modifications, extensively identified the levels of phosphorylation and O-glycosylation modifications in HB tissues compared to normal liver tissues, and analyzed the molecular processes involved in these two modifications.
Researchers initially conducted Western blotting (WB) and immunohistochemistry (IHC) experiments using samples of hepatoblastoma (HB) tissues and normal liver tissues. Employing an O-GlcNAc modification pan-antibody (PTM-952, PTM Biolabs), they observed a higher level of O-glycosylation modification on proteins in HB tissues compared to normal liver tissues. Considering the potential co-occurrence of O-glycosylation and phosphorylation on the same or different serine or threonine residues, researchers utilized the proteomics platform to conduct O-glycosylation modification proteomics based on O-glycosylation antibody enrichment and phosphoproteomics based on IMAC enrichment (mass spectrometry strategy).
Results of WB and IHC experiments and modificationomics workflow
Select Services
In this project, a total of 114 O-glycosylation sites on 78 proteins and 3494 phosphorylation sites on 2088 proteins were successfully identified. Through bioinformatics analysis, researchers found that O-glycosylation sites exhibited a characteristic motif of ...T..S... and were inclined to participate in functions such as nucleic acid binding, transcription regulation, mRNA splicing, and processing within the cell nucleus.
Types of glycosylated proteins and the molecular biological processes they are involved in
The impact of different modifications on protein function varies, thus the crosstalk between modifications is crucial for understanding protein function regulation. Researchers systematically analyzed proteins identified with both phosphorylation and O-glycosylation sites in this proteomic dataset. They discovered that 52% of the identified O-glycosylated proteins were also identified with phosphorylation modifications. These proteins are mainly associated with chromatin regulation, transcription, translation, transport, and organelle composition. Through protein-protein interaction (PPI) network analysis, it was found that proteins with dual modifications may play a synergistic role in certain cell signaling processes.
Among the dually modified proteins, researchers focused on NPM1, HSPE1, and HSPB1, three proteins involved in tumor development. Through mass spectrometry confirmation and IP-WB validation, the differential changes in phosphorylation and O-glycosylation modification levels of these three proteins in tumor tissues compared to normal tissues were confirmed.
MS/MS modification spectra of three proteins, NPM1/HSPE1/HSPB1, and IP-WB modification validation results
Of these, the protein HSPB1 is associated with the survival and chemoresistance of HB tumor cell lines. Early studies have shown abnormal expression of Heat Shock Protein B-1 (HSPB-1) in various human cancers including glioma, hepatocellular carcinoma, non-small cell lung cancer, and breast cancer. However, the expression and function of HSPB-1 protein in HB cell lines have yet to be elucidated. Researchers experimentally confirmed the presence of O-glycosylation and phosphorylation on HSPB-1 in HB cell lines, and competition between O-glycosylation and phosphorylation was observed at the serine residue at position 82. Furthermore, in vitro drug experiments revealed that the O-glycosylation level of HSPB-1 can affect the tolerance of HB cells to cisplatin, further indicating that HSPB-1 may be a potential therapeutic target for hepatoblastoma.
HSPB-1 protein O glycosylation modification affects drug resistance in HB cell lines
In summary, this study, by comparing tumor tissues with normal tissues and employing big data and bioinformatics analysis, revealed for the first time the comprehensive modification landscape of the well-studied phosphorylation modification and the less-studied O-glycosylation modification in tumor tissues, as well as the crosstalk between the two modifications. Through bioinformatics analysis and background research screening, potential key regulatory proteins affecting the mechanism of tumor occurrence, such as HSPB-1, were identified. Through experimental verification of proteomic data and exploration of the mechanism of modification, theoretical basis for potential tumor therapeutic targets was provided.
References
- Yang, Weiming, et al. "Quantitative mapping of the in vivo O-GalNAc glycoproteome in mouse tissues identifies GalNAc-T2 O-glycosites in metabolic disorder." Proceedings of the National Academy of Sciences 120.43 (2023): e2303703120.
- Song, Hang, et al. "Global profiling of O-GlcNAcylated and/or phosphorylated proteins in hepatoblastoma." Signal transduction and targeted therapy 4.1 (2019): 40.
