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Exploring Pathogenic Mechanisms and Growth Mechanisms via N-Glycoproteome Analysis

Glycosylation modification plays a crucial role in the formation of protein function and structure, with over half of the proteins in the organism undergoing glycosylation. Processes such as cell adhesion, molecular recognition, and signal transduction involve the participation of glycosylated proteins. Based on the different linkage patterns of glycan side chains, protein glycosylation modifications are generally categorized into O-linked glycosylation and N-linked glycosylation.

N-glycosylated proteins refer to glycoproteins formed by the covalent attachment of glycans to the asparagine (Asn) residue's amino (NH2) group. N-linked glycosylation proteins have a characteristic sequence of Asn-X-Ser/Thr (X represents any amino acid except proline). N-linked glycosylation is a widespread and essential modification in protein glycosylation, participating in vital biological processes such as cell recognition, signal transduction, adhesion, and immunity. The efficient detection of N-glycosylated proteome holds significant research utility and promising applications.

Below are two cases of quantitative analysis of N-glycosylated proteome.

Case 1. N-Glycoproteome Analysis Unveils Pathogenic Mechanisms and Therapeutic Targets in Osteoarthritis and Kashin-Beck Disease ⁽¹⁾

Glycoproteins are involved in the development of numerous diseases, and the types and abundance of N-glycosylated proteins in the cartilage of osteoarthritis (OA) and Kashin-Beck disease (KBD) remain unclear. This study aims to identify N-glycosylated proteins in the knee cartilage of OA and KBD patients and compare them with normal adult controls to elucidate the pathological mechanisms and potential therapeutic targets of OA and KBD.

Cartilage samples were collected from age- and gender-matched individuals with OA (n = 9), KBD (n = 9), and normal adults (n = 9). N-glycosylated proteome quantitative analysis based on LC-MS/MS was performed to obtain differentially expressed N-glycosylated proteins in KBD and OA.

Experimental results:

A total of 594 N-glycosylated proteins and 1,146 N-glycosylated peptide segments were identified.
GO analysis revealed that N-glycosylated proteins are primarily involved in protein metabolic processes, single-cell to multicellular and multicellular organism processes, cell adhesion, biological adhesion, and multicellular organism development.
KEGG and PPI analysis showed that key N-glycosylated proteins are closely associated with pathways related to autophagy, lysosomes, protein digestion and absorption, which are relevant to OA and KBD.
Key significantly differentially expressed N-glycosylated proteins and pathways primarily disrupt the synthesis and degradation metabolism of extracellular matrix (ECM) and fundamental components of chondrocytes, as well as interfere with the transmission of substances or information, leading to degeneration and degradation of cartilage in OA and KBD. These key N-glycosylated proteins or pathways identified in this study represent potential targets for understanding the pathological mechanisms and treatment of OA and KBD.

Case 2. N-Glycoproteome Analysis Reveals Growth Mechanisms of Wheat Seedling Leaves under Drought Stress ⁽²⁾

Cell membrane proteins are involved in many complex physiological processes. Currently, research on cell membrane proteomics mainly focuses on humans and animals, with limited reports on plant cell membrane glycoproteins. Wheat is an important cereal crop. Studying cell membrane glycoproteins in wheat seedling leaves under drought stress can help identify the potential role of glycoproteins in drought stress response.

In this study, the authors conducted a comprehensive analysis of the wheat plasma membrane N-glycoproteome under drought stress for the first time, using glycopeptide HILIC enrichment and LC-MS/MS identification. A total of 414 glycosylation sites, corresponding to 407 glycopeptides and 312 glycoproteins, were identified. Among them, 173 cell membrane proteins exhibited significant changes in 215 N-glycosylation sites under drought stress. Functional enrichment analysis revealed that differentially glycosylated proteins are involved in extracellular signal reception and transduction, closely associated with protein kinase activity related to plant cell wall remodeling. Motif analysis showed that 79.5% of the motifs are located in the disordered and flexible regions on the protein surface. By reducing the flexibility of the protein, glycosylation modification can promote protein stability and enhance protein structural stability.

References

Han, Jing, et al. "Identification of N-Glycoproteins of Knee Cartilage from Adult Osteoarthritis and Kashin-Beck Disease Based on Quantitative Glycoproteomics, Compared with Normal Control Cartilage." Cells 11.16 (2022): 2513.
Chang, Yanan, et al. "Plasma membrane N-glycoproteome analysis of wheat seedling leaves under drought stress." International Journal of Biological Macromolecules 193 (2021): 1541-1550.

* For Research Use Only. Not for use in diagnostic procedures.

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