Comparative N-Glycoproteomics Reveals Subtype-Specific N-Glycosylation Signatures and Immune Associations in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) comprises intrahepatic (iCCA) and extrahepatic (eCCA) subtypes, each exhibiting distinct molecular characteristics. Understanding these differences is critical for identifying subtype-specific therapeutic targets and advancing precision medicine. Protein glycosylation, a key post-translational modification, regulates immune evasion and metastasis, yet the glycoproteomic difference between iCCA and eCCA remains unexplored. Here we presented the first comprehensive N-glycoproteomic profile of eCCA and compared it with iCCA using a publicly available dataset. Our N-glycoproteomic analysis of paired eCCA tumors and normal adjacent tissues (NATs) identified 8,372 N-glycopeptides, 3,467 N-glycosites, and 2,627 N-glycoproteins. Comparative analysis revealed distinct N-glycosylation signature, with eCCA exhibiting higher fucosylated glycans and iCCA showing increased sialylation. Pathway enrichment analysis of N-glycoproteins revealed a more prominent lysosome-related enrichment in eCCA, whereas pathways related to immune modulation, cytoskeletal components, and the extracellular matrix were significantly enriched in both subtypes. Immune profiling revealed an immunosuppressive microenvironment in both eCCA and iCCA, characterized by reduced natural killer cell infiltration and subtype-specific fibroblast and endothelial cell remodeling. DPM1, a glycosylation enzyme highly expressed in eCCA, was associated with tumor-specific N-glycopeptides and reduced immune cell infiltration. Its knockdown impaired cell migration, and glycoproteomic analysis implicated DPM1 in regulating adhesion, proteostasis, and immune pathways, highlighting its potential as a therapeutic target in eCCA. Our findings provide insights into N-glycosylation alterations in CCA subtypes, underscoring N-glycosylation-related mechanisms as potential biomarkers and therapeutic targets, particularly in eCCA.