O-GlcNAcylation is a key post-translational modification that modifies the functions of proteins. Associations between O-GlcNAcylation, shorter survival of cholangiocarcinoma (CCA) patients, and increased migration/invasion of CCA cell lines have been reported. However, the specific O-GlcNAcylated proteins (OGPs) that participate in promotion of CCA progression are poorly understood. OGPs were isolated from human CCA cell lines, KKU-213 and KKU-214, using a click chemistry-based enzymatic labeling system, identified using LC-MS/MS, and searched against an OGP database. From the proteomic analysis, a total of 21 OGPs related to cancer progression were identified, of which 12 have not been previously reported. Among these, hnRNP-K, a multifaceted RNA- and DNA-binding protein known as a pre-mRNA-binding protein, was one of the most abundantly expressed, suggesting its involvement in CCA progression. O-GlcNAcylation of hnRNP-K was further verified by anti-OGP/anti-hnRNP-K immunoprecipitations and sWGA pull-down assays. The perpetuation of CCA by hnRNP-K was evaluated using siRNA, which revealed modulation of cyclin D1, XIAP, EMT markers, and MMP2 and MMP7 expression. In native CCA cells, hnRNP-K was primarily localized in the nucleus; however, when O-GlcNAcylation was suppressed, hnRNP-K was retained in the cytoplasm. These data signify an association between nuclear accumulation of hnRNP-K and the migratory capabilities of CCA cells. In human CCA tissues, expression of nuclear hnRNP-K was positively correlated with high O-GlcNAcylation levels, metastatic stage, and shorter survival of CCA patients. This study demonstrates the significance of O-GlcNAcylation on the nuclear translocation of hnRNP-K and its impact on the progression of CCA.

The leading cause of death in cancer patients is metastasis, for which an effective treatment is still necessary. During metastasis, cancer cells aberrantly express several glycans that are correlated with poor patient outcome. This study was aimed toward exploring the effects of O-GlcNAcylation on membranous N-glycans that are associated with the progression of cholangiocarcinoma (CCA). Global O-GlcNAcylation in CCA cells was depleted using specific siRNA against O-GlcNAc transferase (OGT), which transfers GlcNAc to the acceptor proteins. Using an HPLC-Chip/Time-of-Flight (Chip/TOF) MS system, the N-glycans associated with O-GlcNAcylation were identified by comparing the membranous N-glycans of siOGT-treated cells with those of scramble siRNA-treated cells. In parallel, the membranous N-glycans of the parental cells (KKU-213 and KKU-214) were compared with those of the highly metastatic cells (KKU-213L5 and KKU-214L5). Together, these data revealed that high mannose (Hex₉HexNAc₂) and biantennary complex (Hex₅HexNAc₄Fuc₁NeuAc₁) N-linked glycans correlated positively with metastasis. We subsequently demonstrate that suppression of O-GlcNAcylation decreased the expression of these two N-glycans, suggesting that O-GlcNAcylation mediates their levels in CCA. In addition, the ability of highly metastatic cells to migrate and invade was reduced by the presence of Pisum Sativum Agglutinin (PSA), a mannose-specific lectin, further indicating the association of high mannose type N-glycans with CCA metastasis. The molecular mechanism of O-GlcNAc-mediated progression of CCA was shown to proceed via a series of signaling events, involving the activation of Akt/Erk (i), an increase in FOXO3 phosphorylation (ii), which results in the reduction of MAN1A1 expression (iii) and thus the accumulation of Hex₉HexNAc₂ N-glycans (iv). This study demonstrates for the first time the association between O-GlcNAcylation, high mannose type N-glycans, and the progression of CCA metastasis, suggesting a novel therapeutic target for treatment of metastatic CCA.

Given that unnatural sugar expression is metabolically achieved, the kinetics and disposition of incorporation can lend insight into the temporal and localization preferences of sialylation across the cell surface. However, common detection schemes lack the ability to detail the molecular diversity and distribution of target moieties. Here we employed a mass spectrometric approach to trace the placement of azido sialic acids on membrane glycoconjugates, which revealed substantial variations in incorporation efficiencies between N-/O-glycans, glycosites, and glycosphingolipids. To further explore the propensity for sialylation, we subsequently mapped the native glycome of model epithelial cell surfaces and illustrate that while glycosylation sites span broadly across the extracellular region, a higher number of heterogeneous glycoforms occur on sialylated sites closest to the transmembrane domain. Beyond imaging techniques, this integrative approach provides unprecedented details about the frequency and structure-specific distribution of cell surface sialylation, a critical feature that regulates cellular interactions and homeostatic pathways.

Membrane-bound oligosaccharides form the interfacial boundary between the cell and its environment, mediating processes such as adhesion and signaling. These structures can undergo dynamic changes in composition and expression based on cell type, external stimuli, and genetic factors. Glycosylation, therefore, is a promising target of therapeutic interventions for presently incurable forms of advanced cancer. Here, we show that cholangiocarcinoma metastasis is characterized by down-regulation of the Golgi α-mannosidase I coding gene MAN1A1, leading to elevation of extended high-mannose glycans with terminating α-1,2-mannose residues. Subsequent reshaping of the glycome by inhibiting α-mannosidase I resulted in significantly higher migratory and invasive capabilities while masking cell surface mannosylation suppressed metastasis-related phenotypes. Exclusive elucidation of differentially expressed membrane glycoproteins and molecular modeling suggested that extended high-mannose glycosylation at the helical domain of transferrin receptor protein 1 promotes conformational changes that improve noncovalent interaction energies and lead to enhancement of cell migration in metastatic cholangiocarcinoma. The results provide support that α-1,2-mannosylated N-glycans present on cancer cell membrane proteins may serve as therapeutic targets for preventing metastasis.

Membrane-bound oligosaccharides form the interfacial boundary between the cell and its environment, mediating processes such as adhesion and signaling. These structures can undergo dynamic changes in composition and expression based on cell type, external stimuli, and genetic factors. Glycosylation, therefore, is a promising target of therapeutic interventions for presently incurable forms of advanced cancer. Here, we show that cholangiocarcinoma metastasis is characterized by down-regulation of the Golgi α-mannosidase I coding gene MAN1A1, leading to elevation of extended high-mannose glycans with terminating α-1,2-mannose residues. Subsequent reshaping of the glycome by inhibiting α-mannosidase I resulted in significantly higher migratory and invasive capabilities while masking cell surface mannosylation suppressed metastasis-related phenotypes. Exclusive elucidation of differentially expressed membrane glycoproteins and molecular modeling suggested that extended high-mannose glycosylation at the helical domain of transferrin receptor protein 1 promotes conformational changes that improve noncovalent interaction energies and lead to enhancement of cell migration in metastatic cholangiocarcinoma. The results provide support that α-1,2-mannosylated N-glycans present on cancer cell membrane proteins may serve as therapeutic targets for preventing metastasis.