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An Integrated Proteomic and Glycoproteomic Investigation Reveals Alterations in the N-Glycoproteomic Network Induced by 2-Deoxy-D-Glucose in Colorectal Cancer Cells.

ABSTRACT: As a well-known glycolysis inhibitor for anticancer treatment, 2-Deoxy-D-glucose (2DG) inhibits the growth and survival of cancer cells by interfering with the ATP produced by the metabolism of D-glucose. In addition, 2DG inhibits protein glycosylation in vivo by competing with D-mannose, leading to endoplasmic reticulum (ER) stress and unfolded protein responses in cancer cells. However, the molecular details underlying the impact of 2DG on protein glycosylation remain largely elusive. With an integrated approach to glycoproteomics and proteomics, we characterized the 2DG-induced alterations in N-glycosylation, as well as the cascading impacts on the whole proteome using the HT29 colorectal cancer cell line as a model system. More than 1700 site-specific glycoforms, represented by unique intact glycopeptides (IGPs), were identified. The treatment of 2DG had a broad effect on the N-glycoproteome, especially the high-mannose types. The glycosite occupancy of the high-mannose N-glycans decreased the most compared with the sialic acid and fucose-containing N-glycans. Many of the proteins with down-regulated high-mannose were implicated in functional networks related to response to topologically incorrect protein, integrin-mediated signaling, lysosomal transport, protein hydroxylation, vacuole, and protein N-glycosylation. The treatment of 2DG also functionally disrupted the global cellular proteome, evidenced by significant up-regulation of the proteins implicated in protein folding, endoplasmic reticulum, mitochondrial function, cellular respiration, oxidative phosphorylation, and translational termination. Taken together, these findings reveal the complex changes in protein glycosylation and expression underlying the various effects of 2DG on cancer cells, and may provide insightful clues to inform therapeutic development targeting protein glycosylation.

SUBMITTER: Ma C

PROVIDER: S-EPMC9331968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer.

Project description:Whether the relationship between type 2 diabetes mellitus (T2DM) and osteoarthritis (OA) can be solely attributed to the shared risk factors, such as obesity, remains controversial. Several studies have revealed the critical role of abnormal glycosylation in the pathogenesis of OA and T2DM. Therefore, we speculate that T2DM may contribute to the pathogenesis of OA through the intrinsic mechanisms of N-glycosylation aberrations. Using N-glycoproteomics, we compared the changes in N-glycosylated protein abundance in cartilage samples from patients with OA without and with T2DM (DM-OA), and from patients with traumatic joint injury (NC) as controls. We identified 847 N-glycosylation sites corresponding to 729 peptides fragments from 374 proteins. The number of N-glycosylated proteins in the DM-OA group tended to decrease compared with that in the OA and NC groups. We identified 22 upregulated and 1 down-regulated N-glycosylated peptides in the OA group compared to the NC group, while only fibronectin 1 (FN1) at position N1007, cartilage intermediate layer protein 1 (CILP) at N346, and collagen type VI alpha 1 chain (COL6A1) at N804, were also identified in the DM-OA group. Compared to the OA group, the downregulation of secreted protein acidic and rich in cysteine (SPARC) at N116, collagen type VI alpha 1 chain (COL6A2) at N785, and asporin (ASPN) at N282, and the upregulation of complement component C8 alpha chain (C8α) at N437, were the most remarkable alterations in the DM-OA group. The differentially expressed N-glycosylated proteins between the OA and DM-OA groups were mainly located extracellularly and enriched in the KEGG pathways involving PI3K/Akt signaling, focal adhesion, and ECM-receptor interaction. Their predicted protein-protein interactions were also depicted. We were thus able to show the general characteristics of N-glycosylation aberrations in OA and DM-OA. Moreover, the upregulated glycosylated complement C8α in the DM-OA group might augment membrane attack complex activity, thereby exacerbating cartilage destruction. Although further confirmation is required, our hypothesis proposes a possible explanation for the deduction that T2DM is an independent risk factor for OA.

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An Integrated Proteomic and Glycoproteomic Investigation Reveals Alterations in the N-Glycoproteomic Network Induced by 2-Deoxy-D-Glucose in Colorectal Cancer Cells.

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