Identification of N-Glycosylation in Hepatocellular Carcinoma Patients’ Serum with a Comparative Proteomic Approach

Huang, Yingnan; Wu, Hao; Xue, Ruyi; Liu, Taotao; Dong, Ling; Yao, Jun; Zhang, Yang; Shen, Xin

doi:10.1371/journal.pone.0077161

Cited by 9 publications

(9 citation statements)

References 35 publications

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“…Glycosylation-site analysis is critical to reveal these modifications. It provides overall insights into the number and identity of proteins which may change their glycosylation in response to specific diseases [7,8]. Moreover, site-specific glycan structural analysis becomes more straightforward once the glycosylation sites are determined.…”

Section: Introductionmentioning

confidence: 99%

From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins

Bai

Zhu

et al. 2015

Anal Bioanal Chem

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O-glycosylation-site characterization of individual glycoproteins is a major challenge because of the heterogeneity of O-glycan core structures. In proteomic studies, O-glycosylation-site analysis is even more difficult because of the complexity of the sample. In this work, we designed a rapid and convenient workflow for characterizing the O-glycosylation sites of individual proteins and the human-plasma proteome. A mixture of exoglycosidases was used to partially remove O-glycan chains and leave an N-acetylgalacosamine (GalNAc) residue attached to the Ser or Thr residues. The O-glycosylated peptides could then be identified by using liquid chromatography-tandem mass spectrometry (LC-MS-MS) to detect the 203 Da mass increase. Jacalin was used to selectively isolate O-GalNAc glycopeptides before LC-MS-MS analysis, which is optional for individual proteins and necessary for complex human-plasma proteins. Bovine fetuin and human chorionic gonadotropin (hCG) were used to test the analytical workflow. The workflow indicated superior sensitivity by not only covering most previously known O-glycosylation sites but also discovering several novel sites. Using only one drop of blood, a total of 49 O-GalNAc-linked glycopeptides from 36 distinctive glycoproteins in human plasma were identified unambiguously. The approach described herein is simple, sensitive, and global for site analysis of core 1 through core 4 O-glycosylated proteins.

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Section: Introductionmentioning

confidence: 99%

From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins

Bai

Zhu

et al. 2015

Anal Bioanal Chem

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“…Fucose‐specific lectins have shown to play an important role in identifying aberrant fucosylation in pathological conditions such as cancer 5 . AFP is an important serum glycoprotein, which is aberrantly glycosylated and secreted by HCC 27‐29 . Hence, AFP is the most reliable widely used tumour marker for the diagnosis of HCC.…”

Section: Discussionmentioning

confidence: 99%

The fucose‐specific lectin ANL from Aspergillus niger possesses anti‐cancer activity by inducing the intrinsic apoptosis pathway in hepatocellular and colon cancer cells

Jagadeesh

Belur

Campbell

2021

Cell Biochemistry & Function

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The L‐fucose‐specific lectin from Aspergillus niger (ANL), isolated from the corneal smears of a keratitis patient was reported earlier. Here, we examined the interaction of ANL with human hepatocellular and colon cancer cells, evaluated its anti‐cancer activity and diagnostic potential to detect aberrantly glycosylated tumour‐associated serum glycoproteins such as alpha‐fetoprotein (AFP). We observed that ANL strongly bound to both HepG2 and HT‐29 cell‐lines and this interaction was effectively blocked with L‐fucose and mucin in a dose and time‐dependent manner with an IC50 of 1.25 and 5 μg/mL for HepG2 and HT‐29 cells respectively at 48 hours. ANL treatment increased hypodiploidy and decreased the number of HepG2 cell in G0‐G1 phase at both 24 and 48 hours. Furthermore, ANL increased the level of apoptosis in both HepG2 and HT‐29 cells in a time‐dependent manner via enhanced production of reactive oxygen species and altered mitochondrial membrane potential, indicative of intrinsic apoptotis pathway activation. Immunoblot analysis confirmed the time‐dependent elevation of levels of cytochrome c, initiator caspase‐9 and activation of caspase‐3. ANL immunohistochemistry on colon cancer tissue and quantification of AFP in HCC patient serum samples by developing an ANL‐anti‐AFP antibody sandwich enzyme‐linked immunosorbent assay confirmed the diagnostic potential of ANL. Here, interaction of ANL with AFP could be effectively blocked in the presence of competing fucose‐bearing glycans. We found ANL to be more sensitive than Lens culinaris lectin, a well‐known fucose‐specific lectin and currently used diagnostic agent. ANL can be further explored as a diagnostic and anti‐cancer agent.

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“…Aberrant serum total N -glycome is a prominently serological marker of chronic and end-stage liver diseases [ 22 , 23 , 24 , 25 , 26 ]. Being the most abundant circulating N -glycoprotein, IgG takes charge of the alternation in serum total N -glycome in disease status, even though it is not originated from the liver [ 27 ].…”

Section: Discussionmentioning

confidence: 99%

Fucosyl-Agalactosyl IgG1 Induces Cholangiocarcinoma Metastasis and Early Recurrence by Activating Tumor-Associated Macrophage

Chang

Tsai

2018

Cancers

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Concern over roles of serum IgG agalactosylation in chronic inflammatory diseases has been mounting for years but less touched in cancers. The present study addressed the underlying role of agalactosylated IgG beyond tumorigenesis. Liquid-chromatography-tandem mass spectrometry was leveraged for the analysis of IgG1 and IgG2 N-glycomes. We found that a high percentage of serum fucosyl-agalactosyl IgG1 (IgG1-G0F) in patients with cholangiocarcinoma was associated with poor tumor differentiation and tumor metastasis. Results from Kaplan–Meier analyses and a stepwise Cox regression analysis showed that patients with serum IgG1-G0F ≥40% were highly correlated with poor recurrence-free survivals and overall survivals. Interestingly, patients with cholangiocarcinoma whose serum IgG1-G0F ≥40% had more CD163+ tumor-associated macrophages in cancerous tissues than adjacent non-cancerous counterparts. In vitro assays revealed that agalactosylated IgG upregulated tumor-associated macrophage markers CD163 and CD204 in human U-937 cells and peripheral macrophages. Moreover, a positive and a negative feedback loop of transforming growth factor-β1 and interferon-γ, respectively, on IgG agalactosylation was identified using hybridoma cells and verified in sera of the patients. In conclusion, agalactosylated IgG activates tumor-associated macrophages, thereby promoting tumor metastasis and recurrence of cholangiocarcinoma.

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Identification of N-Glycosylation in Hepatocellular Carcinoma Patients’ Serum with a Comparative Proteomic Approach

Cited by 9 publications

References 35 publications

From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins

From individual proteins to proteomic samples: characterization of O-glycosylation sites in human chorionic gonadotropin and human-plasma proteins

The fucose‐specific lectin ANL from Aspergillus niger possesses anti‐cancer activity by inducing the intrinsic apoptosis pathway in hepatocellular and colon cancer cells

Fucosyl-Agalactosyl IgG1 Induces Cholangiocarcinoma Metastasis and Early Recurrence by Activating Tumor-Associated Macrophage

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