Ganglong Yang

Altered N-Glycan Expression Profile in Epithelial-to-Mesenchymal Transition of NMuMG Cells Revealed by an Integrated Strategy Using Mass Spectrometry and Glycogene and Lectin Microarray Analysis

Epithelial-to-mesenchymal transition (EMT) is an essential biological process that occurs in embryonic development, metastatic diseases, and cancer progression. Altered expression of glycans is known to be associated with cancer progression. No studies to date have presented global analysis of the precise variation of N-glycans in EMT. We describe here the profile of N-glycans and glycogene expression in the EMT process induced by transforming growth factor-β1 (TGFβ1) in a normal mouse mammary gland epithelial (NMuMG) cell model. An integrated strategy with a combination of mass spectrometry, glycogene microarray analysis, and lectin microarray analysis was applied, and results were confirmed by lectin histochemistry and quantitative real-time PCR. In TGFβ-induced EMT, levels of high-mannose-type N-glycans were enhanced, antennary N-glycans, and fucosylation were suppressed, and bisecting GlcNAc N-glycans were greatly suppressed. The expression of seven N-glycan-related genes was significantly changed. The products of glycogenes ALG9, MGAT3, and MGAT4B appeared to contribute to the observed alteration of N-glycans. The findings indicate that dysregulation of N-glycan synthesis plays a role in the EMT process. Systematic glycomic analysis based on the combination of techniques described here is expected to facilitate the discovery of the aberrant N-glycosylation in tumor progression and provide essential information in systems glycobiology.

Quantitative Glycome Analysis of N-Glycan Patterns in Bladder Cancer vs Normal Bladder Cells Using an Integrated Strategy

Diagnosis of bladder cancer, one of the most common types of human cancer, at an early (nonmuscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy in general is closely associated with alterations of glycan expression. Glycosylation status, particularly global glycomes, in bladder cancer has not been well studied. We integrated lectin microarray and mass spectrometry (MS) methods to quantitatively analyze and compare glycan expression in four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed using lectin microarray analysis and confirmed by lectin staining and lectin blotting. Associations of glycopatterns with diverging stages were evaluated by lectin histochemistry on tissue microarrays. N-Glycans were derivatized by amidation of sialylated glycans with acetohydrazide and reductive amination with the stable isotope tags [(12)C6]- and [(13)C6]-aniline, and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). N-Glycan biosynthesis-associated proteins were quantitatively analyzed by a stable isotope labeling by amino acids in cell culture (SILAC) proteomics method, which revealed significant differences in expression of 13 glycosyltransferases and 4 glycosidases. Our findings indicate that sialyl Lewis X (sLe(x)), terminal GalNAc and Gal, and high mannose-type N-glycans were more highly expressed in bladder cancer cells and tissues than in normal cells. Bladder cancer cells showed high expression of core-fucosylated N-glycans but low expression of terminally fucosylated N-glycans. Each of these glycome changes may be directly related to bladder cancer progression.

Alteration of N-glycans and Expression of Their Related Glycogenes in the Epithelial-Mesenchymal Transition of HCV29 Bladder Epithelial Cells

The epithelial-mesenchymal transition (EMT) is an essential step in the proliferation and metastasis of solid tumor cells, and glycosylation plays a crucial role in the EMT process. Certain aberrant glycans have been reported as biomarkers during bladder cancer progression, but global variation of N-glycans in this type of cancer has not been previously studied. We examined the profiles of N-glycan and glycogene expression in transforming growth factor-beta (TGFβ)-induced EMT using non-malignant bladder transitional epithelium HCV29 cells. These expression profiles were analyzed by mass spectrometry, lectin microarray analysis, and GlycoV4 oligonucleotide microarray analysis, and confirmed by lectin histochemistry and real-time RT-PCR. The expression of 5 N-glycan-related genes were notably altered in TGFβ-induced EMT. In particular, reduced expression of glycogene man2a1, which encodes α-mannosidase 2, contributed to the decreased proportions of bi-, tri- and tetra-antennary complex N-glycans, and increased expression of hybrid-type N-glycans. Decreased expression of fuca1 gene, which encodes Type 1 α-L-fucosidase, contributed to increased expression of fucosylated N-glycans in TGFβ-induced EMT. Taken together, these findings clearly demonstrate the involvement of aberrant N-glycan synthesis in EMT in these cells. Integrated glycomic techniques as described here will facilitate discovery of glycan markers and development of novel diagnostic and therapeutic approaches to bladder cancer.

Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method.

The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia), KK47 (low grade nonmuscle invasive bladder cancer, NMIBC), and YTS1 (metastatic bladder cancer) have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC) progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO) term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer.

Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase‑4 gene expression by Smads during transforming growth factor β‑induced epithelial‑mesenchymal transition

The process of epithelial‑mesenchymal transition (EMT) is essential for the proliferation and metastasis of tumor cells. Our previous study demonstrated that the expression of gangliotetraosylceramide (Gg4) and the transcription of UDP‑Gal: β1,3-galactosyltransferase‑4 (β3GalT4), a gene which controls the expression of Gg4, are reduced during transforming growth factor (TGF)‑β‑induced EMT in normal murine mammary gland (NMuMG) cells. The present study revealed that the transcription level of β3GalT4 in patients with breast cancer was decreased compared with healthy control subjects. However, the molecular basis underlying these effects remains to be elucidated. Analysis of the β3GalT4 promoter sequence revealed a putative Smad‑binding element (SBE) of Smad4, which is a transcriptional factor in TGFβ responses and forms a complex with Smad3. To clarify the association between the Smad3/4 complex and the β3GalT4 gene during EMT in NMuMG cells, an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay were performed. The Smad3/4 complex was found to bind directly to the SBE of the β3GalT4 promoter. Overexpression of Smad3 and Smad4 through stable transfection had no notable effect on cell phenotype, but did alter the expression of various EMT protein markers. Treatment with TGFβ reduced the expression of Gg4 and the mRNA levels of the β3GalT4 gene in Smad3‑ and Smad4‑overexpressing cells compared with vector‑transfected cells. Expression of the epithelial markers E‑cadherin and β‑catenin decreased in parallel with the reduction in Gg4. These findings suggested that the activated Smad3/4 complex downregulated the expression of Gg4 and the β3GalT4 gene through translocation into the nucleus and binding to the β3GalT4 promoter.

Alteration of N-glycan expression profile and glycan pattern of glycoproteins in human hepatoma cells after HCV infection

BACKGROUND Hepatitis C virus (HCV) infection causes chronic liver diseases, liver fibrosis and even hepatocellular carcinoma (HCC). However little is known about any information of N-glycan pattern in human liver cell after HCV infection. METHODS The altered profiles of N-glycans in HCV-infected Huh7.5.1 cell were analyzed by using mass spectrometry. Then, lectin microarray, lectin pull-down assay, reverse transcription-quantitative real time PCR (RT-qPCR) and western-blotting were used to identify the altered N-glycosylated proteins and glycosyltransferases. RESULTS Compared to uninfected cells, significantly elevated levels of fucosylated, sialylated and complex N-glycans were found in HCV infected cells. Furthermore, Lens culinaris agglutinin (LCA)-binding glycoconjugates were increased most. Then, the LCA-agarose was used to precipitate the specific glycosylated proteins and identify that fucosylated modified annexin A2 (ANXA2) and heat shock protein 90 beta family member 1 (HSP90B1) was greatly increased in HCV-infected cells. However, the total ANXA2 and HSP90B1 protein levels remained unchanged. Additionally, we screened the mRNA expressions of 47 types of different glycosyltransferases and found that α1,6-fucosyltransferase 8 (FUT8) was the most up-regulated and contributed to strengthen the LCA binding capability to fucosylated modified ANXA2 and HSP90B1 after HCV infection. CONCLUSIONS HCV infection caused the altered N-glycans profiles, increased expressions of FUT8, fucosylated ANXA2 and HSP90B1 as well as enhanced LCA binding to Huh7.5.1. GENERAL SIGNIFICANCE Our results may lay the foundation for clarifying the role of N-glycans and facilitate the development of novel diagnostic biomarkers and therapeutic targets based on the increased FUT8, fucosylated ANXA2 and HSP90B1 after HCV infection.

A lectin-based isolation/enrichment strategy for improved coverage of N-glycan analysis

Glycomics provides an increasingly useful research tool as the genomes and proteomes of more and more animal species are elucidated. In view of the general complexity and heterogeneity of glycans, improved depth-of-coverage and sensitivity are required for glycosylation analysis. In this study, we established the lectin-based isolation/enrichment strategy for total glycomic information. Specific lectins are added onto the filter to capture corresponding glycans prior to release of N-glycans by peptide N-glycosidase F (PNGase F). Non-bound glycans and bound glycans are released and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Application of the strategy to chicken ovalbumin, normal mouse mammary epithelial cells (NMuMG), and human serum resulted in detection of 5, 6, and 11 additional N-glycan structures, respectively. The strategy facilitates identification of intact N-glycans in biological samples, and can be extended to detailed analysis of O-glycome or glycoproteome.

Identification of aberrantly expressed glycans in gastric cancer by integrated lectin microarray and mass spectrometric analyses

Cancer progression is usually associated with alterations of glycan expression patterns. Little is known regarding global glycomics in gastric cancer, the most common type of epithelial cancer. We integrated lectin microarray and mass spectrometry (MS) methods to profile glycan expression in three gastric cancer cell lines (SGC-7901, HGC-27, and MGC-803) and one normal gastric epithelial cell line (GES-1). Significantly altered glycans were confirmed by lectin staining and MALDI-TOF/TOF-MS. The three cancer cell lines showed increased levels of core-fucosylated N-glycans, GalNAcα-Ser/Thr (Tn antigen), and Sia2-6Galβ1-4GlcNAc N-glycans, but reduced levels of biantennary N-glycans, Galβ1-3GalNAcα-Ser/Thr (T antigen), and (GlcNAc)n N-glycans. Lectin histochemistry was used to validate aberrant expression of four representative glycans (core-fucosylation, Sia2-6Galβ1-4GlcNAc, biantennary N-glycans, T antigen, recognized respectively by lectins LCA, SNA, PHA-E+L, and ACA) in clinical gastric cancer samples. Lower binding capacity for ACA was correlated with significantly poorer patient prognosis. Our findings indicate for the first time that glycans recognized by LCA, ACA, and PHA-E+L are aberrantly expressed in gastric cancer, and suggest that ACA is a potential prognostic factor for gastric cancer.

Quantitative Analysis of Differential Proteome Expression in Epithelial-to-Mesenchymal Transition of Bladder Epithelial Cells Using SILAC Method

Epithelial-to-mesenchymal transition (EMT) is an essential biological process involved in embryonic development, cancer progression, and metastatic diseases. EMT has often been used as a model for elucidating the mechanisms that underlie bladder cancer progression. However, no study to date has addressed the quantitative global variation of proteins in EMT using normal and non-malignant bladder cells. We treated normal bladder epithelial HCV29 cells and low grade nonmuscle invasive bladder cancer KK47 cells with transforming growth factor-beta (TGF-β) to establish an EMT model, and studied non-treated and treated HCV29 and KK47 cells by the stable isotope labeling amino acids in cell culture (SILAC) method. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography/LTQ Orbitrap mass spectrometry. Among a total of 2994 unique identified and annotated proteins in HCV29 and KK47 cells undergoing EMT, 48 and 56 proteins, respectively, were significantly upregulated, and 106 and 24 proteins were significantly downregulated. Gene ontology (GO) term analysis and pathways analysis indicated that the differentially regulated proteins were involved mainly in enhancement of DNA maintenance and inhibition of cell-cell adhesion. Proteomes were compared for bladder cell EMT vs. bladder cancer cells, revealing 16 proteins that displayed similar changes in the two situations. Studies are in progress to further characterize these 16 proteins and their biological functions in EMT.

Ganglioside-magnetosome complex formation enhances uptake of gangliosides by cells

Bacterial magnetosomes, because of their nano-scale size, have a large surface-to-volume ratio and are able to carry large quantities of bioactive substances such as enzymes, antibodies, and genes. Gangliosides, a family of sialic acid-containing glycosphingolipids, function as distinctive cell surface markers and as specific determinants in cellular recognition and cell-to-cell communication. Exogenously added gangliosides are often used to study biological functions, transport mechanisms, and metabolism of their endogenous counterparts. Absorption of gangliosides into cells is typically limited by their tendency to aggregate into micelles in aqueous media. We describe here a simple strategy to remove proteins from the magnetosome membrane by sodium dodecyl sulfate treatment, and efficiently immobilize a ganglioside (GM1 or GM3) on the magnetosome by mild ultrasonic treatment. The maximum of 11.7±1.2 µg GM1 and 11.6±1.5 μg GM3 was loaded onto 1 mg magnetosome, respectively. Complexes of ganglioside-magnetosomes stored at 4°C for certain days presented the consistent stability. The use of GM1-magnetosome complex resulted in the greatest enhancement of ganglioside incorporation by cells. GM3-magnetosome complex significantly inhibited EGF-induced phosphorylation of the epidermal growth factor receptor. Both of these effects were further enhanced by the presence of a magnetic field.