Cell surface-specific N-glycan profiling in breast cancer

Aberrant changes in specific glycans have been shown to be associated with immunosurveillance, tumorigenesis, tumor progression and metastasis. In this study, the N-glycan profiling of membrane proteins from human breast cancer cell lines and tissues was detected using modified DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). The N-glycan profiles of membrane proteins were analyzed from 7 breast cancer cell lines and MCF 10A, as well as from 100 pairs of breast cancer and corresponding adjacent tissues. The results showed that, compared with the matched adjacent normal tissue samples, two biantennary N-glycans (NA2 and NA2FB) were significantly decreased (p <0.0001) in the breast cancer tissue samples, while the triantennary glycan (NA3FB) and a high-mannose glycan (M8) were dramatically increased (p = 0.001 and p <0.0001, respectively). Moreover, the alterations in these specific N-glycans occurred through the oncogenesis and progression of breast cancer. These results suggested that the modified method based on DSA-FACE is a high-throughput detection technology that is suited for analyzing cell surface N-glycans. These cell surface-specific N-glycans may be helpful in recognizing the mechanisms of tumor cell immunologic escape and could be potential targets for new breast cancer drugs

Specific N-glycans of Hepatocellular Carcinoma Cell Surface and the Abnormal Increase of Core-α-1, 6-fucosylated Triantennary Glycan via N-acetylglucosaminyltransferases-IVa Regulation

Glycosylation alterations of cell surface proteins are often observed during the progression of malignancies. The specific cell surface N-glycans were profiled in hepatocellular carcinoma (HCC) with clinical tissues (88 tumor and adjacent normal tissues) and the corresponding serum samples of HCC patients. The level of core-α-1,6-fucosylated triantennary glycan (NA3Fb) increased both on the cell surface and in the serum samples of HCC patients (p \u3c 0.01). Additionally, the change of NA3Fb was not influenced by Hepatitis B virus (HBV)and cirrhosis. Furthermore, the mRNA and protein expression of N-acetylglucosaminyltransferase IVa (GnT-IVa), which was related to the synthesis of the NA3Fb, was substantially increased in HCC tissues. Knockdown of GnT-IVa leads to a decreased level of NA3Fb and decreased ability of invasion and migration in HCC cells. NA3Fb can be regarded as a specific cell surface N-glycan of HCC. The high expression of GnT-IVa is the cause of the abnormal increase of NA3Fb on the HCC cell surface, which regulates cell migration. This study demonstrated the specific N-glycans of the cell surface and the mechanisms of altered glycoform related with HCC. These findings lead to better understanding of the function of glycan and glycosyltransferase in the tumorigenesis, progression and metastasis of HCC

Specific N-glycans of Hepatocellular Carcinoma Cell Surface and the Abnormal Increase of Core-α-1, 6-fucosylated Triantennary Glycan via N-acetylglucosaminyltransferases-IVa Regulation

Glycosylation alterations of cell surface proteins are often observed during the progression of malignancies. The specific cell surface N-glycans were profiled in hepatocellular carcinoma (HCC) with clinical tissues (88 tumor and adjacent normal tissues) and the corresponding serum samples of HCC patients. The level of core-α-1,6-fucosylated triantennary glycan (NA3Fb) increased both on the cell surface and in the serum samples of HCC patients (p \u3c 0.01). Additionally, the change of NA3Fb was not influenced by Hepatitis B virus (HBV)and cirrhosis. Furthermore, the mRNA and protein expression of N-acetylglucosaminyltransferase IVa (GnT-IVa), which was related to the synthesis of the NA3Fb, was substantially increased in HCC tissues. Knockdown of GnT-IVa leads to a decreased level of NA3Fb and decreased ability of invasion and migration in HCC cells. NA3Fb can be regarded as a specific cell surface N-glycan of HCC. The high expression of GnT-IVa is the cause of the abnormal increase of NA3Fb on the HCC cell surface, which regulates cell migration. This study demonstrated the specific N-glycans of the cell surface and the mechanisms of altered glycoform related with HCC. These findings lead to better understanding of the function of glycan and glycosyltransferase in the tumorigenesis, progression and metastasis of HCC

Comparative Analysis of the Transcriptional Response of Tolerant and Sensitive Wheat Genotypes to Drought Stress in Field Conditions

Drought stress is one of the most adverse environmental limiting factors for wheat (Triticum aestivum L.) productivity worldwide. For better understanding of the molecular mechanism of wheat in response to drought, a comparative transcriptome approach was applied to investigate the gene expression change of two wheat cultivars, Jimai No. 47 (drought-tolerant) and Yanzhan No. 4110 (drought-sensitive) in the field under irrigated and drought-stressed conditions. A total of 3754 and 2325 differential expressed genes (DEGs) were found in Jimai No. 47 and Yanzhan No. 4110, respectively, of which 377 genes were overlapped, which could be considered to be the potential drought-responsive genes. GO (Gene Ontology) analysis showed that these DEGs of tolerant genotype were significantly enriched in signaling transduction and MAP (mitogen-activated protein) kinase activity, while that of sensitive genotype was involved in photosynthesis, membrane protein complex, and guard cell differentiation. Furthermore, 32 and 2 RNA editing sites were identified in drought-tolerant and sensitive genotypes under drought compared to irrigation, demonstrating that RNA editing also plays an important role in response to drought in wheat. This study investigated the gene expression pattern and RNA editing sites of two wheat cultivars with contrasting tolerance in field condition, which will contribute to a better understanding of the molecular mechanism of drought tolerance in wheat and beyond

Human serum N-glycan profiles are age and sex dependent

Design, setting and participants: a total of 265 healthy northern Chinese men and women were grouped by age and gender. The mean age in males and females was similar. Objective: the study is aimed to evaluate the effects of the age and gender on the human serum N-glycans profiles in the clinical diagnose of ageing and disease. Methods: the 265 human serum N-glycan profiles were obtained by DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Comparison of N-glycan profiles was carried out among the different genders and age groups and the data were analysed with the GeneMapper software. Results: age-related changes in the three N-glycan structures (NGA2F, NGA2FB and NA2F) were observed. Interestingly, fucosylation of N-glycans was significantly different (P < 0.0001) between men and women: more core-alpha-1,6-fucosylated glycans were detected in women, whereas more branching-alpha-1,3-fucosylated N-glycans were seen in men. Conclusions: the N-glycome profile in serum is gender and age dependent. This should be taken into consideration in the development of serum glycome markers

The representative N-glycan profiles of membrane proteins from human breast cancer cell line and tissue.

<p>The desialylated N-glycan profiles of membrane proteins from cell lines (n=8) and tumor tissues (n=100). Oligomaltose is used as a sugar mass reference. The number of glucose units (degree of polymerization, DP) in these structures is indicated. N-glycan profiles from RNaseB and serum were used as N-glycan profile controls. RNaseB contained high mannose from M5 to M9. Serum contained various complex N-glycans and the most abundant glycans detected in serum are marked. The vertical axis represents the glycan intensity of the peaks as a percent of the relative fluorescence level. The X-axis represents the retention time of the peaks. The N-glycan structures of the corresponding peaks are shown below the panels. NGA2F is an agalacto core-α-1, 6-fucosylated biantennary glycan; NA2 is a bigalacto biantennary glycan; NA2F is a bigalacto core-α-1, 6-fucosylated biantennary glycan; NA2FB is a bigalacto core-α-1, 6-fucosylated bisecting biantennary glycan; NA3 is tri-antennary; NA3FB is a core-α-1, 6-fucosylated triantennary glycan. The symbols used in the structural formulas are as follows: (○) β-linked N-acetylglucosamine; (●) β-linked galactose; (□) α-linked mannose; (■) β-linked mannose; (▲) α-1, 6-linked fucose.</p

Establishment of the DSA-FACE method for analyzing N-glycan profiling in breast cancer.

<p>(A) Equal amounts (20 μg and 100 μg for cell line and tissue specimens, respectively) of cytoplasmic and membrane proteins (CP and MP) from Bcap 37 cells and tumor tissue were determined by Western blotting using γ-tubulin and Na⁺/K⁺ ATPase-α1 antibody, respectively. Na⁺/K⁺ ATPase-α1 (an integral membrane protein) and γ-tubulin (a core component of the centrosome) were chosen as the cytomembrane and cytoplasm markers, respectively. (B) The representative N-glycan profiles of cytosolic and membrane proteins from cell line (upper panel, n=8) and tissue (lower panel, n=200).</p

The exoglycosidase sequencing of the membrane protein N-glycans from tumor tissues.

<p>(A) Exoglycosidase sequencing of N-glycans of membrane proteins from breast cancer tissue (lower panel) and glycoproteins from healthy human serum as reference (upper panel) to verify the structures of peaks B1, B4 and B5. The structures of peaks B2 and B3 are confirmed in (B) and (C), respectively. The total N-glycans were treated with single or combined exoglycosidase arrays as indicated in context. The arrow lines indicate the changes in glycan peaks that underwent glycosidase digestion. The nomenclature of N-glycans and symbolic representations correspond to those in Figure 2.</p

The significantly changed N-glycans of the membrane proteins of human breast cancer cell lines.

<p>(A) The representative N-glycan profiles of membrane proteins from human breast cancer cell lines (n=7). MCF 10A: from breast fibrocystic disease as a non-tumorigenic epithelial cell line; SK-BR-3, MCF-7, Bcap 37, MDA-MB-231, T-47D, Hs 578T and ZR-75-30: cancer cell lines; Serum: from healthy humans. (B–D) The statistically significant difference in N-glycan peaks B1, B2, B4 and B5 between MCF 10A and various cancer cell lines. Peak B2 is significantly increased in cancer cell lines compared with MCF 10A. On the contrary, peaks B1 and B4 are dramatically decreased in cancer cell lines. Particularly, peak B4 is completely lost in Bcap 37, MDA-MB-231 and Hs 578T cell lines. The percentages of each specific peak height in the total peak heights are expressed as mean ± SD. Asterisks indicate statistically significant differences between the various cancer cell lines and the MCF 10A cell line (* <i>p</i> < 0.05, ** <i>p</i> < 0.01). Five major glycan peaks B1-B5 in normal control and breast cancer groups were detected. Experiments were repeated three times.</p