Interleukin-22 regulates B3GNT7 expression to induce fucosylation of glycoproteins in intestinal epithelial cells.

Interleukin (IL)-22 is a cytokine that plays a critical role in intestinal epithelial homeostasis. Its downstream functions are mediated through interaction with the heterodimeric IL-22 receptor and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL-22 signaling can induce transcription of genes necessary for intestinal epithelial cell proliferation, tissue regeneration, tight junction fortification, and antimicrobial production. Recent studies have also implicated IL-22 signaling in the regulation of intestinal epithelial fucosylation in mice. However, whether IL-22 regulates intestinal fucosylation in human intestinal epithelial cells and the molecular mechanisms that govern this process are unknown. Here, in experiments performed in human cell lines and human-derived enteroids, we show that IL-22 signaling regulates expression of the B3GNT7 transcript, which encodes a ß1-3-N-acetylglucosaminyltransferase that can participate in the synthesis of poly-N-acetyllactosamine (polyLacNAc) chains. Additionally, we find that IL-22 signaling regulates levels of the α1-3-fucosylated Lewis X (Lex) blood group antigen, and that this glycan epitope is primarily displayed on O-glycosylated intestinal epithelial glycoproteins. Moreover, we show that increased expression of B3GNT7 alone is sufficient to promote increased display of Lex-decorated carbohydrate glycan structures primarily on O-glycosylated intestinal epithelial glycoproteins. Together, these data identify B3GNT7 as an intermediary in IL-22-dependent induction of fucosylation of glycoproteins and uncover a novel role for B3GNT7 in intestinal glycosylation.

Down-regulation of OGT promotes cisplatin resistance by inducing autophagy in ovarian cancer.

Cisplatin resistance significantly affects the survival rate of patients with ovarian cancer. However, the main mechanism underlying cisplatin resistance in ovarian cancer remains unclear. Methods: Immunohistochemistry was used to determine the expression of OGT, OGA and O-GlcNAc in chemoresistant and chemosensitive ovarian cancer tissues. Functional analyses (in vitro and in vivo) were performed to confirm the role of OGT in cisplatin resistance. Autophagy-related proteins were tested by western blot. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus reporter were used for autophagy flux analysis. Immunoprecipitation assay was utilized to detect protein-protein interactions. Results: We found that O-GlcNAc and O-GlcNAc transferase (OGT) levels were significantly lower in chemoresistant ovarian cancer tissues than in chemosensitive tissues, whereas O-GlcNAcase (OGA) levels did not differ. The down-regulation of OGT increased cisplatin resistance in ovarian cancer cells but had no effect on the efficacy of paclitaxel. The down-regulation of OGT improved tumor resistance to cisplatin in a mouse xenograft tumor model. OGT knockdown enhanced cisplatin-induced autophagy, which reduced apoptotic cell death induced by cisplatin, and promoted autolysosome formation. A reduction in O-GlcNAcylated SNAP-29 levels caused by the down-regulation of OGT promoted the formation of the SNARE complex and autophagic flux. Conclusion: Our findings suggest that down-regulation of OGT enhances cisplatin-induced autophagy via SNAP-29, resulting in cisplatin-resistant ovarian cancer. OGT may represent a novel target for overcoming cisplatin resistance in ovarian cancer.

A Conserved Splicing Silencer Dynamically Regulates O-GlcNAc Transferase Intron Retention and O-GlcNAc Homeostasis.

Modification of nucleocytoplasmic proteins with O-GlcNAc regulates a wide variety of cellular processes and has been linked to human diseases. The enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) add and remove O-GlcNAc, but the mechanisms regulating their expression remain unclear. Here, we demonstrate that retention of the fourth intron of OGT is regulated in response to O-GlcNAc levels. We further define a conserved intronic splicing silencer (ISS) that is necessary for OGT intron retention. Deletion of the ISS in colon cancer cells leads to increases in OGT, but O-GlcNAc homeostasis is maintained by concomitant increases in OGA protein. However, the ISS-deleted cells are hypersensitive to OGA inhibition in culture and in soft agar. Moreover, growth of xenograft tumors from ISS-deleted cells is compromised in mice treated with an OGA inhibitor. Thus, ISS-mediated regulation of OGT intron retention is a key component in OGT expression and maintaining O-GlcNAc homeostasis.

Membrane topology of human monoacylglycerol acyltransferase-2 and identification of regions important for its localization to the endoplasmic reticulum.

Acyl CoA:2-monoacylglycerol acyltransferase (MGAT)-2 has an important role in dietary fat absorption in the intestine. MGAT2 resides in the endoplasmic reticulum and catalyzes the synthesis of diacylglycerol which is then utilized as a substrate for triacylglycerol synthesis. This triacylglycerol is then incorporated into chylomicrons which are released into the circulation. In this study, we determined the membrane topology of human MGAT2. Protease protection experiments showed that the C-terminus is exposed to the cytosol, while the N-terminus is partially buried in the ER membrane. MGAT2, like murine DGAT2, was found to have two transmembrane domains. We also identified a region of MGAT2 associated with the ER membrane that contains the histidine-proline-histidine-glycine sequence present in all DGAT2 family members that is thought to comprise the active site. Proteolysis experiments demonstrated that digestion of total cellular membranes from cells expressing MGAT2 with trypsin abolished MGAT activity, indicating that domains that are important for catalysis face the cytosol. We also explored the role that the five cysteines residues present in MGAT2 have in catalysis. MGAT activity was sensitive to two thiol modifiers, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). Furthermore, mutation of four cysteines resulted in a reduction in MGAT activity. However, when the C-terminal cysteine (C334) was mutated, MGAT activity was actually higher than that of wild-type FL-MGAT2. Lastly, we determined that both transmembrane domains of MGAT2 are important for its ER localization, and that MGAT2 is present in mitochondrial-associated membranes.

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch.

Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the ß3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.

Radiosensitisation of human glioma cells by inhibition of ß1,6-GlcNAc branched N-glycans.

Gliomas are the most prevalent type of primary brain tumors and are resistant to radiation therapy. ß1,6-GlcNAc branched N-glycans, which are encoded by N-acetylglucosaminyltransferase V (GnT-V), play important roles in glioma progression. However, the relationship between ß1,6-GlcNAc branched expression and radiosensitivity in glioma cells is still unknown. In this study, the expression of ß1,6-GlcNAc branched N-glycans in nonneoplastic brain and glioma samples was characterized by lectin histochemistry. The radiosensitivity of glioma cells was evaluated by colony formation assay. We found that ß1,6-GlcNAc branches were highly expressed in glioblastoma specimens, compared with diffuse astrocytomas and nonneoplastic brain. In addition, ß1,6-GlcNAc branched expression was negatively correlated with the radiosensitivity of glioblastoma cells. Furthermore, the inhibition of N-linked ß1,6-GlcNAc branches by GnT-V silencing in U251 cells could reduce the cell clonogenic survival after X-irradiation. Meanwhile, the G2/M checkpoint was impaired and there was an increase in the number of apoptotic cells. Tunicamycin, an inhibitor of N-glycan biosynthesis, was also able to enhance the radiosensitivity of U251 cells. Thus, our results suggest that development of therapeutic approaches targeting N-linked ß1,6-GlcNAc branches may be a promising strategy in glioblastoma treatment.

B3GNT3 Expression Is a Novel Marker Correlated with Pelvic Lymph Node Metastasis and Poor Clinical Outcome in Early-Stage Cervical Cancer.

BACKGROUND: The ß1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) encodes a member of the B3GNT family that functions as the backbone structure of dimeric sialyl-Lewis A and is involved in L-selectin ligand biosynthesis, lymphocyte homing and lymphocyte trafficking. B3GNT3 has been implicated as an important element in the development of certain cancers. However, the characteristics of B3GNT3 in the development and progression of cancer remain largely unknown. Thus, our study aimed to investigate the expression pattern and the prognostic value of B3GNT3 in patients with early-stage cervical cancer. METHODS: The mRNA and protein levels of B3GNT3 expression were examined in eight cervical cancer cell lines and ten paired cervical cancer tumors, using real-time PCR and western blotting, respectively. Immunohistochemistry (IHC) was used to analyze B3GNT3 protein expression in paraffin-embedded tissues from 196 early-stage cervical cancer patients. Statistical analyses were applied to evaluate the association between B3GNT3 expression scores and clinical parameters, as well as patient survival. RESULTS: B3GNT3 expression was significantly upregulated in cervical cancer cell lines and lesions compared with normal cells and adjacent noncancerous cervical tissues. In the 196 cases of tested early-stage cervical cancer samples, the B3GNT3 protein level was positively correlated with high risk TYPES of human papillomavirus (HPV) infection (P = 0.026), FIGO stage (P < 0.001), tumor size (P = 0.025), tumor recurrence (P = 0.004), vital status (P < 0.001), concurrent chemotherapy and radiotherapy (P = 0.016), lymphovascular space involvement (P = 0.003) and most importantly, lymph node metastasis (P = 0.003). Patients with high B3GNT3 expression had a shorter overall survival (OS) and disease-free survival (DFS) compared with those with low expression of this protein. Multivariate analysis suggested that B3GNT3 expression is an independent prognostic indicator for cervical cancer patients. CONCLUSIONS: Our study demonstrated that elevated B3GNT3 expression is associated with pelvic lymph node metastasis and poor outcome in early-stage cervical cancer patients. B3GNT3 may be a novel prognostic marker and therapeutic target for the treatment of cervical cancer.

E2F1 Transcription Factor Regulates O-linked N-acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Expression.

Protein O-GlcNAcylation, which is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), has emerged as an important posttranslational modification that may factor in multiple diseases. Until recently, it was assumed that OGT/OGA protein expression was relatively constant. Several groups, including ours, have shown that OGT and/or OGA expression changes in several pathologic contexts, yet the cis and trans elements that regulate the expression of these enzymes remain essentially unexplored. Here, we used a reporter-based assay to analyze minimal promoters and leveraged in silico modeling to nominate several candidate transcription factor binding sites in both Ogt (i.e. the gene for OGT protein) and Mgea5 (i.e. the gene for OGA protein). We noted multiple E2F binding site consensus sequences in both promoters. We performed chromatin immunoprecipitation in both human and mouse cells and found that E2F1 bound to candidate E2F binding sites in both promoters. In HEK293 cells, we overexpressed E2F1, which significantly reduced OGT and MGEA5 expression. Conversely, E2F1-deficient mouse fibroblasts had increased Ogt and Mgea5 expression. Of the known binding partners for E2F1, we queried whether retinoblastoma 1 (Rb1) might be involved. Rb1-deficient mouse embryonic fibroblasts showed increased levels of Ogt and Mgea5 expression, yet overexpression of E2F1 in the Rb1-deficient cells did not alter Ogt and Mgea5 expression, suggesting that Rb1 is required for E2F1-mediated suppression. In conclusion, this work identifies and validates some of the promoter elements for mouse Ogt and Mgea5 genes. Specifically, E2F1 negatively regulates both Ogt and Mgea5 expression in an Rb1 protein-dependent manner.

Silencing ß-linked N-acetylglucosamine transferase induces apoptosis in human gastric cancer cells through PUMA and caspase-3 pathways.

ß-linked N-acetylglucosamine (GlcNAc) is a monosaccharide that is catalyzed by O-GlcNAcylation transferase (OGT) to bind serine or threonine hydroxyl moieties of numerous nuclear and cytoplasmic proteins. Recent studies have shown that O-GlcNAcylation is elevated in various cancer types, which is associated with oncogenesis and tumor progression. However, whether OGT is expressed and/or plays a role in gastric cancer is unknown. In the present study, we used qPCR to determine that OGT mRNA levels are significantly elevated in gastric cancer tissues compared with that in corresponding adjacent tissues. In addition, in vivo silencing of OGT in nude mice suppressed tumor proliferation and decreased tumor burden. Furthermore, in vitro OGT knockdown induced more cell apoptosis through increasing PUMA and caspase-3 expression. We used a glycan-binding protein gene microarray to identify potential downstream target genes of OGT, and found that apoptosis-related genes such as galectin and HBEGF were decreased after OGT suppression, suggesting that OGT silencing induces apoptosis in gastric cancer tissues. We concluded that OGT plays a key role in gastric cancer proliferation and survival, and could be a potential target for therapy.

Alteration of O-GlcNAcylation affects serine phosphorylation and regulates gene expression and activity of pyruvate kinase M2 in colorectal cancer cells.

O-GlcNAcylation is a dynamic post-translational modification that has extensive crosstalk with phosphorylation either at the same or adjacent sites of various proteins. We have previously reported that O-GlcNAcylation level was increased in primary breast and colorectal cancer, but the interplay of the two modifications remains unclear. Therefore, we explored crosstalk of the modifications by RNA interference against O-GlcNAc transferase (OGT) in colorectal cancer cells. Two-dimensional immunoblotting and mass spectrometric analysis showed that the levels of O-GlcNAc and serine phosphorylation of many proteins including serine hydroxymethyltransferase, cytokeratin-8, pyruvate kinase M2 (PKM2), heterogeneous nuclear ribonucleoprotein L, and lamin-B1, were reduced in siOGT cells compared to siScramble cells. In HT29 cells, immunoprecipitated PKM2 revealed decreased O-GlcNAc and serine phosphorylation levels after siOGT knockdown, but increased levels after treatment with Thiamet-G, an inhibitor of O-GlcNAcase (OGA). In addition, when global O-GlcNAcylation was enhanced by treating cells with Thiamet-G, PKM2 expression level was upregulated, but PKM2-specific activity was decreased. On the other hand, in OGT knockdown cells, PKM2 expression level was downregulated, but PKM2-specific activity was increased. Moreover, the metastatic colorectal cancer cells, SW620, had more O-GlcNAc-PKM2 and showed lower PKM2-specific activity compared to the non-metastatic colorectal cancer SW480 cells. These results suggested roles of O-GlcNAcylation in modulating serine phosphorylation, as well as in regulating PKM2 activity and expression. Interfering levels of O-GlcNAcylation of PKM2 may be a novel target in controlling cancer metabolism and tumorigenesis of colorectal cancer.

mTOR/MYC Axis Regulates O-GlcNAc Transferase Expression and O-GlcNAcylation in Breast Cancer.

UNLABELLED: Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-ß-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K-mTOR-MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade. IMPLICATIONS: Evidence indicates OGT as a therapeutic target in c-MYC-amplified cancers.

Glutathione depletion and acute exercise increase O-GlcNAc protein modification in rat skeletal muscle.

Post-translational modification of intracellular proteins with O-linked ß-N-acetylglucosamine (O-GlcNAc) profoundly affects protein structure, function, and metabolism. Although many skeletal muscle proteins are O-GlcNAcylated, the modification has not been extensively studied in this tissue, especially in the context of exercise. This study investigated the effects of glutathione depletion and acute exercise on O-GlcNAc protein modification in rat skeletal muscle. Diethyl maleate (DEM) was used to deplete intracellular glutathione and rats were subjected to a treadmill run. White gastrocnemius and soleus muscles were analyzed for glutathione status, O-GlcNAc and O-GlcNAc transferase (OGT) protein levels, and mRNA expression of OGT, O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase. DEM and exercise both reduced intracellular glutathione and increased O-GlcNAc. DEM upregulated OGT protein expression. The effects of the interventions were significant 4 h after exercise (P < 0.05). The changes in the mRNA levels of O-GlcNAc enzymes were different in the two muscles, potentially resulting from different rates of oxidative stress and metabolic demands between the muscle types. These findings indicate that oxidative environment promotes O-GlcNAcylation in skeletal muscle and suggest an interrelationship between cellular redox state and O-GlcNAc protein modification. This could represent one mechanism underlying cellular adaptation to oxidative stress and health benefits of exercise.

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro.

AIM: To evaluate the biochemical features and activities of a glyco-engineered form of the anti-human epidermal growth factor receptor monoclonal antibody (EGFR mAb) cetuximab in vitro. METHODS: The genes encoding the Chinese hamster bisecting glycosylation enzyme (GnTIII) and anti-human EGFR mAb were cloned and coexpressed in CHO DG44 cells. The bisecting-glycosylated recombinant EGFR mAb (bisec-EGFR mAb) produced by these cells was characterized with regard to its glycan profile, antiproliferative activity, Fc receptor binding affinity and cell lysis capability. The content of galactose-α-1,3-galactose (α-Gal) in the bisec-EGFR mAb was measured using HPAEC-PAD. RESULTS: The bisec-EGFR mAb had a higher content of bisecting N-acetylglucosamine residues. Compared to the wild type EGFR mAb, the bisec-EGFR mAb exhibited 3-fold higher cell lysis capability in the antibody-dependent cellular cytotoxicity assay, and 1.36-fold higher antiproliferative activity against the human epidermoid carcinoma line A431. Furthermore, the bisec-EGFR mAb had a higher binding affinity for human FcγRIa and FcγRIIIa-158F than the wild type EGFR mAb. Moreover, α-Gal, which was responsible for cetuximab-induced hypersensitivity reactions, was not detected in the bisec-EGFR mAb. CONCLUSION: The glyco-engineered EGFR mAb with more bisecting modifications and lower α-Gal content than the approved therapeutic antibody Erbitux shows improved functionality in vitro, and requires in vivo validations.

Inhibition of N-acetylglucosaminyltransferase V enhances sensitivity of radiotherapy in human prostate cancer.

The purpose of this study was to investigate the relationship between N-acetylglucosaminyltransferase V (GnT-V) and radiation sensitivity of prostate cancer (PCa) cells both in vitro and in vivo. Firstly, the GnT-V expression was studied in 84 cases of PCa tissues, in which higher level of GnT-V was detected more frequently in the advanced tumors. Secondly, the GnT-V stably suppressed cell lines PCa/1079 (Lncap/1079 and PC3/1079) were constructed from PCa cell lines (Lncap and PC3) in vitro. Attenuation of GnT-V inhibited cell proliferation, migration and increased apoptosis, which resulted in enhanced radiation sensitivity of PCa cells. The underlying mechanism may be relevant to the increasing ratio of Bax/Bcl-2, the blocking transcription of NF-κB and the reduction of cell cycle G2-M arrest. Finally, in in vivo study, compared with control groups, the irradiated PCa xenograft nude mice of PCa/1079 indicated to reduce tumor-growth rate and enhance survival time. Summary, our studies showed that inhibition of GnT-V probably improved PCa cells' radiation sensitivity.

Silibinin inhibits ICAM-1 expression via regulation of N-linked and O-linked glycosylation in ARPE-19 cells.

To evaluate the effects of silibinin on intercellular adhesion molecule-1 (ICAM-1) expression, we used ARPE-19 cells as a model in which tumor necrosis factor (TNF-α) and interferon (IFN-γ) enhanced ICAM-1 expression. This upregulation was inhibited by silibinin. In an adherence assay using ARPE-19 and THP-1 cells, silibinin inhibited the cell adhesion function of ICAM-1. The inhibitory effects of silibinin on ICAM-1 expression were mediated via the blockage of nuclear translocation of p65 proteins in TNF-α and phosphorylation of STAT1 in IFN-γ-stimulated cells. In addition, silibinin altered the degree of N-linked glycosylation posttranslationally in ARPE-19 cells by significantly enhancing MGAT3 gene expression. Silibinin can increase the O-GlcNAc levels of glycoproteins in ARPE-19 cells. In a reporter gene assay, PUGNAc, which can also increase O-GlcNAc levels, inhibited NF-κB reporter activity in TNF-α-induced ARPE-19 cells and this process was augmented by silibinin treatment. Overexpression of OGT gene was associated with reduced TNF-α-induced ICAM-1 levels, which is consistent with that induced by silibinin treatment. Taken together, silibinin inhibits ICAM-1 expression and its function through altered O-linked glycosylation in NF-κB and STAT1 signaling pathways and decreases the N-linked glycosylation of ICAM-1 transmembrane protein in proinflammatory cytokine-stimulated ARPE-19 cells.

Differential expression patterns of N-acetylglucosaminyl transferases and polylactosamines in uterine lesions.

Polylactosamine (polyLacNAc) is a fundamental structure in glycoconjugates and it is expressed in specific cells/tissues associated with the development and carcinogenesis. ß1,3-N-acetylglucosaminyl transferases (ß3GnTs) play an important role in polyLacNAc synthesis, however the roles of these glycosyltransferases and their products in cancer progression are still unclear. In this sense, this work aimed to evaluate differential expression pattern of the N-acetylglucosaminyl transferases and polylactosamines in invasive and premalignant lesions of the uterus cervix. The expression of ß3GnT2 and ß3GnT3 were evaluated in normal (n=10) and uterine cervix lesions (n= 120) malignant (squamous carcinoma - SC) and premalignant (cervical intraepithelial neoplasia - CIN - grades 1, 2 and 3) using immunohistochemistry. Besides, lectin histochemistry with Phytolacca americana lectin (PWM) and Wheat germ agglutinin (WGA) was also carried out to observe the presence of polyLacNAc chains and N-acetylglucosamine (GlcNAc), respectively. The ß3GnT3 was expressed in almost all samples (99%) and ß3GnT2 was higher expressed in disease samples mainly in CIN 3, when compared with normal (P=0.002), CIN 1 (P=0.009) and CIN 2 (P=0.03). The expression of polyLacNAc was higher is SC samples, when compared with normal (P=0.03), CIN 1 (P=0.02) and CIN 3 (P=0.004), and was observed only nuclear expression in nearly 50% of the SC samples, showing a statistically significant when compared with normal (P=0.01), CIN 1 (P=0.002), CIN 2 (P=0.007) and CIN 3 (P=0.04). Deferring from transferases and polyLacNAc chains, GlcNAc (WGA ligand) reveals a gradual staining pattern decrease with the increase of the lesion degree, being more expressed in CIN 1 lesions when compared with normal (P<0.0001), CIN 2 (P<0.0001), SC (P<0.0001) and CIN 3 (P=0.0003). Our data reveals ß3GnT2 and polyLacNAc may be involved in the progression of the pre-malignant lesions of human the uterine cervix. In addition, polyLacNAc expression only in the nucleus can be associated a poor prognostic in uterine lesions.

Let-7c inhibits metastatic ability of mouse hepatocarcinoma cells via targeting mannoside acetylglucosaminyltransferase 4 isoenzyme A.

Aberrant glycosylation may promote tumor invasion and metastasis. To investigate whether microRNA (miRNA) is involved in glycosylation-related metastasis, we examined the role of let-7c, a well-known tumor-suppressor miRNA, in glycosylation in murine hepatocarcinoma cell lines Hca-F and Hca-P. We found that let-7c level was higher in Hca-P cells (with lower lymphatic metastasis potential) than in Hca-F cells (with higher lymphatic metastasis potential). Overexpression of let-7c decreased hyper-N-glycosylation of Hca-F cells and repressed their metastatic and invasive ability. Mannoside acetylglucosaminyltransferase 4, isoenzyme A (Mgat4a) is a key glycosyltransferase in the pathway of synthesizing complex N-glycans. Bioinformatics analysis indicates that Mgat4a may be a target of let-7c, which has been verified by dual-luciferase reporter gene assay. Furthermore, the anti-metastatic effect of overexpressed let-7c is similar to that of Mgat4a siRNAs transfection. Hence, our results suggest that let-7c may inhibit the metastatic ability of Hca-F cells, at least partially, via repressing Mgat4a activity.

Epigenetic regulation of a brain-specific glycosyltransferase N-acetylglucosaminyltransferase-IX (GnT-IX) by specific chromatin modifiers.

Expression of glycosyltransferase genes is essential for glycosylation. However, the detailed mechanisms of how glycosyltransferase gene expression is regulated in a specific tissue or during disease progression are poorly understood. In particular, epigenetic studies of glycosyltransferase genes are limited, although epigenetic mechanisms, such as histone and DNA modifications, are central to establish tissue-specific gene expression. We previously found that epigenetic histone activation is essential for brain-specific expression of N-acetylglucosaminyltransferase-IX (GnT-IX, also designated GnT-Vb), but the mechanism of brain-specific chromatin activation around GnT-IX gene (Mgat5b) has not been clarified. To reveal the mechanisms regulating the chromatin surrounding GnT-IX, we have investigated the epigenetic factors that are specifically involved with the mouse GnT-IX locus by comparing their involvement with other glycosyltransferase loci. We first found that a histone deacetylase (HDAC) inhibitor enhanced the expression of GnT-IX but not of other glycosyltransferases tested. By overexpression and knockdown of a series of HDACs, we found that HDAC11 silenced GnT-IX. We also identified the O-GlcNAc transferase (OGT) and ten-eleven translocation-3 (TET3) complex as a specific chromatin activator of GnT-IX gene. Moreover, chromatin immunoprecipitation (ChIP) analysis in combination with OGT or TET3 knockdown showed that this OGT-TET3 complex facilitates the binding of a potent transactivator, NeuroD1, to the GnT-IX promoter, suggesting that epigenetic chromatin activation by the OGT-TET3 complex is a prerequisite for the efficient binding of NeuroD1. These results reveal a new epigenetic mechanism of brain-specific GnT-IX expression regulated by defined chromatin modifiers, providing new insights into the tissue-specific expression of glycosyltransferases.

Suppression of B3GNT7 gene expression in colon adenocarcinoma and its potential effect in the metastasis of colon cancer cells.

The cell surface sialyl Lewis a (sLe(a)) and sialyl Lewis x (sLe(x)) antigens, which are built on the terminals of glyco-structures called poly-N-acetyllactosamine (LacNAc) chains, have been shown to play a critical role in the metastasis of colon cancer. In the present investigation, expression of the B3GNT7 gene, which encodes a ß-1,3-N-acetylglucosaminyltransferase that mainly acts on and extends sulfated poly-LacNAc chains, was found to be markedly suppressed during the oncogenetic processes associated with colon cancer. DNA methylation in the promoter region of the B3GNT7 gene was found to play a significant role in the suppression of the B3GNT7 gene in colon cancer cells. The results obtained from Transwell experiments and the nude mice xenograft model demonstrated that ectopic expression of the B3GNT7 gene in colon cancer cells diminished the migration capability and the liver-metastasis potential, respectively, of colon cancer cells. Flow cytometric analysis showed that expression of cell surface sLe(a) and sLe(x) antigens was decreased in colon cancer cells when the B3GNT7 gene was ectopically expressed. Taken together, the results of the present investigation suggest a link between suppression of B3GNT7 gene expression and elevation of sLe(a)/sLe(x) antigen expressions on the surface of cells and that this consequently promotes the metastasis potential of cancer cells as part of the colon cancer oncogenetic process.

Expression of N-acetylglucosaminyltransferase V in gastric cancer correlates with metastasis and prognosis.

N-acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes ß1-6 branching of N-acetylglucosamine on asparagine (N)-linked oligosaccharides (N-glycan) of cell proteins and the dysfunction of which is a common feature of various carcinomas. Nevertheless, the role of GnT-V remains controversial. Therefore, the clinical implication of GnT-V expression may differ in each cancer type. The implication of GnT-V status in patients with gastric cancer has not been studied extensively. In the present study, we examined GnT-V expression in gastric cancer specimen both at protein and mRNA levels. We compared GnT-V expression with clinical and pathologic variables. Kaplan-Meier survival curves were generated to show the cause-specific survival. Furthermore, the small interfering RNA was devised to downregulate the GnT-V mRNA expression in SGC7901 and BGC 823 cells. We characterized the function implication of GnT-V by cell proliferation and invasiveness analysis. Analysis in gastric cancer specimen revealed that GnT-V expression correlated with tumor grade and stage. The overall survival time of positive GnT-V expression in gastric cancer was significantly shorter than that of negative GnT-V expression. Moreover, the downregulation of GnT-V expression by small interfering RNA resulted in a decrease of cell proliferation and invasiveness in SGC7901 and BGC 823 cells accompanied by morphological change. This supports that GnT-V correlates with metastasis and prognosis in gastric cancer. These results contribute to new insight into the underlying molecular mechanisms of GnT-V regulation in gastric cancer with potential translational clinical applications.