MGAT3 Expression Research Articles

Remodelling of the glycome of B-cell precursor acute lymphoblastic leukemia cells developing drug-tolerance.

Reduced responsiveness of precursor B-acute lymphoblastic leukemia (BCP-ALL) to chemotherapy can be first detected in the form of minimal residual disease leukemia cells that persist after 28 days of initial treatment. The ability of these cells to resist chemotherapy is partly due to the microenvironment of the bone marrow, which promotes leukemia cell growth and provides protection, particularly under these conditions of stress. It is unknown if and how the glycocalyx of such cells is remodelled during the development of tolerance to drug treatment, even though glycosylation is the most abundant cell surface post-translational modification present on the plasma membrane. To investigate this, we performed omics analysis of BCP-ALL cells that survived a 30-day vincristine chemotherapy treatment while in co-culture with bone marrow stromal cells. Proteomics showed decreased levels of some metabolic enzymes. Overall glycocalyx changes included a shift from Core-2 to less complex Core-1 O-glycans, and reduced overall sialylation, with a shift from α2-6 to α2-3 linked Neu5Ac. Interestingly, there was a clear increase in bisecting complex N-glycans with a concomitant increased mRNA expression of MGAT3 , the only enzyme known to form bisecting N-glycans. These small but reproducible quantitative differences suggest that individual glycoproteins become differentially glycosylated. Glycoproteomics confirmed glycosite-specific modulation of cell surface and lysosomal proteins in drug-tolerant BCP-ALL cells, including HLA-DRA, CD38, LAMP1 and PPT1. We conclude that drug-tolerant persister leukemia cells that grow under continuous chemotherapy stress have characteristic glycotraits that correlate with and perhaps contribute to their ability to survive and could be tested as neoantigens in drug-resistant leukemia.

Circ_0070934 promotes MGAT3 expression and inhibits epithelial-mesenchymal transition in bronchial epithelial cells by sponging miR-199a-5p

BackgroundCircular RNA (circRNA) has the potential to serve as a crucial regulator in the progression of bronchial asthma. The objective of this investigation was to elucidate the functional dynamics of the circ_0070934/miR-199a-5p/Mannoside acetylglucosaminyltransferase 3 (MGAT3) axis in the development of asthma.MethodsCirc_0070934, miR-199a-5p and MGAT3 in peripheral venous blood of 38 asthmatic patients and 43 healthy controls were detected by qRT-PCR, and the expression of MGAT3 protein was examined by ELISA. The GSE148000 dataset was analyzed for differences in MGAT3. The BEAS-2B cells were transfected with circ_0070934 plasmid and small interfering RNA, miR-199a-5p mimics and inhibitors. The apoptosis level was detected by flow cytometry and MGAT3 was detected by qRT-PCR and Western blot. The expression of E-cadherin, N-cadherin, Vimentin was examined by Western blot. Interleukin-4 (IL-4) and IL-13 were used to co-stimulate BEAS-2B cells as an asthmatic airway epithelial cell model. BEAS-2B cells exposed to type 2 cytokines (IL-4 and IL-13) were treated with circ_0070934 plasmid, and the expression of E-cadherin, N-cadherin, and Vimentin was detected by Western blot. The binding relationships were verified using dual-luciferase reporter assay and miRNA pull-down assay.ResultsThe expression of circ_0070934 and MGAT3 in peripheral venous blood of asthmatic patients was down-regulated, and the expression of miR-199a-5p was up-regulated. And the expression of MGAT3 was reduced in sputum of asthma patients. Down-regulating the expression of circ_0070934 could promote apoptosis of BEAS-2B cells and increase epithelial-mesenchymal transition (EMT), and this effect can be partially reversed by down-regulating miR-199a-5p. Circ_0070934 could inhibit the process of epithelial mesenchymal transition induced by IL-4 and IL-13 in BEAS-2B cells. In addition, miR-199a-5p could respectively bind to circ_0070934 and MGAT3.ConclusionThe findings of this study indicate that circ_0070934 may function as a competitive endogenous RNA (ceRNA) of miR-199a-5p, thereby modulating the expression of MGAT3 and impacting the process of EMT in bronchial epithelial cells. These results contribute to the establishment of a theoretical framework for advancing the prevention and treatment strategies for asthma.

N-glycosylation Regulates Intrinsic IFN-γ Resistance in Colorectal Cancer: Implications for Immunotherapy

Background & AimsAdvanced colorectal carcinoma (CRC) is characterized by a high frequency of primary immune evasion and refractoriness to immunotherapy. Given the importance of interferon (IFN)-γ in CRC immunosurveillance, we investigated whether and how acquired IFN-γ resistance in tumor cells would promote tumor growth, and whether IFN-γ sensitivity could be restored. MethodsSpontaneous and colitis-associated CRC development was induced in mice with a specific IFN-γ pathway inhibition in intestinal epithelial cells. The influence of IFN-γ pathway gene status and expression on survival was assessed in patients with CRC. The mechanisms underlying IFN-γ resistance were investigated in CRC cell lines. ResultsThe conditional knockout of the IFN-γ receptor in intestinal epithelial cells enhanced spontaneous and colitis-associated colon tumorigenesis in mice, and the loss of IFN-γ receptor α (IFNγRα) expression by tumor cells predicted poor prognosis in patients with CRC. IFNγRα expression was repressed in human CRC cells through changes in N-glycosylation, which decreased protein stability via proteasome-dependent degradation, inhibiting IFNγR-signaling. Downregulation of the bisecting N-acetylglucosaminyltransferase III (MGAT3) expression was associated with IFN-γ resistance in all IFN-γ–resistant cells, and highly correlated with low IFNγRα expression in CRC tissues. Both ectopic and pharmacological reconstitution of MGAT3 expression with all-trans retinoic acid increased bisecting N-glycosylation, as well as IFNγRα protein stability and signaling. ConclusionsTogether, our results demonstrated that tumor-associated changes in N-glycosylation destabilize IFNγRα, causing IFN-γ resistance in CRC. IFN-γ sensitivity could be reestablished through the increase in MGAT3 expression, notably via all-trans retinoic acid treatment, providing new prospects for the treatment of immune-resistant CRC.

Glycoproteomic analysis reveals the effects of bisecting GlcNAc in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second major subtype of primary liver cancer and has caused more and more attention with increasing incidence and mortality worldwide. Our previous study found that bisecting N-glycans are commonly increased in ICC, while the effects and potential functions of bisecting GlcNAc in ICC are still largely unclear. In this study, we further confirmed that the structures of bisecting GlcNAc were significantly up-regulated in ICC compared with paracancer tissues by glycoproteomic data and lectin histochemistry. The expression of its glycosyltransferase MGAT3 was also up-regulated in ICC tissues at both mRNA and protein levels, and expression of MGAT3 is negatively correlated with overall survival explored by bioinformatic analyses and published datasets from 255 patients. Next, the silencing of MGAT3 could inhibit the growth and invasion of ICC cells, and overexpressing of MGAT3 only promoted ICC cell invasion. Further glycoproteomic analysis showed that the commonly glycoproteins modified by bisecting GlcNAc after MGAT3-overexpression in two ICC cell lines were mainly involved in cell movement-related biological processes, such as cell adhesion, integrin-related and ECM-receptor interaction. This study sheds light on the potential effects of bisecting GlcNAc in ICC cells and suggests that MGAT3 might be used as a potential target in the therapy of ICC.

Exosomal miR‑663b exposed to TGF‑β1 promotes cervical cancer metastasis and epithelial‑mesenchymal transition by targeting MGAT3.

Transforming growth factor (TGF)-β1 is a key cytokine affecting the pathogenesis and progression of cervical cancer. Tumor-derived exosomes contain microRNAs (miRNAs/miRs) that interact with cancer and stromal cells, thereby contributing to tissue remodeling in the tumor microenvironment (TME). The present study was designed to clarify how TGF-β1 affects tumor biological functions through exosomes released by cervical cancer cells. Deep RNA sequencing found that TGF-β1 stimulated cervical cancer cells to secrete more miR-663b-containing exosomes, which could be transferred into new target cells to promote metastasis. Further studies have shown that miR-663b directly targets the 3′-untranslated regions (3′-UTR) of mannoside acetylglucosaminyltransferase 3 (MGAT3) and is involved in the epithelial-mesenchymal transition (EMT) process. Remarkably, the overexpression of MGAT3 suppressed cervical cancer cell metastasis promoted by exosomal miR-663b, causing increased expression of epithelial differentiation marker E-cadherin and decreased expression of mesenchymal markers N-cadherin and β-catenin. Throughout our study, online bioinformation tools and dual luciferase reporter assay were applied to identify MGAT3 as a novel direct target of miR-663b. Exosome PKH67-labeling experiment verified that exosomal miR-663b could be endocytosed by cervical cancer cells and subsequently influence its migration and invasion functions which were measured by wound healing and Transwell assays. The expression of miR-663b and MGAT3 and the regulation of the EMT pathway caused by MGAT3 were detected by quantitative real-time transcription-polymerase chain reaction (qPCR) and western blot analysis. These results, thus, provide evidence that cancer cell-derived exosomal miR-663b is endocytosed by cervical cancer cells adjacent or distant after TGF-β1 exposure and inhibits the expression of MGAT3, thereby accelerating the EMT process and ultimately promoting local and distant metastasis.

Suppression of G6PD induces the expression and bisecting GlcNAc-branched N-glycosylation of E-Cadherin to block epithelial-mesenchymal transition and lymphatic metastasis

BackgroundAs the rate-limit enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) plays important roles in tumour progression, but the exact mechanism through which G6PD controls cancer metastasis remains unclear.MethodsG6PD expression in resected oral squamous cell carcinoma (OSCC) samples was analysed by immunohistochemistry. The effects and mechanism of G6PD suppression on OSCC cell lines were measured by transwell assay, wound healing assay, western and lectin blot, mass spectrometer analysis, ChIP-PCR, and luciferase reporter assay. BALB/c-nude mice were used to establish orthotopic xenograft model.ResultsG6PD expression in the tumours of 105 OSCC patients was associated with lymphatic metastasis and prognosis. In vitro cellular study suggested that G6PD suppression impaired cell migration, invasion, and epithelial-mesenchymal transition. Furtherly, G6PD knockdown activated the JNK pathway, which then blocked the AKT/GSK-3β/Snail axis to induce E-Cadherin expression and transcriptionally regulated MGAT3 expression to promote bisecting GlcNAc-branched N-glycosylation of E-Cadherin. An orthotopic xenograft model further confirmed that dehydroepiandrosterone reduced lymphatic metastatic rate of OSCC, which was partially reversed by JNK inhibition.ConclusionsSuppression of G6PD promoted the expression and bisecting GlcNAc-branched N-glycosylation of E-Cadherin via activating the JNK pathway, which thus acted on OSCC metastasis.

Bisecting N-Acetylglucosamine on EGFR Inhibits Malignant Phenotype of Breast Cancer via Down-Regulation of EGFR/Erk Signaling.

Glycosylation, the most prevalent and diverse post-translational modification of protein, plays crucial biological roles in many physiological and pathological events. Alteration of N-glycan has been detected during breast cancer progression. Among the specific N-glycan structures, bisecting N-Acetylglucosamine (GlcNAc) is a β1,4-linked GlcNAc attached to the core β-mannose residue, and is catalyzed by glycosyltransferase MGAT3. Bisecting GlcNAc levels were commonly dysregulated in different types of cancer. In this study, we utilized mass spectrometry and lectin microarray analysis to investigate aberrant N-glycans in breast cancer cells. Our data showed the decreased levels of bisecting GlcNAc and down-regulated expression of MGAT3 in breast cancer cells than normal epithelial cells. Using PHA-E (a plant lectin recognizing and combining bisecting GlcNAc) based enrichment coupled with nanoLC-MS/MS, we analyzed the glycoproteins bearing bisecting GlcNAc in various breast cancer cells. Among the differentially expressed glycoproteins, levels of bisecting GlcNAc on EGFR were significantly decreased in breast cancer cells, confirmed by immunostaining and immunoprecipitation. We overexpressed MGAT3 in breast cancer MDA-MB-231 cells, and overexpression of MGAT3 significantly enhanced the bisecting N-GlcNAc on EGFR and suppressed the EGFR/Erk signaling, which further resulted in the reduction of migratory ability, cell proliferation, and clonal formation. Taken together, we conclude that bisecting N-GlcNAc on EGFR inhibits malignant phenotype of breast cancer via down-regulation of EGFR/Erk signaling.

Suppression of MGAT3 expression and the epithelial–mesenchymal transition of lung cancer cells by miR-188-5p

BackgroundTo investigate the effect of miR-188-5p overexpression on the invasion and migration of cultured lung cancer cells, and on related cellular mechanisms that underlie epithelial mesenchymal transition (EMT). MethodsHuman lung cancer cell line 95D was transfected with miR-188-5p mimic. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to quantify the expression levels of genes including E-cadherin, Snail, α-SMA, and MGAT3. Changes in cell motility, invasion and proliferation were studied using scratch migration assay, transwell invasion assay, and colony formation assay, respectively. The expression levels of EMT-related proteins and MGAT3 protein were also determined via immunofluorescent staining. The ability of miR-188-5p to regulate its target gene, MGAT3, was assessed using dual luciferase activity assay. ResultsLung cancer cell line 95D showed the lowest miR-188-5p expression level thus was used in this study. Transfection with miR-188-5p mimic significantly suppressed migration, invasion and clonal formation potency of 95D cells. Dual luciferase activity assay implicated that miR-188-5p exerts its negative regulatory effect on MGAT3 expression through recognizing the 3′ untranslated region (3′UTR) of the MGAT3 gene. Over-expression of miR-188-5p in 95D cells also remarkably increased E-cadherin protein expression and decreased the expression levels of Snail and α-SMA, which suppressed the EMT process. ConclusionMiR-188-5p reduces the expression of MGAT3 and inhibits the metastatic properties of a highly invasive lung cancer cell line, probably via targeted regulation of EMT process. Further research to explore the potential therapeutic value of miR-188-5p, both as a biomarker and as a drug candidate for the management of metastatic lung cancer may be warranted.

Low MGAT3 expression identifies long-term survivors with high grade serous ovarian cancer

Introduction: MGAT3 gene expression is implicated in oncogenic signaling pathways. We recently found that MGAT3 expression correlated with the presence of bisecting GlcNAc on N-glycoproteins in ovarian cancer cells. MGAT3 encodes the enzyme attaching bisecting GlcNAc to these glycoproteins. We investigated (i) how MGAT3 expression is regulated, (ii) whether DNA methylation, MGAT3 expression, and bisecting GlcNAc presence are functionally linked, and (iii) whether MGAT3 expression has any predictive value.

Epigenetic activation of MGAT3 and corresponding bisecting GlcNAc shortens the survival of cancer patients.

Bisecting GlcNAc on N-glycoproteins is described in E-cadherin-, EGF-, Wnt- and integrin- cancer-associated signaling pathways. However, the mechanisms regulating bisecting GlcNAc expression are not clear. Bisecting GlcNAc is attached to N-glycans through beta 1-4 N-acetylglucosaminyl transferase III (MGAT3), which is encoded by two exons flanked by high-density CpG islands. Despite a recently described correlation of MGAT3 and bisecting GlcNAc in ovarian cancer cells, it remains unknown whether DNA methylation is causative for the presence of bisecting GlcNAc. Here, we narrow down the regulatory genomic region and show that reconstitution of MGAT3 expression with 5-Aza coincides with reduced DNA methylation at the MGAT3 transcription start site. The presence of bisecting GlcNAc on released N-glycans was detected by mass spectrometry (LC-ESI-qTOF-MS/MS) in serous ovarian cancer cells upon DNA methyltransferase inhibition. The regulatory impact of DNA methylation on MGAT3 was further evaluated in 18 TCGA cancer types (n = 6118 samples) and the results indicate an improved overall survival in patients with reduced MGAT3 expression, thereby identifying long-term survivors of high-grade serous ovarian cancers (HGSOC). Epigenetic activation of MGAT3 was also confirmed in basal-like breast cancers sharing similar molecular and genetic features with HGSOC. These results provide novel insights into the epigenetic regulation of MGAT3/bisecting GlcNAc and demonstrate the importance of N-glycosylation in cancer progression.

Structural Changes in N-Glycans on Induced Pluripotent Stem Cells Differentiating Toward Cardiomyocytes.

Cell-surface glycans vary widely, depending on cell properties. Previously, we reported that the pattern of N-glycan expression on murine induced pluripotent stem cells (iPSCs) changed toward that of the cardiac tissue during cardiomyogenic differentiation. In this study, N-glycans were isolated from human iPSCs, iPSC-derived cardiomyocytes (iPSC-CMs), and human cardiomyocytes (hCMCs). Their structures were analyzed by a mapping technique based on high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometric data. Of 52 isolated N-glycans, the structures of 38 were clearly identified. In addition, 11 structures were partially identified because the binding style and fucose binding site at the nonreduced terminal could not be identified. Quantitation of each type of N-glycan, based on the terminal glycosylation process, revealed that the exposed N-acetylglucosamine (GlcNAc) and the nonreduced terminal fucose types decreased, whereas the exposed galactose or the α2-3 NeuAc types increased in the iPSCs during cardiomyogenic differentiation. However, the bisecting GlcNAc and the triantennary structures were found in relative abundance in the iPSC-CMs in comparison with hCMCs or iPSCs. Expression of MGAT3, a glycosyltransferase-encoding gene that produces the bisecting GlcNAc structures, was higher in iPSCs and iPSC-CMs than in hCMCs. These findings will prove useful in understanding the directional precision of cardiomyogenic differentiation in vitro. This study focused on N-glycans produced in human induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes to investigate their change on cardiomyogenic differentiation in vitro. This shows that the expression pattern of N-glycans in human iPSCs changed toward the pattern observed in human cardiomyocytes upon cardiomyogenic differentiation. Structural differences were also observed in the bisecting N-acetylglucosamine and the triantennary structures upon cardiomyogenic differentiation. The findings of this study will help in understanding the directional precision of cardiomyogenic differentiation in vitro.

Abstract POSTER-BIOL-1323: Elevated MGAT3 expression in ovarian cancer cells is epigenetically regulated and correlates with expression of bisecting GlcNAc-modified proteins

Abstract Changes in the glycosylation of membrane proteins are a common feature of malignant transformation including ovarian cancer development. Of particular interest are N-linked glycans because these cell surface glycans mediate various cellular functions in cell-cell communication, cell adhesion, and cell motility. However the nature of these changes, their underlying molecular mechanisms, and the biological function of these glycans are poorly understood. In the present study, N-glycans were released from extracted membrane glycoproteins from ovarian cancer and normal ovarian epithelial cell lines and analysed using electrospray ionisation mass spectrometry; RT-qPCR measured gene expression of related glycosyltransferases; bisulfite sequencing analysed DNA methylation of the corresponding genes. We identified characteristic glycan features unique to membrane proteins of ovarian cancer cells. Among those ovarian cancer specific N-glycans were the ‘bisecting N-acetyl-glucosamine (GlcNAc)’ type. The bisecting GlcNAc is the product of the enzyme MGAT3 (beta-1,4-N-acetylglucosaminyltransferase 3) and the expression of the MGAT3 gene was elevated in ovarian cancer cell lines (SKOV3, IGROV1, A2780, OVCAR3) as compared to normal ovary surface epithelial (HOSE) cells. This elevated MGAT3 expression in ovarian cancer cells correlated with DNA hypomethylation at the transcription start site (TSS) of the MGAT3 promotor, which contains a large CpG island with 204 CpG dinucleotides located -600/+800bp relative to the TSS. This finding suggests that MGAT3 expression is epigenetically regulated. This was validated by the finding that the treatment of HOSE cells with the DNA-methyltransferase inhibitor 5-Aza increased MGAT3 expression and corresponded to a reduction in DNA methylation at the TSS. Taken together, we conclude that the elevated MGAT3 expression in ovarian cancer cells correlates with the presence of the resultant specific N-glycan substructures and with the epigenetic regulation of the associated enzymes by DNA methylation. These findings may stimulate the development of novel anti-glycan targeted drugs and clinical diagnostic tools for ovarian cancer Citation Format: Francis Jacob, Merrina Anugraham, Reto Kohler, Sheri Nixdorf, Arun Vijay Everest-Dass, André Fedier, Viola Heinzelmann-Schwarz, Nicolle H. Packer. Elevated MGAT3 expression in ovarian cancer cells is epigenetically regulated and correlates with expression of bisecting GlcNAc-modified proteins [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1323.

Elevated MGAT3 expression in ovarian cancer cells is epigenetically regulated by DNA hypomethylation

Elevated MGAT3 expression in ovarian cancer cells is epigenetically regulated by DNA hypomethylation

Specific Glycosylation of Membrane Proteins in Epithelial Ovarian Cancer Cell Lines: Glycan Structures Reflect Gene Expression and DNA Methylation Status

Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. N-glycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the "bisecting N-acetyl-glucosamine" type N-glycans, increased levels of α 2-6 sialylated N-glycans and "N,N'-diacetyl-lactosamine" type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique "bisecting GlcNAc" type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association with epigenetic programming of their associated synthetic enzymes in ovarian cancer could potentially be used for the development of novel anti-glycan drug targets and clinical diagnostic tools.

Epigenetic inhibitors upregulate expression of MGAT3 in HepG2 cells and cause significant changes in glycosylation of secreted proteins (608.2)

Protein glycosylation is a ubiquitous modification which affects protein structure and function. The majority of blood proteins are synthesized and glycosylated in the liver. Changes in plasma protein glycosylation are often associated with different types of liver disease, including hepatocellular carcinoma (HCC). Interestingly, secretomes of HCC patients and of HepG2 cells in culture are comparable, making this cell line an appropriate model to study how induced epigenetic changes can affect glycosylation of secreted proteins. We have correlated global changes in the expression of glyco‐genes with preferential appearance of particular glycan structures in the HepG2 secretome following the treatment of HepG2 cells with DNA methylation and histone deacetylation inhibitors. Using Human RT Profiler glycosylation qPCR array that contains 94 known glyco‐genes we identified that 20% of these genes are regulated by DNA methylation, and this regulation was reflected on glycan profile of secreted proteins from Hepg2 cells. The majority of changes in the glycosylation profile could be attributed to changed expression of Mgat3 responsible for addition of bisecting GlcNAc, which prevents further fucosylation and branching. We further confirmed its role in plasma glycome by targeted modulation of Mgat3 expression with epigenetic inhibitors. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, the presented work contributes to our understanding of their efficiency in altering the N‐glycan profiles of secreted proteins.Grant Funding Source: Supported by European Commission grants IBD‐BIOM and IntegraLife

Glycogenes mediate the invasive properties and chemosensitivity of human hepatocarcinoma cells

Aberrant cell-surface glycosylation patterns are present on tumors and have been linked to tumor progression. This study aimed to identify the alterations of glycogene and N-glycan involved in tumor invasion, tumorigenicity and drug resistance in MHCC97-H and MHCC97-L human hepatocarcinoma cell lines, which have high, low metastatic potential, respectively. Using real-time PCR for quantification of glycogene and FITC-lectin binding for glycan profiling, we found that the expression of glycogenes and glycan profiling were different in MHCC97-H cells, as compared to those in MHCC97-L cells. We silenced the expression levels of glycogenes MGAT3 and MGAT5, which were over-expressed in MHCC97-L and MHCC97-H cells. Knockdown of MGAT3 expression promoted MHCC97-L cells invasion and increased resistance to 5-fluorouracil in vitro. The silencing of MGAT5 in MHCC97-H cells inhibited invasion and increased sensitivity to 5-fluorouracil in vitro. Further analysis of the N-glycan regulation by tunicamycin application or PNGase F treatment in MHCC97-H and MHCC97-L cells showed partial inhibition of N-glycan glycosylation, decreased invasion, tumorigenicity and increased sensitivity to 5-fluorouracil both in vitro and in vivo. These findings suggest that alterations of glycogene and N-glycan in human hepatocarcinoma cells correlate with tumor invasion, tumorigenicity and sensitivity to chemotherapeutic drug, and have significant implications for the development of treatment strategies.

Loss and Recovery of Mgat3 and GnT-III Mediated E-cadherin N-glycosylation Is a Mechanism Involved in Epithelial-Mesenchymal-Epithelial Transitions

BackgroundN-acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase encoded by Mgat3 that catalyzes the addition of β1,4-bisecting-N-acetylglucosamine on N-glycans. GnT-III has been pointed as a metastases suppressor having varying effects on cell adhesion and migration. We have previously described the existence of a functional feedback loop between E-cadherin expression and GnT-III-mediated glycosylation. The effects of GnT-III-mediated glycosylation on E-cadherin expression and cellular phenotype lead us to evaluate Mgat3 and GnT-III-glycosylation role during Epithelial-Mesenchymal-Transition (EMT) and the reverted process, Mesenchymal-Epithelial-Transition (MET).Methodology/Principal FindingsWe analyzed the expression profile and genetic mechanism controlling Mgat3 expression as well as GnT-III-mediated glycosylation, in general and specifically on E-cadherin, during EMT/MET. We found that during EMT, Mgat3 expression was dramatically decreased and later recovered when cells returned to an epithelial-like phenotype. We further identified that Mgat3 promoter methylation/demethylation is involved in this expression regulation. The impact of Mgat3 expression variation, along EMT/MET, leads to a variation in the expression levels of the enzymatic product of GnT-III (bisecting GlcNAc structures), and more importantly, to the specific modification of E-cadherin glycosylation with bisecting GlcNAc structures.Conclusions/SignificanceAltogether, this work identifies for the first time Mgat3 glycogene expression and GnT-III-mediated glycosylation, specifically on E-cadherin, as a novel and major component of the EMT/MET mechanism signature, supporting its role during EMT/MET.

Carnitine Precursor γ-Butyrobetaine is a Novel Substrate of the Na+- and Cl−-dependent GABA Transporter Gat2

To study transport of the carnitine precursor gamma-butyrobetaine (GBB) by rat liver-specific GABA transporter 2 (rGat2), we measured the uptake of deuterated GBB (d(3)-GBB) by Xenopus oocytes expressing rGat2. rGat2-mediated d(3)-GBB uptake was Na(+) and Cl(-) dependent, and was saturable with a K(m) value of 70.6 ± 34.8 µM. In carnitine transporter Octn2-deficient Jvs mice, mRNA expression of mGat3, the mouse ortholog of GAT2, decreased in liver, but increased in brain, while mRNA expression of mBbox1, which hydroxylates GBB to carnitine, increased in kidney and brain. These results indicate that GAT2 plays a role in transmembrane transport of GBB.

Identification of Acyl Coenzyme A:Monoacylglycerol Acyltransferase 3, an Intestinal Specific Enzyme Implicated in Dietary Fat Absorption

Acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol using 2-monoacylglycerol and fatty acyl coenzyme A. This enzymatic reaction is believed to be an essential and rate-limiting step for the absorption of fat in the small intestine. Although the first MGAT-encoding cDNA, designated MGAT1, has been recently isolated, it is not expressed in the small intestine and hence cannot account for the high intestinal MGAT enzyme activity that is important for the physiology of fat absorption. In the current study, we report the identification of a novel MGAT, designated MGAT3, and present evidence that it fulfills the criteria to be the elusive intestinal MGAT. MGAT3 encodes a approximately 36-kDa transmembrane protein that is highly homologous to MGAT1 and -2. In humans, expression of MGAT3 is restricted to gastrointestinal tract with the highest level found in the ileum. At the cellular level, recombinant MGAT3 is localized to the endoplasmic reticulum. Recombinant MGAT3 enzyme activity produced in insect Sf9 cells selectively acylates 2-monoacylglycerol with higher efficiency than other stereoisomers. The molecular identification of MGAT3 will facilitate the evaluation of using intestinal MGAT as a potential point of intervention for antiobesity therapies.