Interleukin-10 Directly Inhibits CD8+ T Cell Function by Enhancing N-Glycan Branching to Decrease Antigen Sensitivity.

Chronic viral infections remain a global health concern. The early events that facilitate viral persistence have been linked to the activity of the immunoregulatory cytokine IL-10. However, the mechanisms by which IL-10 facilitates the establishment of chronic infection are not fully understood. Herein, we demonstrated that the antigen sensitivity of CD8+ T cells was decreased during chronic infection and that this was directly mediated by IL-10. Mechanistically, we showed that IL-10 induced the expression of Mgat5, a glycosyltransferase that enhances N-glycan branching on surface glycoproteins. Increased N-glycan branching on CD8+ T cells promoted the formation of a galectin 3-mediated membrane lattice, which restricted the interaction of key glycoproteins, ultimately increasing the antigenic threshold required for T cell activation. Our study identified a regulatory loop in which IL-10 directly restricts CD8+ T cell activation and function through modification of cell surface glycosylation allowing the establishment of chronic infection.

N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates.

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan ß1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans.

Tumor cell E-selectin ligands determine partialefficacy of bortezomib on spontaneous lung metastasis formation of solid human tumors in vivo.

Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.

Complex N-Linked Glycosylation: A Potential Modifier of Niemann-Pick Disease, Type C1 Pathology.

Complex asparagine-linked glycosylation plays key roles in cellular functions, including cellular signaling, protein stability, and immune response. Previously, we characterized the appearance of a complex asparagine-linked glycosylated form of lysosome-associated membrane protein 1 (LAMP1) in the cerebellum of Npc1-/- mice. This LAMP1 form was found on activated microglia, and its appearance correlated both spatially and temporally with cerebellar Purkinje neuron loss. To test the importance of complex asparagine-linked glycosylation in NPC1 pathology, we generated NPC1 knock-out mice deficient in MGAT5, a key Golgi-resident glycosyl transferase involved in complex asparagine-linked glycosylation. Our results show that Mgat5-/-:Npc1-/- mice were smaller than Mgat5+/+:Npc1-/- mice, and exhibited earlier NPC1 disease onset and reduced lifespan. Western blot and lectin binding analyses of cerebellar extracts confirmed the reduction in complex asparagine-linked glycosylation, and the absence of the hyper-glycosylated LAMP1 previously observed. Western blot analysis of cerebellar extracts demonstrated reduced calbindin staining in Mgat5-/-:Npc1-/- mice compared to Mgat5+/+:Npc1-/- mutant mice, and immunofluorescent staining of cerebellar sections indicated decreased levels of Purkinje neurons and increased astrogliosis in Mgat5-/-:Npc1-/- mice. Our results suggest that reduced asparagine-linked glycosylation increases NPC1 disease severity in mice, and leads to the hypothesis that mutations in genes involved in asparagine-linked glycosylation may contribute to disease severity progression in individuals with NPC1. To examine this with respect to MGAT5, we analyzed 111 NPC1 patients for two MGAT5 SNPs associated with multiple sclerosis; however, we did not identify an association with NPC1 phenotypic severity.

Energetic adaptations: Metabolic control of endocytic membrane traffic.

Endocytic membrane traffic controls the access of myriad cell surface proteins to the extracellular milieu, and thus gates nutrient uptake, ion homeostasis, signaling, adhesion and migration. Coordination of the regulation of endocytic membrane traffic with a cell's metabolic needs represents an important facet of maintenance of homeostasis under variable conditions of nutrient availability and metabolic demand. Many studies have revealed intimate regulation of endocytic membrane traffic by metabolic cues, from the specific control of certain receptors or transporters, to broader adaptation or remodeling of the endocytic membrane network. We examine how metabolic sensors such as AMP-activated protein kinase, mechanistic target of rapamycin complex 1 and hypoxia inducible factor 1 determine sufficiency of various metabolites, and in turn modulate cellular functions that includes control of endocytic membrane traffic. We also examine how certain metabolites can directly control endocytic traffic proteins, such as the regulation of specific protein glycosylation by limiting levels of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) produced by the hexosamine biosynthetic pathway. From these ideas emerge a growing appreciation that endocytic membrane traffic is orchestrated by many intrinsic signals derived from cell metabolism, allowing alignment of the functions of cell surface proteins with cellular metabolic requirements. Endocytic membrane traffic determines how cells interact with their environment, thus defining many aspects of nutrient uptake and energy consumption. We examine how intrinsic signals that reflect metabolic status of a cell regulate endocytic traffic of specific proteins, and, in some cases, exert broad control of endocytic membrane traffic phenomena. Hence, endocytic traffic is versatile and adaptable and can be modulated to meet the changing metabolic requirements of a cell.

The polymorphisms of FGFR2 and MGAT5 affect the susceptibility to COPD in the Chinese people.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by incomplete reversible airflow limitation and chronic inflammatory response lesions. This study mainly explored whether FGFR2 and MGAT5 polymorphisms affected the risk of COPD in the Chinese people. METHODS: Five variants in FGFR2 and MGAT5 were chosen and genotyped using Agena MassARRAY platform from 315 COPD patients and 314 healthy controls. The correlation of FGFR2 and MGAT5 with COPD susceptibility was evaluated with odds ratio (OR) and 95% confidence interval (CI) via logistic regression. RESULTS: We found rs2420915 enhanced the risk of COPD, while rs6430491, rs2593704 reduced the susceptibility of COPD (p < 0.05). Rs2420915 could promote the incidence of COPD in the elderly and nonsmokers. Rs1907240 and rs2257129 also increased the susceptibility to COPD in nonsmokers (p < 0.05). MGAT5-rs2593704 played a protective role in COPD development in different subgroups (age ≤ 70, male, smokers, and individuals with BMI ≤ 24 kg/m2). Meanwhile, rs6430491 was linked with a lower risk of COPD in nonsmoking and BMI ≤ 24 kg/m2 subgroups. CONCLUSIONS: We concluded that FGFR2 and MGAT5 genetic polymorphisms are correlated with the risk of COPD in the Chinese people. These data underscored the important role of FGFR2 and MGAT5 gene in the occurrence of COPD and provided new biomarkers for COPD treatment. TRIAL REGISTRATION: NA.

MicroRNA-424 Predicts a Role for ß-1,4 Branched Glycosylation in Cell Cycle Progression.

MicroRNA regulation of protein expression plays an important role in mediating many cellular processes, from cell proliferation to cell death. The human microRNA miR-424 is up-regulated in response to anti-proliferative cytokines, such as transforming growth factor ß (TGFß), and directly represses cell cycle progression. Our laboratory recently established that microRNA can be used as a proxy to identify biological roles of glycosylation enzymes (glycogenes). Herein we identify MGAT4A, OGT, and GALNT13 as targets of miR-424. We demonstrate that MGAT4A, an N-acetylglucosaminyltransferase that installs the ß-1,4 branch of N-glycans, is directly regulated by miR-424 in multiple mammary epithelial cell lines and observe the loss of MGAT4A in response to TGFß, an inducer of miR-424. Knockdown of MGAT4A induces cell cycle arrest through decreasing CCND1 levels. MGAT4A does not affect levels of ß-1,6 branched N-glycans, arguing that this effect is specific to ß-1,4 branching and not due to gross changes in overall N-linked glycosylation. This work provides insight into the regulation of cell cycle progression by specific N-glycan branching patterns.

Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation.

Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N-glycosylation of recombinant erythropoietin (rEPO), a human α2,6-sialyltransferase (ST6Gal1) was expressed in Chinese hamster ovary (CHO-K1) cells. Sialylation increased on both EPO and CHO cellular proteins as observed by SNA lectin analysis, and HPLC profiling revealed that the sialic acid content of total glycans on EPO increased by 26%. The increase in sialic acid content was further verified by detailed profiling of the N-glycan structures using mass spectra (MS) analysis. In order to enhance antennarity/branching, UDP-N-acetylglucosamine: α-1,3-D-mannoside ß1,4-N-acetylglucosaminyltransferase (GnTIV/Mgat4) and UDP-N-acetylglucosamine:α-1,6-D-mannoside ß1,6-N-acetylglucosaminyltransferase (GnTV/Mgat5), was incorporated into CHO-K1 together with ST6Gal1. Tri- and tetraantennary N-glycans represented approximately 92% of the total N-glycans on the resulting EPO as measured using MS analysis. Furthermore, sialic acid content of rEPO from these engineered cells was increased ∼45% higher with tetra-sialylation accounting for ∼10% of total sugar chains compared to ∼3% for the wild-type parental CHO-K1. In this way, coordinated overexpression of these three glycosyltransferases for the first time in model CHO-K1 cell lines provides a mean for enhancing both N-glycan branching complexity and sialylation with opportunities to generate tailored complex N-glycan structures on therapeutic glycoproteins in the future.

Structure and function of N-acetylglucosaminyltransferase V (GnT-V).

BACKGROUND: The ß1,6-GlcNAc branch in N-glycans, produced by a glycosyltransferase N-acetylglucosaminyltransferase V (GnT-V or MGAT5), is associated with cancer and autoimmune diseases. SCOPE: Here, we summarize the structure and activity regulation of GnT-V. We also describe the roles of the ß1,6-GlcNAc branch on glycoproteins in cells and the phenotypes of Mgat5-deficient mice, focusing on cancer and the immune system. MAJOR CONCLUSIONS: GnT-V has a unique structure for substrate recognition, and its activity and function are regulated by shedding. The glycans produced by GnT-V play pivotal roles in the differentiation of neural cells, cancer malignancy and immunotherapy, and the development of autoimmune diseases by regulating the functions and cell surface residency of glycoproteins. GENERAL SIGNIFICANCE: Controlling the expression or activity of GnT-V could be a therapeutic option against cancer and autoimmune diseases. Future work should clarify how GnT-V selectively modifies the specific glycoproteins or N-glycosylation sites in vivo.

N-acetylglucosaminyltransferase V confers hepatoma cells with resistance to anoikis through EGFR/PAK1 activation.

Elevated expression and activity of N-acetylglucosaminyltransferase V (Mgat5) in hepatocellular carcinoma (HCC) is a common early event involved in tumor invasion during hepatocarcinogenesis. A better understanding of the functional role and the molecular mechanism for Mgat5-targeted protein and downstream signaling pathway behind hepatoma invasion and metastasis is urgently needed. Here, we show that Mgat5 overexpression promoted anchorage-independent growth and inhibited anoikis in hepatoma cells. This effect was reversed by glycosyltransferase inactive mutant Mgat5 L188R transfection, α-mannosidase II inhibitor swainsonine treatment and N-acetyl glucosamine (GlcNAc) phosphotransferase (GPT) inhibitor tunicamycin administration. Mgat5 overexpression increased p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase inactivation with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor P21-activated kinase inhibitor III (IPA3) treatment reversed anoikis resistance in Mgat5-overexpressed hepatoma cells. Furthermore, Mgat5 overexpression upregulated ß-1-6-GlcNAc branched N-glycosylation and following phosphorylation of epidermal growth factor receptor (EGFR) in hepatoma cells. EGFR tyrosine kinase inhibitors AG1478 and Iressa treatment declined anchorage-independent growth and anoikis resistance, which could be rescued by constitutive active mutant PAK1 T423E coexpression in Mgat5-overexpressed hepatoma cells. Conversely, knockdown of Mgat5 reduced EGFR/PAK1-dependent anoikis resistance, which could be reversed by PAK1 T423E. These results identified Mgat5-mediated ß-1-6-GlcNAc branched N-glycosylation and following activation of EGFR as a potential novel upstream molecular event for PAK1-induced anoikis resistance in hepatoma cells, implicating that molecular targeted therapeutics against Mgat5/EGFR/PAK1 might open a new avenue for personalized medicine in advanced-stage HCC patients.

Aberrant N-glycosylation in cancer: MGAT5 and ß1,6-GlcNAc branched N-glycans as critical regulators of tumor development and progression.

BACKGROUND: Changes in protein glycosylation are widely observed in tumor cells. N-glycan branching through adding ß1,6-linked N-acetylglucosamine (ß1,6-GlcNAc) to an α1,6-linked mannose, which is catalyzed by the N-acetylglucosaminyltransferase V (MGAT5 or GnT-V), is one of the most frequently observed tumor-associated glycan structure formed. Increased levels of this branching structure play a pro-tumoral role in various ways, for example, through the stabilization of growth factor receptors, the destabilization of intercellular adhesion, or the acquisition of a migratory phenotype. CONCLUSION: In this review, we provide an updated and comprehensive summary of the physiological and pathophysiological roles of MGAT5 and ß1,6-GlcNAc branched N-glycans, including their regulatory mechanisms. Specific emphasis is given to the role of MGAT5 and ß1,6-GlcNAc branched N-glycans in cellular mechanisms that contribute to the development and progression of solid tumors. We also provide insight into possible future clinical implications, such as the use of MGAT5 as a prognostic biomarker.

RUNX2 promotes gastric cancer progression through the transcriptional activation of MGAT5 and MMP13.

Introduction: RUNX2 is overexpressed in gastric cancer but the mechanism(s) through which it promotes tumor progression remain undefined. Here, we investigated the role of RUNX2 on gastric cancer pathogenesis at the molecular level. Methods: The qRT-PCR and western bolt were utilized to examine the mRNA and protein levels. CCK-8, Transwell and wound healing assays were used to measure cell proliferation, invasion and migration. CHIP-PCR gel electrophoresis was used to verify RUNX2 as a transcription factor for MMP13 and MGAT5. The in vivo assay was utilized to assess tumor growth. In vivo assay was used to evaluate tumor growth, aberrant expression of RUNX2 and lung metastasis of gastric cancer. Results: RUNX2 is overexpressed in MKN-45 and AGS cells. Genetic RUNX2 silencing reduced the proliferation, invasion and migration of MKN-45 and AGS cells. Analysis of the gastric cancer samples from the database revealed a significant positive correlation between MGAT5, MMP13, and RUNX2 expression. JASPAR analysis revealed that there was a potential binding site of RUNX2 in the promoter regions of MGAT5 and MMP13, and the experimental results confirmed that RUNX2 could regulate the expression of MGAT5 and MMP13 respectively. In vivo assays confirmed the aberrant expression of RUNX2 in mouse models of gastric cancer and reduced growth and lung metastasis in RUNX2 silenced xenograft tumors assessed. Conclusion: Collectively, these data reveal that RUNX2 enhances MGAT5 and MMP13 expression in gastric cancer cells and represents a biomarker and potential therapeutic target for gastric cancer therapy.

ISLR interacts with MGAT5 to promote the malignant progression of human gastric cancer AGS cells.

Objectives: Gastric cancer is a common malignant tumor with high morbidity and mortality. The present study aimed to investigate the role of the immunoglobulin superfamily containing leucine-rich repeat (ISLR) gene in gastric cancer and examine whether ISLR could interact with N-acetylglucosaminyltransferase V (MGAT5) to affect the malignant progression of gastric cancer. Materials and Methods: The expression of ISLR and MGAT5 in human normal gastric epithelial cells and human gastric cancer cells, and the transfection efficiency of ISLR interference plasmids and MGAT5 overexpression plasmids were all detected by reverse transcription-quantitative PCR (RT-qPCR) and western blot. The viability, proliferation, migration and invasion, and epithelial-mesenchymal transition (EMT) of gastric cancer cells after indicated transfection were detected by Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, wound healing assay, and transwell assay. The interaction between ISLR and MGAT5 was confirmed by co-immunoprecipitation. The expression of proteins related to migration, invasion, and EMT was detected by immunofluorescence and western blot. Results: As a result, ISLR was highly expressed in gastric cancer and was associated with poor prognosis. Interference with ISLR inhibited the viability, proliferation, migration, invasion, and EMT of gastric cancer cells. ISLR interacted with MGAT5 in gastric cancer cells. MGAT5 overexpression weakened the effects of ISLR knockdown on suppressing the viability, proliferation, migration, invasion, and EMT of gastric cancer cells. Conclusion: ISLR interacted with MGAT5 to promote the malignant progression of gastric cancer.

The cancer-associated glycosyltransferase GnT-V (MGAT5) recognizes the N-glycan core via residues outside its catalytic pocket.

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) is a glycosyltransferase involved in cancer metastasis that creates the ß1,6-branch on N-glycans of target proteins such as cell adhesion molecules and cell surface receptors. The 3D structure of GnT-V and its catalytic site, which are critical for the interaction with the N-glycan terminal, have already been revealed. However, it remains unclear how GnT-V recognizes the core part of N-glycan or the polypeptide part of the acceptor. Using molecular dynamics simulations and biochemical experiments, we found that several residues outside the catalytic pocket are likely involved in the recognition of the core part of N-glycan. Furthermore, our simulation suggested that UDP binding affects the orientation of the acceptor due to the conformational change at the Manα1,6-Man linkage. These findings provide new insights into how GnT-V recognizes its glycoprotein substrates.

Regulation of neural stem cell differentiation and brain development by MGAT5-mediated N-glycosylation.

Undifferentiated neural stem and progenitor cells (NSPCs) encounter extracellular signals that bind plasma membrane proteins and influence differentiation. Membrane proteins are regulated by N-linked glycosylation, making it possible that glycosylation plays a critical role in cell differentiation. We assessed enzymes that control N-glycosylation in NSPCs and found that loss of the enzyme responsible for generating ß1,6-branched N-glycans, N-acetylglucosaminyltransferase V (MGAT5), led to specific changes in NSPC differentiation in vitro and in vivo. Mgat5 homozygous null NSPCs in culture formed more neurons and fewer astrocytes compared with wild-type controls. In the brain cerebral cortex, loss of MGAT5 caused accelerated neuronal differentiation. Rapid neuronal differentiation led to depletion of cells in the NSPC niche, resulting in a shift in cortical neuron layers in Mgat5 null mice. Glycosylation enzyme MGAT5 plays a critical and previously unrecognized role in cell differentiation and early brain development.

Loss of N-acetylglucosaminyl transferase V is involved in the impaired osteogenic differentiation of bone marrow mesenchymal stem cells.

The imbalance of bone resorption and bone formation causes osteoporosis (OP), a common skeletal disorder. Decreased osteogenic activity was found in the bone marrow cultures from N-acetylglucosaminyl transferase V (MGAT5)-deficient mice. We hypothesized that MGAT5 was associated with osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and involved in the pathological mechanisms of osteoporosis. To test this hypothesis, the mRNA and protein expression levels of MGAT5 were determined in bone tissues of ovariectomized (OVX) mice, a well-established OP model, and the role of MGAT5 in osteogenic activity was investigated in murine BMSCs. As expected, being accompanied by the loss of bone mass density and osteogenic markers (runt-related transcription factor 2, osteocalcin and osterix), a reduced expression of MGAT5 in vertebrae and femur tissues were found in OP mice. In vitro, knockdown of Mgat5 inhibited the osteogenic differentiation potential of BMSCs, as evidenced by the decreased expressions of osteogenic markers and less alkaline phosphatase and alizarin red S staining. Mechanically, knockdown of Mgat5 suppressed the nuclear translocation of ß-catenin, thereby downregulating the expressions of downstream genes c-myc and axis inhibition protein 2, which were also associated with osteogenic differentiation. In addition, Mgat5 knockdown inhibited bone morphogenetic protein (BMP)/transforming growth factor (TGF)-ß signaling pathway. In conclusion, MGAT5 may modulate the osteogenic differentiation of BMSCs via the ß-catenin, BMP type 2 (BMP2) and TGF-ß signals and involved in the process of OP.

Identification of glycogene signature as a tool to predict the clinical outcome and immunotherapy response in breast cancer.

Background: Breast cancer is one of the most important diseases in women around the world. Glycosylation modification correlates with carcinogenesis and roles of glycogenes in the clinical outcome and immune microenvironment of breast cancer are unclear. Methods: A total of 1297 breast cancer and normal cases in the TCGA and GTEx databases were enrolled and the transcriptional and survival information were extracted to identify prognostic glycogenes using Univariate Cox, LASSO regression, Multivariate Cox analyses and Kaplan-Meier method. The immune infiltration pattern was explored by the single sample gene set enrichment method. The HLA and immune checkpoint genes expression were also compared in different risk groups. The expressions of a glycogene MGAT5 as well as its products were validated by immunohistochemistry and western blotting in breast cancer tissues and cells. Results: A 19-glycogene signature was identified to separate breast cancer patients into high- and low-risk groups with distinct overall survival rates (P < 0.001). Compared with the high-risk group, proportion of naive B cells, plasma cells and CD8+ T cells increased in the low-risk group (P < 0.001). Besides, expressions of HLA and checkpoint genes, such as CD274, CTLA4, LAG3 and TIGIT3, were upregulated in low-risk group. Additionally, highly expressed MGAT5 was validated in breast cancer tissues and cells. Downstream glycosylation products of MGAT5 were all increased in breast cancer. Conclusions: We identified a 19-glycogene signature for risk prediction of breast cancer patients. Patients in the low-risk group demonstrated a higher immune infiltration and better immunotherapy response. The validation of MGAT5 protein suggests a probable pathway and target for the development and treatment of breast cancer.

IGF2BP1 Promotes the Liver Cancer Stem Cell Phenotype by Regulating MGAT5 mRNA Stability by m6A RNA Methylation.

The aim of this study was to elucidate the mechanism of action of the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) on the phenotype of the liver cancer stem cells (LCSCs). To gain insight into the mechanism of action of the IGF2BP1 on LCSCs, the IGF2BP1 shRNA sequences were transfected into hepatocellular carcinoma (HCC) cells. The LCSC phenotypes were measured by stemness gene expressions, spheroid formations, percentages of the CD133+ cells, colony formations, and tumorigenesis in vivo. Next, we screened for possible molecular mechanisms from the Cancer Genome Atlas (TCGA) database, and a methylated RNA immunoprecipitation-quantitative polymerase chain reaction (MeRIP-qPCR) was used to adjust the binding of IGF2BP1 to the target gene, alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase (MGAT5). The MeRIP-qPCR was used to detect the binding of IGF2BP1 and MGAT5 through N6 methyladenosine (m6A) modification. Furthermore, we adjusted the attenuation of the mRNA of the MGAT5 using quantitative real-time PCR (qRT-PCR). The IGF2BP1 was upregulated in the LCSCs. Furthermore, the IGF2BP1 promoted self-renewal and chemoresistance in human LCSCs and tumorigenesis in mice and it enhanced the expression of stemness genes in the LCSCs compared with the HCC cells. Further exploration indicated that the IGF2BP1 binds directly to the MGAT5 and inhibits its mRNA attenuation, suggesting that the IGF2BP1 impacts MGAT5 mRNA stability through m6A modification. Thus, it can be concluded that the IGF2BP1 facilitated the LCSC phenotypes by promoting the MGAT5 mRNA stability through the upregulation of m6A modification of the MGAT5 mRNA.

Glioma stem cells invasive phenotype at optimal stiffness is driven by MGAT5 dependent mechanosensing.

BACKGROUND: Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context. METHOD: To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9. RESULTS: We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (ß1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (ß1-6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression. CONCLUSION: MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer.

Galectins and Their Ligand Glycoconjugates in the Central Nervous System Under Physiological and Pathological Conditions.

Galectins are ß-galactoside-binding lectins consisting of 15 members in mammals. Galectin-1,-3,-4,-8, and -9 are predominantly expressed in the central nervous system (CNS) and regulate various physiological and pathological events. This review summarizes the current knowledge of the cellular expression and role of galectins in the CNS, and discusses their functions in neurite outgrowth, myelination, and neural stem/progenitor cell niches, as well as in ischemic/hypoxic/traumatic injuries and neurodegenerative diseases such as multiple sclerosis. Galectins are expressed in both neurons and glial cells. Galectin-1 is mainly expressed in motoneurons, whereas galectin-3-positive neurons are broadly distributed throughout the brain, especially in the hypothalamus, indicating its function in the regulation of homeostasis, stress response, and the endocrine/autonomic system. Astrocytes predominantly contain galectin-1, and galectin-3 and-9 are upregulated along with its activation. Activated, but not resting, microglia contain galectin-3, supporting its phagocytic activity. Galectin-1,-3, and -4 are characteristically expressed during oligodendrocyte differentiation. Galectin-3 from microglia promotes oligodendrocyte differentiation and myelination, while galectin-1 and axonal galectin-4 suppress its differentiation and myelination. Galectin-1- and- 3-positive cells are involved in neural stem cell niche formation in the subventricular zone and hippocampal dentate gyrus, and the migration of newly generated neurons and glial cells to the olfactory bulb or damaged lesions. In neurodegenerative diseases, galectin-1,-8, and -9 have neuroprotective and anti-inflammatory activities. Galectin-3 facilitates pro-inflammatory action; however, it also plays an important role during the recovery period. Several ligand glycoconjugates have been identified so far such as laminin, integrins, neural cell adhesion molecule L1, sulfatide, neuropilin-1/plexinA4 receptor complex, triggering receptor on myeloid cells 2, and T cell immunoglobulin and mucin domain. N-glycan branching on lymphocytes and oligodendroglial progenitors mediated by ß1,6-N-acetylglucosaminyltransferase V (Mgat5/GnTV) influences galectin-binding, modulating inflammatory responses and remyelination in neurodegenerative diseases. De-sulfated galactosaminoglycans such as keratan sulfate are potential ligands for galectins, especially galectin-3, regulating neural regeneration. Galectins have multitudinous functions depending on cell type and context as well as post-translational modifications, including oxidization, phosphorylation, S-nitrosylation, and cleavage, but there should be certain rules in the expression patterns of galectins and their ligand glycoconjugates, possibly related to glucose metabolism in cells.