Glycomic Alterations Research Articles

Evaluation of the context of downstream N- and free N-glycomic alterations induced by swainsonine in HepG2 cells

Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.

Changes in Serum N-Glycome for Risk Drinkers: A Comparison with Standard Markers for Alcohol Abuse in Men and Women.

Background and aim: Glycomic alterations serve as biomarker tools for different diseases. The present study aims to evaluate the diagnostic capability of serum N-glycosylation to identify alcohol risk drinking in comparison with standard markers. Methods: We included 1516 adult individuals (age range 18–91 years; 55.3% women), randomly selected from a general population. A total of 143 (21.0%) men and 50 (5.9%) women were classified as risk drinkers after quantification of daily alcohol consumption and the Alcohol Use Disorders Identification Test (AUDIT). Hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) was used for the quantification of 46 serum N-glycan peaks. Serum gamma-glutamyltransferase (GGT), carbohydrate-deficient transferrin (CDT), and red blood cell mean corpuscular volume (MCV) were measured by standard clinical laboratory methods. Results: Variations in serum N-glycome associated risk drinking were more prominent in men compared to women. A unique combination of N-glycan peaks selected by the selbal algorithm shows good discrimination between risk-drinkers and non-risk drinkers for men and women. Receiver operating characteristics (ROC) curves show accuracy for the diagnosis of risk drinking, which is comparable to that of the golden standards, GGT, MCV and CDT markers for men and women. Additionally, the inclusion of N-glycan peaks improves the diagnostic accuracy of the standard markers, although it remains relatively low, due to low sensitivity. For men, the area under the ROC curve using N-glycome data is 0.75, 0.76, and 0.77 when combined with GGT, MCV, and CDT, respectively. In women, the areas were 0.76, 0.73, and 0.73, respectively. Conclusion: Risk drinking is associated with significant variations in the serum N-glycome, which highlights its potential diagnostic utility.

Immunoglobulin G GlycosylationChanges in Aging and Other Inflammatory Conditions.

Among the multiple roles played by protein glycosylation, the fine regulation of biological interactions is one of the most important. The asparagine 297 (Asn297) of IgG heavy chains is decorated by a diantennary glycan bearing a number of galactose and sialic acid residues on the branches ranging from 0 to 2. In addition, the structure can present core-linked fucose and/or a bisecting GlcNAc. In many inflammatory and autoimmune conditions, as well as in metabolic, cardiovascular, infectious, and neoplastic diseases, the IgG Asn297-linked glycan becomes less sialylated and less galactosylated, leading to increased expression of glycans terminating with GlcNAc. These conditions alter also the presence of core-fucose and bisecting GlcNAc. Importantly, similar glycomic alterations are observed in aging. The common condition, shared by the above-mentioned pathological conditions and aging, is a low-grade, chronic, asymptomatic inflammatory state which, in the case of aging, is known as inflammaging. Glycomic alterations associated with inflammatory diseases often precede disease onset and follow remission. The aberrantly glycosylated IgG glycans associated with inflammation and aging can sustain inflammation through different mechanisms, fueling a vicious loop. These include complement activation, Fcγ receptor binding, binding to lectin receptors on antigen-presenting cells, and autoantibody reactivity. The complex molecular bases of the glycomic changes associated with inflammation and aging are still poorly understood.

Interferon-α alters host glycosylation machinery during treated HIV infection.

BackgroundA comprehensive understanding of host factors modulated by the antiviral cytokine interferon-α (IFNα) is imperative for harnessing its beneficial effects while avoiding its detrimental side-effects during HIV infection. Cytokines modulate host glycosylation which plays a critical role in mediating immunological functions. However, the impact of IFNα on host glycosylation has never been characterized.MethodsWe assessed the impact of pegylated IFNα2a on IgG glycome, as well as CD8+ T and NK cell-surface glycomes, of 18 HIV-infected individuals on suppressive antiretroviral therapy. We linked these glycomic signatures to changes in inflammation, CD8+ T and NK cell phenotypes, and HIV DNA.FindingsWe identified significant interactions that support a model in which a) IFNα increases the proportion of pro-inflammatory, bisecting GlcNAc glycans (known to enhance FcγR binding) within the IgG glycome, which in turn b) increases inflammation, which c) leads to poor CD8+ T cell phenotypes and poor IFNα-mediated reduction of HIV DNA. Examining cell-surface glycomes, IFNα increases levels of the immunosuppressive GalNAc-containing glycans (T/Tn antigens) on CD8+ T cells. This induction is associated with lower HIV-gag-specific CD8+ T cell functions. Last, IFNα increases levels of fucose on NK cells. This induction is associated with higher NK functions upon K562 stimulation.InterpretationIFNα causes host glycomic alterations that are known to modulate immunological responses. These alterations are associated with both detrimental and beneficial consequences of IFNα. Manipulating host glycomic interactions may represent a strategy for enhancing the positive effects of IFNα while avoiding its detrimental side-effects.FundingNIH grants R21AI143385, U01AI110434.

A lectin-based glycomic approach identifies FUT8 as a driver of radioresistance in oesophageal squamous cell carcinoma.

Radio-resistance is recognized as a main factor in the failure of radiotherapy in oesophageal squamous cell carcinoma (ESCC). Aberrant cell surface glycosylation has been reported to correlate with radio-resistance in different kinds of tumours. However, glycomic alterations and the corresponding enzymes associated with ESCC radio-resistance have not yet been defined. Two radioresistant cell lines, EC109R and TE-1R, were established from parental ESCC cell lines EC109 and TE-1 by fractionated irradiation. A lectin microarray was used to screen for altered glycan patterns. RNA-sequencing (RNA-seq) was employed to identify differentially expressed glycosyltransferases. Cell Counting Kit-8, colony formation and flow cytometry assays were used to measure cell viability and radiosensitivity. Expression of glycosyltransferase in ESCC tissues was assessed by immunohistochemistry. In vivo radiosensitivity was analysed using a nude mouse xenograft model. Downstream effectors of the enzyme were verified using a lectin-based pull-down assay combined with mass spectrometry. We found that EC109R and TE-1R cells were more resistant to irradiation than the parental EC109 and TE-1 cells. Using lectin microarrays combined with RNA sequencing, we found that α1, 6-fucosyltransferase (FUT8) was overexpressed in the radioresistant ESCC cell lines. Both gain- and loss-of-function studies confirmed that FUT8 regulates the sensitivity of ESCC cells to irradiation. Importantly, we found that high FUT8 expression was positively linked to radio-resistance and a poor prognosis in ESCC patients who received radiation therapy. Moreover, FUT8 inhibition suppressed the growth and formation of xenograft tumours in nude mice after irradiation. Using a lectin-based pull-down assay and mass spectrometry, we found that CD147 could be glycosylated by FUT8. As expected, inhibition of CD147 partly reversed FUT8-induced radio-resistance in ESCC cells. Our results indicate that FUT8 functions as a driver of radio-resistance in ESCC by targeting CD147. Therefore, FUT8 may serve as a marker for predicting the response to radiation therapy in patients with ESCC.

Discovery of Pancreatic Ductal Adenocarcinoma-Related Aberrant Glycosylations: A Multilateral Approach of Lectin Microarray-Based Tissue Glycomic Profiling With Public Transcriptomic Datasets.

Aberrant protein glycosylation is one of the most notable features in cancerous tissues, and thereby glycoproteins with disease-relevant glycosylation alterations are fascinating targets for the development of biomarkers and therapeutic agents. For this purpose, a reliable strategy is needed for the analysis of glycosylation alterations occurring on specific glycoproteins during the progression of cancer. Here, we propose a bilateral approach combining lectin microarray-based tissue glycomic profiling and database-derived transcriptomic datasets. First, lectin microarray was used to perform differential glycomic profiling of crude extracts derived from non-tumor and tumor regions of frozen tissue sections from pancreatic ductal adenocarcinoma (PDAC). This analysis revealed two notable tissue glycome alterations in PDAC samples: increases in sialylated glycans and bisecting N-acetylglucosamine and a decrease in ABO blood group antigens. To examine aberrations in the glycosylation machinery related to these glycomic alterations, we next employed public datasets of gene expression profiles in cancerous and normal pancreases provided by The Cancer Genome Atlas and the Genotype-Tissue Expression projects, respectively. In this analysis, glycosyltransferases responsible for the glycosylation alterations showed aberrant gene expression in the cancerous tissues, consistent with the tissue glycomic profiles. The correlated alterations in glycosyltransferase expression and tissue glycomics were then evaluated by differential glycan profiling of a membrane N-glycoprotein, basigin, expressed in tumor and non-tumor pancreatic cells. The focused differential glycomic profiling for endogenous basigin derived from non-tumor and cancerous regions of PDAC tissue sections demonstrated that PDAC-relevant glycan alterations of basigin closely reflected the notable features in the disease-specific alterations in the tissue glycomes. In conclusion, the present multi-omics strategy using public transcriptomic datasets and experimental glycomic profiling using a tiny amount of clinical specimens successfully demonstrated that basigin is a representative N-glycoprotein that reflects PDAC-related aberrant glycosylations. This study indicates the usefulness of large public data sets such as the gene expression profiles of glycosylation-related genes for evaluation of the highly sensitive tissue glycomic profiling results. This strategy is expected to be useful for the discovery of novel glyco-biomarkers and glyco-therapeutic targets.

Dynamic analysis of N-glycomic and transcriptomic changes in the development of ovarian cancer cell line A2780 to its three cisplatin-resistant variants.

BackgroundPlatinum resistance development is a dynamic process that occurs during continuous chemotherapy and contributes to high mortality in ovarian cancer. Abnormal glycosylation has been reported in platinum resistance. Many studies on platinum resistance have been performed, but few of them have investigated platinum resistance-associated glycans based on N-glycomics. Moreover, glycomic alterations during platinum resistance development in ovarian cancer are rarely reported. Therefore, the objective of this study was to determine platinum resistance-related N-glycans in ovarian cancer cells during continuous exposure to cisplatin. These glycans might be involved in the mechanism of platinum resistance and serve as biomarkers to monitor its development.MethodsThis study mimicked the development of platinum resistance in ovarian cancer by continuously exposing A2780 cells to cisplatin. Cisplatin-resistant variants were confirmed by higher half maximal inhibitory concentration (IC50) values and increased P-glycoprotein (ABCB1, P-gp) expression compared to A2780 cells. Analysis of dynamic N-glycomic changes during the development of platinum resistance in cisplatin-resistant variants was performed with MALDI-time-of-flight (TOF)-MS combined with ethyl esterification derivatization, which were used to discriminate between α2,3- and α2,6-linkage N-acetylneuraminic acid. N-glycan alterations were further validated on a glycotransferase level via transcriptome sequencing and real-time PCR (RT-PCR).ResultsCompared to the A2780 cells, MS analysis indicated that α2,3-linked sialic structures and N-glycan gal-ratios were significantly higher, while fucosylated glycans were lower in three cisplatin-resistant variants. Transcriptome sequencing and RT-PCR showed that gene expression of ST3GAL6 and MGAT4A increased, while gene expression of FUT11, FUT1, GMDS, and B4GALT5 decreased in three cisplatin-resistant variants.ConclusionsAnalysis of N-glycans and glycogene expression showed that α2,3-linked sialic structures might serve as biomarkers to monitor the development of platinum resistance and to guide individualized treatment of ovarian cancer patients.

Frontline Science: Plasma and immunoglobulin G galactosylation associate with HIV persistence during antiretroviral therapy.

Global antibody glycosylation is dynamic and plays critical roles in shaping different immunological outcomes and direct antibody functionality during HIV infection. However, the relevance of global antibody or plasma glycosylation patterns to HIV persistence after antiretroviral therapy (ART) has not been characterized. First, we compared glycomes of total plasma and isolated immunoglobulin G (IgG) from HIV+ ART-suppressed, HIV+ viremic, and HIV-negative individuals. Second, in ART-suppressed individuals, we examined the associations between glycomes and (1) levels of cell-associated HIV DNA and RNA in PBMCs and isolated CD4+ T cells, (2) CD4 count and CD4%, and (3) expression of CD4+ T-cell activation markers. HIV infection is associated with persistent alterations in the IgG glycome including decreased levels of disialylated glycans, which is associated with a lower anti-inflammatory activity, and increased levels of fucosylated glycans, which is associated with lower antibody-dependent cell-mediated cytotoxicity (ADCC). We also show that levels of certain mono- and digalactosylated nonfucosylated glycomic traits (A2G1, A2G2, and A2BG2), which have been reported to be associated with higher ADCC and higher anti-inflammatory activities, exhibit significant negative correlations with levels of cell-associated total HIV DNA and HIV RNA in ART-suppressed individuals. Finally, levels of certain circulating anti-inflammatory glycans are associated with higher levels of CD4 T cells and lower levels of T-cell activation. Our findings represent the first proof-of-concept evidence that glycomic alterations, known to be associated with differential states of inflammation and ADCC activities, are also associated with levels of HIV persistence in the setting of ART suppression.

Impact of the Niemann-Pick c1 Gene Mutation on the Total Cellular Glycomics of CHO Cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive lipid storage disorder, and the majority of cases are caused by mutations in the NPC1 gene. In this study, we clarified how a single gene mutation in the NPC1 gene impacts the cellular glycome by analyzing the total glycomic expression profile of Chinese hamster ovary cell mutants defective in the Npc1 gene (Npc1 KO CHO cells). A number of glycomic alterations were identified, including increased expression of lactosylceramide, GM1, GM2, GD1, various neolacto-series glycosphingolipids, and sialyl-T (O-glycan), which was found to be the major sialylated protein-bound glycan, as well as various N-glycans, which were commonly both fucosylated and sialylated. We also observed significant increases in the total amounts of free oligosaccharides (fOSs), especially in the unique complex- and hybrid-type fOSs. Treatment of Npc1 KO CHO cells with 2-hydroxypropyl-β-cyclodextrin (HPBCD), which can reduce cholesterol and glycosphingolipid (GSL) storage, did not affect the glycomic alterations observed in the GSL-, N-, and O-glycans of Npc1 KO CHO cells. However, HPBCD treatment corrected the glycomic alterations observed in fOSs to levels observed in wild-type cells.

Glycomic and sialoproteomic data of gastric carcinoma cells overexpressing ST3GAL4.

Gastric carcinoma MKN45 cells stably transfected with the full-length ST3GAL4 gene were characterised by glycomic and sialoproteomic analysis. Complementary strategies were applied to assess the glycomic alterations induced by ST3GAL4 overexpression. The N- and O-glycome data were generated in two parallel structural analyzes, based on PGC-ESI-MS/MS. Data on glycan structure identification and relative abundance in ST3GAL4 overexpressing cells and respective mock control are presented. The sialoproteomic analysis based on titanium-dioxide enrichment of sialopeptides with subsequent LC-MS/MS identification was performed. This analysis identified 47 proteins with significantly increased sialylation. The data in this article is associated with the research article published in Biochim Biophys Acta “Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer” [1].

Comprehensive Glycomics of a Multistep Human Brain Tumor Model Reveals Specific Glycosylation Patterns Related to Malignancy.

Cancer cells frequently express glycans at different levels and/or with fundamentally different structures from those expressed by normal cells, and therefore elucidation and manipulation of these glycosylations may provide a beneficial approach to cancer therapy. However, the relationship between altered glycosylation and causal genetic alteration(s) is only partially understood. Here, we employed a unique approach that applies comprehensive glycomic analysis to a previously described multistep tumorigenesis model. Normal human astrocytes were transformed via the serial introduction of hTERT, SV40ER, H-RasV12, and myrAKT, thereby mimicking human brain tumor grades I-IV. More than 160 glycans derived from three major classes of cell surface glycoconjugates (N- and O-glycans on glycoproteins, and glycosphingolipids) were quantitatively explored, and specific glycosylation patterns related to malignancy were systematically identified. The sequential introduction of hTERT, SV40ER, H-RasV12, and myrAKT led to (i) temporal expression of pauci-mannose/mono-antennary type N-glycans and GD3 (hTERT); (ii) switching from ganglio- to globo-series glycosphingolipids and the appearance of Neu5Gc (hTERT and SV40ER); (iii) temporal expression of bisecting GlcNAc residues, α2,6-sialylation, and stage-specific embryonic antigen-4, accompanied by suppression of core 2 O-glycan biosynthesis (hTERT, SV40ER and Ras); and (iv) increased expression of (neo)lacto-series glycosphingolipids and fucosylated N-glycans (hTERT, SV40ER, Ras and AKT). These sequential and transient glycomic alterations may be useful for tumor grade diagnosis and tumor prognosis, and also for the prediction of treatment response.

Detection of Glycomic Alterations Induced by Overexpression of P-Glycoprotein on the Surfaces of L1210 Cells Using Sialic Acid Binding Lectins

P-glycoprotein (P-gp) overexpression is the most frequently observed cause of multidrug resistance in neoplastic cells. In our experiments, P-gp was expressed in L1210 mice leukemia cells (S cells) by selection with vincristine (R cells) or transfection with the gene encoding human P-gp (T cells). Remodeling of cell surface sugars is associated with P-gp expression in L1210 cells as a secondary cellular response. In this study, we monitored the alteration of cell surface saccharides by Sambucus nigra agglutinin (SNA), wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA). Sialic acid is predominantly linked to the surface of S, R and T cells via α-2,6 branched sugars that tightly bind SNA. The presence of sialic acid linked to the cell surface via α-2,3 branched sugars was negligible, and the binding of MAA (recognizing this branch) was much less pronounced than SNA. WGA induced greater cell death than SNA, which was bound to the cell surface and agglutinated all three L1210 cell-variants more effectively than WGA. Thus, the ability of lectins to induce cell death did not correlate with their binding efficiency and agglutination potency. Compared to S cells, P-gp positive R and T cells contain a higher amount of N-acetyl-glucosamine on their cell surface, which is associated with improved WGA binding. Both P-gp positive variants of L1210 cells are strongly resistant to vincristine as P-gp prototypical drug. This resistance could not be altered by liberalization of terminal sialyl residues from the cell surface by sialidase.

Glycomic alterations are associated with multidrug resistance in human leukemia

Correlations of disease phenotypes with glycosylation changes have been analyzed intensively in tumor biology field. In this study we describe glycomic alterations of multidrug resistance in human leukemia cell lines. Using multiple glycan profiling tools: real-time PCR for quantification of glycogenes, FITC–lectin binding for glycan profiling, and mass spectrometry for glycan composition, we compared the glycomics of drug-resistant K562/ADR cells with parental K562 line. The results showed that the expression of glycogenes, glycan profiling and N-glycan composition were different in K562/ADR cells, as compared with those in K562 cells, whereas O-glycans of the two cell lines showed no different mass spectra. Further analysis of the N-glycan regulation by way of tunicamycin application or PNGase F treatment in K562/ADR cells showed partial inhibition of biosynthesis and increased sensitivity to chemotherapeutic drugs in vitro. We targeted glycogene B3GNT8 and ST8SIA4, which were over-expressed in K562/ADR cells, and silenced the expression levels of two glycogenes after using RNA interference approach. The results showed that the silencing of B3GNT8 or ST8SIA4 in K562/ADR cells resulted in increased chemosensitivity to anti-tumor drugs. In conclusion, glycomic alterations are responsible for the overcoming multidrug resistance in human leukemia therapy and the N-linked oligosaccharides are associated with the drug resistance of cancer cells.

Glycomic alterations in the highly-abundant and lesser-abundant blood serum protein fractions for patients diagnosed with hepatocellular carcinoma

Hepatocellular cancer (HCC) is a serious human disease with an unfortunately low survival rate. It further poses a significant epidemic threat to our society through its viral vectors associated with cirrhosis conditions preceding the cancer. A search for biomarkers of these diseases enlists analytical glycobiology, in general, and quantitative biomolecular mass spectrometry (MS), in particular, as valuable approaches to cancer research. The recent advances in quantitative glycan permethylation prior to MALDI-MS oligosaccharide profiling has enabled us to compare the glycan quantitative proportions in the small serum samples of cancer and cirrhotic patients against control individuals. In this investigation, reasoning that some of the observed glycomic changes could be at least partly explained by acute-phase or immune responses, we further fractionated the major serum proteins from the minor components and compared statistically their differential glycosylation, elucidating some causes of quantitatively unusual glycosylation events. Numerous glycan structures were identified and tentatively connected with their originating proteins, with a particular emphasis on sialylated and fucosylated glycans. In particular, for the highly-abundant protein fraction, several smaller, neutral glycans were observed to be different between disease-free individuals and those diagnosed with either HCC or cirrhosis. Further, these types of glycans were also different between the two diseases. In the lesser-abundant protein fraction, a number of sialylated glycans were different between each state-of-health.

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat

BackgroundThere is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model.ResultsRat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory.In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p < 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. Conclusions: We found an increase in core-α-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.