PDF file, 354KB, N-linked glycosylation of total protein from 16 HCC tissue and adjacent liver tissue pairs.

Chromosome 1q often has been observed to be amplified in hepatocellular carcinoma. This review summarizes literature reports of multiple genes that have been proposed as possible 1q amplification drivers. These largely fall within 1q21-1q23. In addition, publicly available copy number alteration data from The Cancer Genome Atlas project were used to identify additional candidate genes involved in carcinogenesis. The most frequent location for gene amplification was 1q22, consistent with the results of the literature search. The genes TPM3 and NUF2 were found to be candidates whose amplification and/or mRNA up-regulation was most highly associated with poorer hepatocellular carcinoma outcomes.

BACKGROUNDChanges in N-linked glycosylation have been observed in the circulation of individuals with hepatocellular carcinoma. In particular, an elevation in the level of core fucosylation has been observed. However, the mechanisms through which core fucose is increased are not well understood. We hypothesized that a review of the literature and related bioinformatic review regarding six genes known to be involved in the attachment of core fucosylation, the synthesis of the fucosylation substrate guanosine diphosphate (GDP)-fucose, or the transport of the substrate into the Golgi might offer mechanistic insight into the regulation of core fucose levels.AIMTo survey the literature to capture the involvement of genes regulating core N-linked fucosylation in hepatocellular carcinomaMETHODSThe PubMed biomedical literature database was searched for the association of hepatocellular carcinoma and each of the core fucose-related genes and their protein products. We also queried The Cancer Genome Atlas Liver hepatocellular carcinoma (LIHC) dataset for genetic, epigenetic and gene expression changes for the set of six genes using the tools at cBioportal.RESULTSA total of 27 citations involving one or more of the core fucosylation-related genes (FPGT, FUK, FUT8, GMDS, SLC35C1, TSTA3) and hepatocellular carcinoma were identified. The same set of gene symbols was used to query the 371 patients with liver cancer in the LIHC dataset to identify the frequency of mRNA over or under expression, as well as non-synonymous mutations, copy number variation and methylation level. Although all six genes trended to more samples displaying over expression relative to under-expression, it was noted that a number of tumor samples had undergone amplification of the genes of the de novo synthesis pathway, GMDS (27 samples) and TSTA3 (78 samples). In contrast, the other four genes had undergone amplification in 2 or fewer samples.CONCLUSIONAmplification of genes involved in the de novo pathway for generation of GDPfucose, GMDS and TSTA3, likely contributes to the elevated core fucose observed in hepatocellular carcinoma.

BACKGROUNDChanges in N-linked glycosylation have been observed in the circulation of individuals with hepatocellular carcinoma.In particular, an elevation in the level of core fucosylation has been observed.However, the mechanisms through which core fucose is increased are not well understood.We hypothesized that a review of the literature and related bioinformatic review regarding six genes known to be involved in the attachment of core fucosylation, the synthesis of the fucosylation substrate guanosine diphosphate (GDP)-fucose, or the transport of the substrate into the Golgi might offer mechanistic insight into the regulation of core fucose levels. AIMTo survey the literature to capture the involvement of genes regulating core Nlinked fucosylation in hepatocellular carcinoma METHODSThe PubMed biomedical literature database was searched for the association of hepatocellular carcinoma and each of the core fucose-related genes and their protein products.We also queried The Cancer Genome Atlas Liver hepatocellular carcinoma (LIHC) dataset for genetic, epigenetic and gene expression changes for the set of six genes using the tools at cBioportal. RESULTSA total of 27 citations involving one or more of the core fucosylation-related genes (FPGT, FUK, FUT8, GMDS, SLC35C1, TSTA3) and hepatocellular carcinoma were identified.The same set of gene symbols was used to query the 371 patients with liver cancer in the LIHC dataset to identify the frequency of mRNA over or under expression, as well as non-synonymous mutations, copy number variation and methylation level.Although all six genes trended to more

The Aleuria aurantia lectin (AAL) derived from orange peel fungus contains five fucose-binding sites that recognizes fucose bound in α-1,2, α-1,3, α-1,4, and α-1,6 linkages to N-acetylglucosamine and galactose. Recently, we have created several recombinant AAL (rAAL) proteins that had altered binding affinity to fucose linkages. In this report, we further characterize the binding specificity of one of the mutated lectins, N224Q lectin. This lectin was characterized by lectin Western blotting, surface plasmon resonance, and glycan microarray and shown to have increased binding to fucosylated glycan. Subsequently, we used this lectin to identify secreted fucosylated glycoproteins from a fetal hepatic cell line. Proteomic analysis revealed several glycoproteins secreted by the fetal cell line that were bound by N224Q lectin. These findings were confirmed by subsequent proteomic analysis of human serum from control patients or patients with hepatocellular carcinoma. These represent candidate oncofetal markers for liver cancer.

Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the β-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers.

The Golgi phosphoprotein GP73 is elevated in the circulation of individuals with a diagnosis of hepatocellular carcinoma. Its usefulness as a biomarker of HCC is questioned, since it has also been reported to be elevated in the circulation of people with liver cirrhosis. Regulation of GP73 by inflammatory cytokines is therefore of interest. The interleukin-6 (IL-6) family cytokines were tested for effects on GP73 mRNA and/or protein levels in human hepatoblastoma HepG2 cells. Levels of GP73 mRNA and protein were up-regulated in HepG2 cells following treatment with either proinflammatory cytokine IL-6 or the related cytokine oncostatin M (OSM). Induction required the shared receptor subunit gp130, and correlated with increased tyrosine phosphorylation of STAT3. Maximal cytokine-mediated induction was not observed in the presence of protein synthesis inhibitor cycloheximide, suggesting additional regulatory factors play an important role. ELISA measurement of GP73 and IL-6 levels in the sera of patients with pre-malignant liver disease revealed a significant correlation between circulating levels of the two proteins. Similarly, a sensitive ELISA assay was developed to measure circulating OSM. OSM levels were elevated 6-7 fold in sera from patients with either cirrhosis or HCC relative to controls without liver disease. Although there was an association between levels of GP73 and OSM in serum from people with liver cirrhosis, there was not a statistically significant correlation in HCC, suggesting that the role of the cytokines in determining circulating levels may be complex. To our knowledge, this is the first report of OSM elevation being associated with liver disease.

Abstract Background: Alterations in glycosylation have long been associated with the development of cancer. In the case of primary hepatocellular carcinoma (HCC), one alteration that has often been associated is increased amounts of fucose attached to the N-glycans of serum proteins secreted by the liver. Methods: In an effort to determine the origin of this increased fucosylation, we have conducted N-linked glycan analysis of HCC tissue, the surrounding nontumor tissue, and compared this to tissue from a nondiseased adult liver. Results: Surprisingly, no difference in the level of fucosylation was observed from the three donor groups, suggesting that the increased levels of fucosylation observed in serum of those with HCC is not the result of increased synthesis of fucosylated proteins in the cancer tissue. On the other hand, increased levels of a tetra-antennary glycan were observed in the HCC tissue as compared with the surrounding tissue or to the nondiseased livers. Conclusions: This represents, to our knowledge, one of the first reports associating increased levels of branching with the development of HCC. Impact: The identification of increased levels of tetra-antennary glycan on liver tumor tissue, as opposed to adjacent or nondiseased tissue may lead to improved detection of HCC. Cancer Epidemiol Biomarkers Prev; 21(6); 925–33. ©2012 AACR.

In this report, the possibility of pharmacologically altering the hepatitis B virus (HBV) epitopes presented by major histocompatibility complex class I on infected cells is demonstrated. The HBV middle envelope glycoprotein (MHBs) maturation appears to require calnexin-mediated folding. This interaction is dependent on glucosidases in the endoplasmic reticulum. Prevention of HBV envelope protein maturation in cultured cells through use of glucosidase inhibitors, such as 6-O-butanoyl castanospermine and N-nonyl deoxynorjirimycin, resulted in MHBs degradation by proteasomes. The de–N-glycosylation associated with polypeptide degradation was predicted to result in conversion of asparagine residues into aspartic acid residues. This prediction was confirmed by showing that peptides corresponding to the N-glycosylation sequons of MHBs, but with aspartic acid replacing asparagine, (1) can prime human cytotoxic T lymphocytes that recognize HBV-producing cells and (2) that the presentation of these envelope motifs by major histocompatibility complex class I is enhanced by incubation with glucosidase inhibitors. Moreover, although peripheral blood mononuclear cells isolated from woodchucks chronically infected with woodchuck hepatitis virus and vaccinated with woodchuck hepatitis virus surface antigen could be induced to recognize the natural MHBs asparagine-containing peptides, only cells isolated from animals treated with glucosidase inhibitor recognized the aspartic acid–containing peptides. Conclusion: These data suggest that pharmacological intervention with glucosidase inhibitors can alter the MHBs epitopes presented. This editing of the amino acid sequence of the polypeptide results in a new epitope, or “editope”, with possible medical significance. (HEPATOLOGY 2010;)

Background: We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C-(HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC).Methodology/Principal Findings: Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (alpha-1,3) fucosylation. Increases in core (alpha-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific.Conclusions/Significance: This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.

Inhibitors of alpha glucosidases prevent the trimming of oligosaccharides on certain nascent glycoproteins, including the hepatitis B virus MHBs envelope glycoprotein. MHBs proteins with untrimmed oligosaccharides do not interact with calnexin, increasing protein misfolding and subsequent degradation by proteasomes. As peptides loaded onto newly synthesized MHC class I complexes are predominantly derived from proteasomes, the possibility that glucosidase inhibition could increase presentation by MHC class I was determined. Using either a model epitope, or a natural MHBs epitope, it was demonstrated that glucosidase inhibitors enhanced presentation by MHC class I and promoted activation of antigen-specific CTLs, suggesting a pharmacologic approach to immune modulation.

The association between elevated circulating levels of GP73 (and fucosylated GP73 in particular) and hepatocellular carcinoma suggests that a thorough analysis of the extent of GP73 glycosylation is warranted. Detailed analysis of the glycosylation patterns of such low abundance proteins are hampered by technical difficulties. Using conventional lectin affinity chromatography, we have established that three quarters of the GP73 secreted from a cell line derived from HCC is fucosylated. Using mass spectrometry, we have established that at least two of three potential sites of N‐linked glycosylation are occupied on most molecules of GP73 secreted from cultured hepatoma cells. Furthermore, the oligosaccharides added to recombinant GP73 resemble those present in the bulk of secreted protein, mostly bi‐antennary with core fucose, with a smaller fraction of tri‐ and tetra‐antennary structures. The frequency of fucosylation observed on the recombinant protein agrees well with the pattern of lectin binding of the endogenous secreted protein. Finally, we have developed a method to interrogate the glycans added to either the near full length protein or at a particular sequon, providing proof of concept that a small peptide embedded in a heterologous context can preserve both fucosylation and a high level of branching of oligosaccharides added. J. Cell. Biochem. 104: 136–149, 2008. © 2007 Wiley‐Liss, Inc.

The likely involvement of inflammation and oxidative stress (IOS) in mental disease has led to advocate anti-oxidant and anti-inflammatory drugs as therapeutic strategies in the treatment of schizophrenia. Since omega-3 fatty acids (ω-3) show anti-inflammatory/neuroprotective properties, we aim to evaluate whether ω-3 treatment during adolescence in the maternal immune stimulation (MIS) animal model of schizophrenia could prevent the brain and behavioural deficits described in adulthood. At gestational day 15, PolyI:C (4 mg/kg) or saline (VH) were injected to pregnant Wistar rats. Male offspring received ω-3 (800 mg/kg) or saline (Sal) daily from postnatal day (PND) 35–49, defining 4 groups: MIS-ω-3; MIS-Sal; VH-ω-3 and VH-Sal. At PND70, rats were submitted to prepulse inhibition test (PPI). FDG-PET and T2-weighted MRI brain studies were performed in adulthood and analyzed by means of SPM12. IOS markers were measured in selected brain areas. MIS-offspring showed a PPI deficit compared with VH-offspring and ω-3 treatment prevented this deficit. Also, ω-3 reduced the brain metabolism in the deep mesencephalic area and prevented the volumetric abnormalities in the hippocampus but not in the ventricles in MIS-offspring. Besides, ω-3 reduced the expression of iNOS and Keap1 and increased the activity/concentration of HO1, NQO1 and GPX. Our study demonstrates that administration of ω-3 during adolescence prevents PPI behavioural deficits and hippocampal volumetric abnormalities, and partially counteracts IOS deficits via iNOS and Nrf2–ARE pathways in the MIS model. This study highlights the need for novel strategies based on anti-inflammatory/anti-oxidant compounds to alter the disease course in high-risk populations at early stages.

Our previous studies demonstrated that urine contains DNA derived from the circulation and that this DNA originated, in part, from organ sites and tumors distal to the urinary tract. To explore the potential use of DNA from urine as compared to other body fluids as a source for circulating DNA for cancer detection, the DNA concentration and the frequency of detection of mutated Kristin‐ras (K‐ras) DNA in serum, plasma, and urine were examined. The concentration of DNA in the urine was similar to that in the serum, but the DNA concentration in plasma was significantly lower than in either urine or serum (P < 0.05). When DNA derived from 10 μL of body fluid was used in each mutation assay, the detection frequency of mutated K‐ras DNA was comparable among serum, plasma, and urine. However, when DNA derived from 200 μL of body fluid was used, the incidence of detecting mutated K‐ras DNA in urine was significant higher (95%) than in either serum (35%) or plasma (40%) (P < 0.0005), suggesting that inhibitory factors in serum/plasma may be more limiting than in urine. The use and practicality of urine as a source of circulating DNA for cancer detection are discussed.

The signal sequences that mediate entry of the hepatitis B virus (HBV) envelope proteins into the endoplasmic reticulum (ER) are located within the S domain at positions 11–32 and at positions 80–98 (from the start of the S domain). In addition, hydrophobic patches at positions 160–184 and 189–210 of the S domain may also be involved in entry into the ER. The role of each of these domains in the entry of the HBV M glycoprotein into the ER was studied by deletion mutations of each of the signal sequences. Glycosylation of proteins was used as a marker of entry into the ER. Our results indicate that association with the ER could not be prevented by the deletion of either individual or combinations of the HBV signal sequences. M protein lacking signal sequence I was able to enter the ER and had limited secretion. In contrast, M protein lacking signal sequence II could not be secreted but still entered the ER. M protein lacking signal sequences I and II, while still associated with the ER, was rapidly degraded by the cytosolic proteasome. The potential use of such a vector as a CTL vaccine was tested through an in vitro antigen presentation assay. In this assay, a DNA vaccine candidate lacking signal sequences I and II lead to a > 6-fold increase in CTL activation, as compared to the vector expressing wild type M protein. These results suggest that increased degradation of the HBV envelope proteins can lead to enhanced antigen presentation.