Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells.

This study determined the effect of brefeldin A (BFA) on the free N-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type free N-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolic endo-ß-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.

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.

Oral Pathobiont-Induced Changes in Gut Microbiota Aggravate the Pathology of Nonalcoholic Fatty Liver Disease in Mice.

Background & Aims: Periodontitis increases the risk of nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanisms are unclear. Here, we show that gut dysbiosis induced by oral administration of Porphyromonas gingivalis, a representative periodontopathic bacterium, is involved in the aggravation of NAFLD pathology. Methods: C57BL/6N mice were administered either vehicle, P. gingivalis, or Prevotella intermedia, another periodontopathic bacterium with weaker periodontal pathogenicity, followed by feeding on a choline-deficient, l-amino acid-defined, high-fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD60). The gut microbial communities were analyzed by pyrosequencing the 16S ribosomal RNA genes. Metagenomic analysis was used to determine the relative abundance of the Kyoto Encyclopedia of Genes and Genomes pathways encoded in the gut microbiota. Serum metabolites were analyzed using nuclear magnetic resonance-based metabolomics coupled with multivariate statistical analyses. Hepatic gene expression profiles were analyzed via DNA microarray and quantitative polymerase chain reaction. Results: CDAHFD60 feeding induced hepatic steatosis, and in combination with bacterial administration, it further aggravated NAFLD pathology, thereby increasing fibrosis. Gene expression analysis of liver samples revealed that genes involved in NAFLD pathology were perturbed, and the two bacteria induced distinct expression profiles. This might be due to quantitative and qualitative differences in the influx of bacterial products in the gut because the serum endotoxin levels, compositions of the gut microbiota, and serum metabolite profiles induced by the ingested P. intermedia and P. gingivalis were different. Conclusions: Swallowed periodontopathic bacteria aggravate NAFLD pathology, likely due to dysregulation of gene expression by inducing gut dysbiosis and subsequent influx of gut bacteria and/or bacterial products.

Profiling of host genetic alterations and intra-tumor microbiomes in colorectal cancer.

Some bacteria are symbiotic in tumor tissues, and metabolites of several bacterial species have been found to cause DNA damage. However, to date, the association between bacteria and host genetic alterations in colorectal cancer (CRC) has not been fully investigated. We evaluated the association between the intra-tumor microbiome and host genetic alterations in 29 Japanese CRC patients. The tumor and non-tumor tissues were extracted from the patients, and 16S rRNA genes were sequenced for each sample. We identified enriched bacteria in tumor and non-tumor tissues. Some bacteria, such as Fusobacterium, which is already known to be enriched in CRC, were found to be enriched in tumor tissues. Interestingly, Bacteroides, which is also known to be enriched in CRC, was enriched in non-tumor tissues. Furthermore, it was shown that certain bacteria that often coexist within tumor tissue were enriched in the presence of a mutated gene or signal pathway with mutated genes in the host cells. Fusobacterium was associated with many mutated genes, as well as cell cycle-related pathways including mutated genes. In addition, the patients with a high abundance of Campylobacter were suggested to be associated with mutational signature 3 indicating failure of double-strand DNA break repairs. These results suggest that CRC development may be partly caused by DNA damage caused by substances released by bacterial infection. Taken together, the identification of distinct gut microbiome patterns and their host specific genetic alterations might facilitate targeted interventions, such as modulation of the microbiome in addition to anticancer agents or immunotherapy.

Identification of novel serum biomarkers of hepatocellular carcinoma using glycomic analysis.

UNLABELLED: The altered N-glycosylation of glycoproteins has been suggested to play an important role in the behavior of malignant cells. Using glycomics technology, we attempted to determine the specific and detailed N-glycan profile for hepatocellular carcinoma (HCC) and investigate the prognostic capabilities. From 1999 to 2011, 369 patients underwent primary curative hepatectomy in our facility and were followed up for a median of 60.7 months. As normal controls, 26 living Japanese related liver transplantation donors were selected not infected by hepatitis B and C virus. Their mean age was 40.0 and 15 (57.7%) were male. We used a glycoblotting method to purify N-glycans from preoperative blood samples from this cohort (10 µL serum) which were then identified and quantified using mass spectrometry (MS). Correlations between the N-glycan levels and the clinicopathologic characteristics and outcomes for these patients were evaluated. Our analysis of the relative areas of all the sugar peaks identified by MS, totaling 67 N-glycans, revealed that a proportion had higher relative areas in the HCC cases compared with the normal controls. Fourteen of these molecules had an area under the curve of greater than 0.80. Analysis of the correlation between these 14 N-glycans and surgical outcomes by univariate and multivariate analysis identified G2890 (m/z value, 2890.052) as a significant recurrence factor and G3560 (m/z value, 3560.295) as a significant prognostic factor. G2890 and G3560 were found to be strongly correlated with tumor number, size, and vascular invasion. CONCLUSION: Quantitative glycoblotting based on whole serum N-glycan profiling is an effective approach to screening for new biomarkers. The G2890 and G3560 N-glycans determined by tumor glycomics appear to be promising biomarkers for malignant behavior in HCCs. (HEPATOLOGY 2013;).

Relationship between ATM and ribosomal protein S6 revealed by the chemical inhibition of Ser/Thr protein phosphatase type 1.

The optimal cellular responses to DNA damage are modulated by kinase and phosphatase. The ataxia telangiectasia mutated (ATM) is a Ser/Thr kinase which is the core of the DNA damage signaling apparatus. The Ser/Thr protein phosphatase type 1 (PP1) inhibitor, tautomycetin (TC) and an antibody to the phospho-(S/T)Q sites of the ATM substrate were used to identify the common substrates for PP1 and ATM in regulating the pathway for DNA damage response. Ribosomal protein S6 (RPS6) was first identified as a substrate for PP1 and ATM. The phosphorylation at Ser247 of RPS6 was then significantly decreased by PP1-mediated dephosphorylation immediately after UV irradiation. These results suggest that PP1 specifically dephosphorylated RPS6 at phospho-Ser247 in vivo. In response to DNA damage, ATM activity was finally required for the phosphorylation of RPS6 at Ser247. We propose from these results a novel mechanism for modulating the RPS6 function by PP1 and ATM which regulates cell growth and survival in response to DNA-damage stimuli.

Functional network of glycan-related molecules: glyco-net in glycoconjugate data bank.

BACKGROUND: Glycans are involved in a wide range of biological process, and they play an essential role in functions such as cell differentiation, cell adhesion, pathogen-host recognition, toxin-receptor interactions, signal transduction, cancer metastasis, and immune responses. Elucidating pathways related to post-translational modifications (PTMs) such as glycosylation are of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids and various biological events are considered extremely valuable and convenient tools for the systematic investigation of PTMs. However, there is no database which dynamically draws functional networks related to glycans. DESCRIPTION: We have created a database called Glyco-Net http://www.glycoconjugate.jp/functions/, with many binary relationships among glycan-related molecules. Using search results, we can dynamically draw figures of the functional relationships among these components with nodes and arrows. A certain molecule or event corresponds to a node in the network figures, and the relationship between the molecule and the event are indicated by arrows. Since all components are treated equally, an arrow is also a node. CONCLUSIONS: In this paper, we describe our new database, Glyco-Net, which is the first database to dynamically show networks of the functional profiles of glycan related molecules. The graphical networks will assist in the understanding of the role of the PTMs. In addition, since various kinds of bio-objects such as genes, proteins, and inhibitors are equally treated in Glyco-Net, we can obtain a large amount of information on the PTMs.

Stereochemical studies of hexylitaconic acid, an inhibitor of p53-HDM2 interaction.

Hexylitaconic acid (1) is an intriguing natural product possessing a chiral carbon, and both its enantiomers have been found in nature. Enantiomeric pure (+)-(1) and (-)-(1) were successfully prepared by racemic synthesis followed by enantiomeric separation in a chiral HPLC system. Their absolute configurations were clarified by the vibrational circular dichroism technique using their methyl esters 2 and lactones 3. Their inhibitory activities against the interaction of p53-HDM2 were also examined.

Structural transition of a 15 amino acid residue peptide induced by GM1.

The ganglioside GM1-binding peptide, p3, with a sequence of VWRLLAPPFSNRLLP, displayed a clear structural alteration depending on the presence or absence of GM1 micelles. The three-dimensional structures of the p3 peptide in the free and GM1 bound states were analyzed using two-dimensional NMR spectroscopic experiments with distance-restrained simulated annealing calculations. The NMR experiments for the p3 peptide alone indicated that the peptide has two conformers derived from the exchange of cis and trans forms at Pro(7)-Pro(8). Further study with theoretical modeling revealed that the p3 peptide has a curb conformation without regular secondary structure. On the other hand, the NMR studies for the p3 peptide with the GM1 micelles elucidated a trans conformer and gave a structure stabilized by hydrophobic interactions of beta- and helical turns. Based on these structural investigations, tryptophan, a core residue of the hydrophobic cluster, might be an essential residue for the recognition of the GM1 saccharides. The dynamic transition of the p3 peptide may play an important role in the function of GM1 as a multiple receptor as in the traditional pathway of the infection by cholera toxin.

Chiral cruciferous phytoalexins: preparation, absolute configuration, and biological activity.

Synthesized by an efficient one-pot spirocyclization method, two chiral cruciferous phytoalexins, 1-methoxyspirobrassinin (2) and 1-methoxyspirobrassinol methyl ether (4a), were prepared through optical resolution using the chiral HPLC method of corresponding racemates. The absolute configuration of natural (+)-2 was elucidated as R by using the direct comparison of ECD and VCD spectra with those of known (S)-(-)-spirobrassinin (1). Another chiral phytoalexin, (-)-4a, had its absolute configuration 2R,3R elucidated through the comparison of observed and calculated VCD. Interestingly, the absolute configurations of natural (S)-(-)-spirobrassinin (1) and (R)-(+)-1-methoxyspirobrassinin (2) were opposite of each other, even though their structures are almost similar, with the exception of an N-methoxy group. A significant difference in the antiproliferative activity between (2R,3R)-(-) and (2S,3S)-(+)-4a was observed.

Azasugar-based MMP/ADAM inhibitors as antipsoriatic agents.

As a part of synthetic studies on MMP (matrix metalloproteinase)/ADAM (a disintegrin and metalloproteinase) inhibitors, we have preliminarily communicated that azasugar-based compound 1a exhibited a potential inhibitory activity on some metalloprotease-catalyzed proteolytic reactions. To find promising candidates for the topical treatment of psoriasis, we investigated stability in aqueous solution of compound 1a and its derivative 1b and then optimized the P1' substuent (2-5). In the present study, we synthesized novel derivatives of compound 1a and evaluated their inhibitory activity toward MMP-1, -3, and -9, TACE, and HB-EGF shedding, from a viewpoint of versatility of azasugars as a functional scaffold. As a result, it was found that compound 1b demonstrated desirable inhibitory activity as an antipsoriatic agent, and some of the derivatives showed selective inhibitory activity. In addition, it was found that compound 1b exhibited a significant therapeutic effect on a mouse TPA-induced epidermal hyperplasia model. Therefore, compound 1b could become a promising candidate as a practical antipsoriatic agent.