ST6GAL1 inhibits metastasis of hepatocellular carcinoma via modulating sialylation of MCAM on cell surface.

The poor prognosis of hepatocellular carcinoma (HCC) is mainly because of its high rate of metastasis. Thus, elucidation of the molecular mechanisms underlying HCC metastasis is of great significance. Glycosylation is an important post-translational modification that is closely associated with tumor progression. Altered glycosylation including the altered sialylation resulting from aberrant expression of ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1) has long been considered as an important feature of cancer cells. However, there is limited information on the roles of ST6GAL1 and α2,6 sialylation in HCC metastasis. Here, we found that ST6GAL1 and α2,6 sialylation were negatively correlated with the metastatic potentials of HCC cells. Moreover, ST6GAL1 overexpression inhibited migration and invasion of HCC cells in vitro and suppressed HCC metastasis in vivo. Using a metabolic labeling-based glycoproteomic strategy, we identified a list of sialylated proteins that may be regulated by ST6GAL1. In particular, an increase in α2,6 sialylation of melanoma cell adhesion molecule (MCAM) inhibited its interaction with galectin-3 and decreased its expression on cell surface. In vitro and in vivo analysis showed that ST6GAL1 exerted its function in HCC metastasis by regulating MCAM expression. Finally, we found the relative intensity of sialylated MCAM was negatively correlated with tumor malignancy in HCC patients. Taken together, these results demonstrate that ST6GAL1 may be an HCC metastasis suppressor by affecting sialylation of MCAM on cell surface, which provides a novel insight into the roles of ST6GAL1 in HCC progression and supports the functional complexity of ST6GAL1 in a cancer type- and tissue type-specific manner.

Glycogene Expression Profiling of Hepatic Cells by RNA-Seq Analysis for Glyco-Biomarker Identification.

Glycans are primarily generated by "glycogenes," which consist of more than 200 genes for glycosynthesis, including sugar-nucleotide synthases, sugar-nucleotide transporters, and glycosyltransferases. Measuring the expression level of glycogenes is one of the approaches to analyze the glycomes of particular biological and clinical samples. To develop an effective strategy for identifying the glycosylated biomarkers, we performed transcriptome analyses using quantitative real-time polymerase chain reaction (qRT-PCR) arrays and RNA sequencing (RNA-Seq). First, we measured and analyzed the transcriptome from the primary culture of human liver cells and hepatocarcinoma cells using RNA-Seq. This analysis revealed similar but distinctive expression profiles of glycogenes among hepatic cells as indicated by the qRT-PCR arrays, which determined a copy number of 186 glycogenes. Both data sets indicated that altered expression of glycosyltransferases affect the glycosylation of particular glycoproteins, which is consistent with the mass analysis data. Moreover, RNA-Seq analysis can uncover mutations in glycogenes and search differently expressed genes out of more than 50,000 distinct human gene transcripts including candidate biomarkers that were previously reported for hepatocarcinoma cells. Identification of candidate glyco-biomarkers from the expression profile of the glycogenes and proteins from liver cancer tissues available from public database emphasized the possibility that even though the expression level of biomarkers might not be altered, the expression of the glycogenes modifying biomarkers, generating glyco-biomarkers, might be different. Pathway analysis revealed that ~20% of the glycogenes exhibited different expression levels in normal and cancer cells. Thus, transcriptome analyses using both qRT-PCR array and RNA-Seq in combination with glycome and glycoproteome analyses can be advantageous to identify "glyco-biomarkers" by reinforcing information at the expression levels of both glycogenes and proteins.

Identification and characterization of sulfated glycoproteins from small cell lung carcinoma cells assisted by management of molecular charges.

Proteins carrying sulfated glycans (i.e., sulfated glycoproteins) are known to be associated with diseases, such as cancer, cystic fibrosis, and osteoarthritis. Sulfated glycoproteins, however, have not been isolated or characterized from complex biological samples due to lack of appropriate tools for their enrichment. Here, we describe a method to identify and characterize sulfated glycoproteins that are involved in chemical modifications to control the molecular charge of the peptides. In this method, acetohydrazidation of carboxyl groups was performed to accentuate the negative charge of the sulfate group, and Girard's T modification of aspartic acid was performed to assist in protein identification by MS tagging. Using this approach, we identified and characterized the sulfated glycoproteins: Golgi membrane protein 1, insulin-like growth factor binding protein-like 1, and amyloid beta precursor-like protein 1 from H2171 cells, a small cell lung carcinoma cell line. These sulfated glycoproteins carry a complex-type N-glycan with a core fucose and 4'-O-sulfated LacdiNAc as the major glycan.

Aberrant methylation of GCNT2 is tightly related to lymph node metastasis of primary CRC.

BACKGROUND: Glycoprotein expression profile is dramatically altered in human cancers; however, specific glycogenes have not been fully identified. MATERIALS AND METHODS: A comprehensive real-time polymerase chain reaction (PCR) system for glycogenes (CRPS-G) identified several outstanding glycogenes. GCNT2 was of particular interest after GCNT2 expression and epigenetics were rigorously investigated in primary colorectal cancer (CRC). RESULTS: The highlights of this work can be summarized as follows: (i) Expression of GCNT2 was remarkably suppressed. (ii) Silenced expression of GCNT2 was reactivated by combined demethylating agents. (iii) Promoter DNA methylation of GCNT2 was silenced in CRC cell lines and tissues. Hypomethylation of GCNT2 variant 2 is tightly associated with lymph node metastasis in primary CRC. (iv) GCNT2 methylation level in the normal tissues also showed a close association with that in the tumor tissues and reflected lymph node metastasis. CONCLUSION: We identified aberrant expression of GCNT2, which can be explained by promoter DNA hypermethylation. Hypomethylation of the GCNT2 variant 2 reflected lymph node metastasis of CRC in the tumor and normal tissues.

JCGGDB: Japan Consortium for Glycobiology and Glycotechnology Database.

The biological significance of glycans has been widely studied and reported in the past. However, most achievements of our predecessors are not readily available in existing databases. JCGGDB is a meta-database involving 15 original databases in AIST and 5 cooperative databases in alliance with JCGG: Japan Consortium for Glycobiology and Glycotechnology. It centers on a glycan structure database and accumulates information such as glycan preferences of lectins, glycosylation sites in proteins, and genes related to glycan syntheses from glycoscience and related fields. This chapter illustrates how to use three major search interfaces (Keyword Search, Structure Search, and GlycoChem Explorer) available in JCGGDB to search across multiple databases.

GlycoRDF: an ontology to standardize glycomics data in RDF.

MOTIVATION: Over the last decades several glycomics-based bioinformatics resources and databases have been created and released to the public. Unfortunately, there is no common standard in the representation of the stored information or a common machine-readable interface allowing bioinformatics groups to easily extract and cross-reference the stored information. RESULTS: An international group of bioinformatics experts in the field of glycomics have worked together to create a standard Resource Description Framework (RDF) representation for glycomics data, focused on glycan sequences and related biological source, publications and experimental data. This RDF standard is defined by the GlycoRDF ontology and will be used by database providers to generate common machine-readable exports of the data stored in their databases. AVAILABILITY AND IMPLEMENTATION: The ontology, supporting documentation and source code used by database providers to generate standardized RDF are available online (http://www.glycoinfo.org/GlycoRDF/).

WURCS: the Web3 unique representation of carbohydrate structures.

In recent years, the Semantic Web has become the focus of life science database development as a means to link life science data in an effective and efficient manner. In order for carbohydrate data to be applied to this new technology, there are two requirements for carbohydrate data representations: (1) a linear notation which can be used as a URI (Uniform Resource Identifier) if needed and (2) a unique notation such that any published glycan structure can be represented distinctively. This latter requirement includes the possible representation of nonstandard monosaccharide units as a part of the glycan structure, as well as compositions, repeating units, and ambiguous structures where linkages/linkage positions are unidentified. Therefore, we have developed the Web3 Unique Representation of Carbohydrate Structures (WURCS) as a new linear notation for representing carbohydrates for the Semantic Web.

Introducing glycomics data into the Semantic Web.

BACKGROUND: Glycoscience is a research field focusing on complex carbohydrates (otherwise known as glycans)a, which can, for example, serve as "switches" that toggle between different functions of a glycoprotein or glycolipid. Due to the advancement of glycomics technologies that are used to characterize glycan structures, many glycomics databases are now publicly available and provide useful information for glycoscience research. However, these databases have almost no link to other life science databases. RESULTS: In order to implement support for the Semantic Web most efficiently for glycomics research, the developers of major glycomics databases agreed on a minimal standard for representing glycan structure and annotation information using RDF (Resource Description Framework). Moreover, all of the participants implemented this standard prototype and generated preliminary RDF versions of their data. To test the utility of the converted data, all of the data sets were uploaded into a Virtuoso triple store, and several SPARQL queries were tested as "proofs-of-concept" to illustrate the utility of the Semantic Web in querying across databases which were originally difficult to implement. CONCLUSIONS: We were able to successfully retrieve information by linking UniCarbKB, GlycomeDB and JCGGDB in a single SPARQL query to obtain our target information. We also tested queries linking UniProt with GlycoEpitope as well as lectin data with GlycomeDB through PDB. As a result, we have been able to link proteomics data with glycomics data through the implementation of Semantic Web technologies, allowing for more flexible queries across these domains.

CA-S27: a novel Lewis a associated carbohydrate epitope is diagnostic and prognostic for cholangiocarcinoma.

Early and specific diagnosis is critical for treatment of cholangiocarcinoma (CCA). In this study, a carbohydrate antigen-S27 (CA-S27) monoclonal antibody (mAb) was established using pooled CCA tissue-extract as immunogen. The epitope recognized by CA-S27-mAb was a new Lewis-a (Le(a)) associated modification of MUC5AC mucin. A Soybean agglutinin/CA-S27-mAb sandwich ELISA to determine CA-S27 in serum was successfully developed. High level of CA-S27 was detected in serum of CCA patients and could differentiate CCA patients from those of gastro-intestinal cancers, hepatomas, benign hepatobiliary diseases and healthy subjects with high sensitivity (87.5%) and high negative predictive value (90.4%). The level of serum CA-S27 was dramatically reduced after tumor removal, indicating tumor origin of CA-S27. Patients with high serum CA-S27 had significantly shorter survivals than those with low serum CA-S27 regardless of serum MUC5AC levels. Fucosyltransferase-III (FUT3) was shown to be a regulator of CA-S27 expression. Suppression of CA-S27 expression with siRNA-FUT3 or neutralization with CA-S27 mAb significantly reduced growth, adhesion, invasion and migration potentials of CCA cells in vitro. In summary, we demonstrate that serum CA-S27, a novel carbohydrate antigen, has potential as diagnostic and prognostic markers for CCA patients. CA-S27 involves in promoting cell growth, adhesion, migration and invasion of CCA cells.

Large-scale identification of N-glycosylated proteins of mouse tissues and construction of a glycoprotein database, GlycoProtDB.

Protein glycosylation is a common post-translational modification that plays important roles in terms of protein function. However, analyzing the relationship between glycosylation and protein function remains technically challenging. This problem arises from the fact that the attached glycans possess diverse and heterogeneous structures. We believe that the first step to elucidate glycan function is to systematically determine the status of protein glycosylation under physiological conditions. Such studies involve analyzing differences in glycan structure on cell type (tissue), sex, and age, as well as changes associated with perturbations as a result of gene knockout of glycan biosynthesis-related enzyme, disease and drug treatment. Therefore, we analyzed a series of glycoproteomes in several mouse tissues to identify glycosylated proteins and their glycosylation sites. Comprehensive analysis was performed by lectin- or HILIC-capture of glycopeptide subsets followed by enzymatic deglycosylation in stable isotope-labeled water (H2¹8O, IGOT) and finally LC-MS analyses. In total, 5060 peptides derived from 2556 glycoproteins were identified. We then constructed a glycoprotein database, GlycoProtDB, using our experimental-based information to facilitate future studies in glycobiology.

Membrane sialidase NEU3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides.

NEU3 is a membrane sialidase specific for gangliosides. Its increased expression and implication in some cancers have been reported. Here, we analyzed NEU3 expression in malignant melanoma cell lines and its roles in the cancer phenotypes. Quantitative RT-PCR revealed that high levels of the NEU3 gene were expressed at almost equivalent levels with those in colon cancers. To examine the effects of overexpression of NEU3, NEU3 cDNA-transfectant cells were established using a melanoma cell line SK-MEL-28 and its mutant N1 lacking GD3. SK-MEL-28 sublines overexpressing both the NEU3 gene and NEU3 enzyme activity showed no changes in both cell growth and ganglioside expression, while N1 cells showed a mild increase in cell proliferation with increased phosphorylation of the EGF receptor and neo-synthesis of Gb3 after NEU3 transfection. In contrast, NEU3 silencing resulted in a definite reduction in cell growth in a melanoma line MeWo, while ganglioside patterns underwent minimal changes. Phosphorylation levels of ERK1/2 with serum stimulation decreased in the NEU3-silenced cells. All these results suggest that NEU3 is highly expressed to enhance malignant phenotypes including apoptosis inhibition in malignant melanomas.

Combination use of anti-CD133 antibody and SSA lectin can effectively enrich cells with high tumorigenicity.

Glycans exhibit characteristic changes in their structures during development and thus have been used as markers for stem/progenitor cells. However, the glycan structures unique to cancer stem cells (CSC) remain unknown. In the present study, we examined glycan structures in CD133+ CD13+ CSC, which were recently found to have a high CSC ability, by means of a lectin microarray. Seven sialylated glycan-recognizing lectins, MAL-I, SNA, SSA, TJA-I, ACG, ABA and MAH, showed higher affinity to CD133+ CD13+ CSC than CD133+ cells with a lower CSC ability. In addition, we demonstrated that CD133+ SSA+ cells isolated from Huh7 cells had a significantly higher ability to form tumors in non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice and spheres under serum-free conditions than CD133+ SSA- cells. These results suggest that hepatic CSC highly express sialylated glycans and that SSA lectin can be used as a tool for isolating CSC. This study is the first report to demonstrate the characteristic glycan structures in CSC and to indicate a new methodology involving lectins for isolating CSC.

A strategy for discovery of cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics.

Detection of cancer at early stages that can be treated through surgery is a difficult task. One methodology for cancer biomarker discovery exploits the fact that glycoproteins produced by cancer cells have altered glycan structures, although the proteins themselves are common, ubiquitous, abundant, and familiar. However, as cancer tissue at the early stage probably constitutes less than 1% of the normal tissue in the relevant organ, only 1% of the relevant glycoproteins in the serum should have altered glycan structures. Here, we describe our strategy to approach the detection of these low-level glycoproteins: (a) a quantitative real-time PCR array for glycogenes to predict the glycan structures of secreted glycoproteins; (b) analysis by lectin microarray to select lectins that distinguish cancer-related glycan structures on secreted glycoproteins; and (c) an isotope-coded glycosylation site-specific tagging high-throughput method to identify carrier proteins with the specific lectin epitope. Using this strategy, we have identified many glycoproteins containing glycan structures that are altered in cancer cells. These candidate glycoproteins were immunoprecipitated from serum using commercially available antibodies, and their glycan alteration was examined by a lectin microarray. Finally, they were analyzed by multistage tandem MS.

Strategy for glycoproteomics: identification of glyco-alteration using multiple glycan profiling tools.

Glycan alterations of proteins, a common feature of cancer cells, are associated with carcinogenesis, invasion and metastasis. Glycomics, the study of glycans and glycan-binding proteins in various biological systems, is an emerging field in the postgenome and postproteomics era. However, systematic and robust strategies for glycomics are still not fully established because the structural analysis of glycans, which comprise different patterns of branching, various possible linkage positions as well as monomer anomericity, is technically difficult. Here, we introduce a new strategy for glyco-alteration analysis of glycoproteins by using multiple glycan profiling tools. To understand glycan alterations of proteins by correlating the glycosyltransferase expression profile with the actual glycan structure, we systematically used three glycan profiling tools: (1) multiplex quantitative PCR (qPCR) array format for profiling the expression pattern of glycogenes, (2) lectin microarray as a multiplex glycan-lectin interaction analysis system for profiling either a pool of cell glycoproteins or a target glycoprotein, and (3) tandem mass spectrometry for identifying the glycan structure connected to a target glycoprotein. Using our system, we successfully identified glycan alterations on alpha-fetoprotein (AFP), including a novel LacdiNAc structure in addition to previously reported alterations such as alpha1,6 fucosylation.

Molecular phylogeny and new taxa in the Archaeosporales (Glomeromycota): Ambispora fennica gen. sp. nov., Ambisporaceae fam. nov., and emendation of Archaeospora and Archaeosporaceae.

The AM fungal family Archaeosporaceae and the genus Archaeospora are rendered paraphyletic by the relationship with the Geosiphonaceae. This problem led to a more detailed study of the Archaeosporales. Members of the Archaeosporaceae were described as forming both glomoid and acaulosporoid spores, or solely acaulosporoid spores. However, we found that Glomus callosum fell into the same phylogenetic clade as A. leptoticha and A. gerdemannii, but exclusively formed glomoid spores. To resolve these inconsistencies, a genus, Ambispora gen. nov., typified by Ambispora fennica sp. nov., is erected based on morphological evidence and SSU and ITS region rDNA data. Ambispora contains three species known to produce both acaulosporoid and glomoid spores: A. fennica, A. leptoticha comb. nov. (basionym G. leptotichum), and A. gerdemannii comb. nov. (basionym G. gerdemannii). Another species, A. callosa comb. nov. (basionym G. callosum), is known only from glomoid spores. Ambispora is placed in a new family, the Ambisporaceae fam. nov. The Archaeosporaceae is maintained with the type species, Archaeospora trappei (basionym Acaulospora trappei), along with Intraspora schenckii (basionym Entrophospora schenckii). Acaulospora nicolsonii, known only from acaulosporoid spores, is discussed and is considered likely to belong in the Ambisporaceae, but is retained within its present genus because of inadequate morphological information and a lack of molecular data.

Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10).

Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isozyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin beta sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isozymes to produce a wide range of O-glycans.

LARGE2 facilitates the maturation of alpha-dystroglycan more effectively than LARGE.

The LARGE gene is thought to encode a putative glycosyltransferase because of its typical topology. However, no enzyme activity has been demonstrated yet, although the gene apparently supports the functional maturation of alpha-dystroglycan by glycosylation when it is transfected into cells. A novel homologous gene to LARGE was identified and named LARGE2. LARGE2 recombinant was co-expressed with alpha-dystroglycan in human embryonic kidney 293T cells to determine its activity to support the maturation of alpha-dystroglycan. The alpha-dystroglycan co-transfected with LARGE2 was more highly glycosylated than that co-transfected with LARGE. Pull-down experiments demonstrated binding activity of LARGE2 as well as LARGE toward alpha-dystroglycan. LARGE2 was found to support the maturation of alpha-dystroglycan more effectively than LARGE. Both of them are ubiquitously expressed in many tissues, except the brain where LARGE2 was not expressed at all. This compensatory function can explain the residual functionally glycosylated alpha-dystroglycan in a patient with MDC1D whose LARGE genes are congenitally null.

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome.

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.