We previously proposed a high-throughput strategy to discover serological biomarker candidates of cancer. This strategy focuses on a series of candidate glycoproteins that are specifically expressed in the original tissues (cells) of the target cancer and that carry glycan structures associated with carcinogenesis [Narimatsu, H., et al. FEBS J.2010, 277(1), 95-105]. Here, we examined the effectiveness of our strategy in identifying biomarkers to assess progression of liver fibrosis and for the early detection of hepatocellular carcinoma (HCC). On the basis of the results of lectin array analyses in culture media of hepatoma cell lines, we captured glycopeptides carrying AAL-ligands (fucosylated glycans) or DSA-ligands (branched glycans) from digests of culture media proteins and sera from HCC patients with a background of liver cirrhosis (LC). Glycoproteins were identified by the IGOT-LC-MS method. In all, 21 candidates were selected from 744 AAL-bound glycoproteins for further verification according to (i) their abundance in serum, (ii) their specific expression in liver, and (iii) the availability of antibodies to the glycoproteins. All selected candidates showed enhancement of AAL-reactivity in sera of HCC patients compared with that of healthy volunteers (HV). These results indicate that our glycoproteomic strategy is effective for identifying multiple glyco-biomarker candidates in a high-throughput manner.

Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. False-negative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glycobiomarker candidates by combined lectin microarray and IGOT-LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT-LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by Western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison with the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases.

Bacterial sialyltransferases (STs) from marine sources were characterized using glycosphingolipids (GSLs). Bacterial STs were found to be beta-galacotoside STs. There were two types of STs: (1) ST obtained from strains such as ishi-224, 05JTC1 (#1), ishi-467, 05JTD2 (#2), and faj-16, 05JTE1 (#3), which form alpha2-3 sialic acid (Sia) linkages, named alpha2-3ST, (2) ST obtained from strains such as ISH-224, N1C0 (#4), pda-rec, 05JTB2 (#5), and pda-0160, 05JTA2 (#6), which form alpha2-6 Sia linkages, named alpha2-6ST. All STs showed affinity to neolacto- and lacto-series GSLs, particularly in neolactotetraosyl ceramide (nLc(4)Cer). No large differences were observed in the pH and temperature profiles of enzyme activities. Kinetic parameters obtained by Lineweaver-Burk plot analysis showed that #3 and #4 STs had practical synthetic activity and thus it became easily possible to achieve large-scale ganglioside synthesis (100-300 muM) using these recombinant enzymes. Gangliosides synthesized from nLc(4)Cer by alpha2-3 and alpha2-6STs were structurally characterized by several analytical and immunological methods, and they were identified as IV(3)alphaNeuAc-nLc(4)Cer(S2-3PG) and IV(6)alphaNeuAc-nLc(4)Cer (S2-6PG), respectively. Further characterization of these STs using lactotetraosylceramide (Lc(4)Cer), neolactohexaosylceramide (i antigen), and IV(6)kladoLc(8)Cer (I antigen) showed the synthesis of corresponding gangliosides as well. Synthesized gangliosides showed binding activity to the influenza A virus [A/panama/2007/99 (H3N2)] at a similar level to purified S2-3PG and S2-6PG from mammalian sources. The above evidence suggests that these STs have unique features, including substrate specificities restricted to lacto- and neolactoseries GSLs, as well as catalytic potentials for ganglioside synthesis. This demonstrates that efficient in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing Sias modifications, thereby permitting the exploration of unknown functions.

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We have established hybridoma cell lines producing monoclonal antibodies (mAbs) directed to N-acetylglucosaminylβ1-3galactose (GlcNAcβ1-3Gal) residue by immunizing BALB/c mice with lactotriaosylceramide (Lc(3)Cer). These obtained hybridoma cells, specific to Lc(3)Cer, were dual immunoglobulin (Ig)-producing cells which secreted both IgM and IgG molecules as antibodies. The established mAbs are able to react with not only Lc(3)Cer but also GlcNAcβ1-3-terminal glycosphingolipids (GSLs) despite branching or lactosamine chain lengths and human transferrin with terminal GlcNAc residues. Comparison of the variable regions of the cloned IgM and IgG by reversed transcription-polymerase chain reaction analysis confirmed that the variable regions determine the specificity, the other amino acids are conserved, and these mAbs are encoded by J558 and Vκ-21family genes. Furthermore, we have analyzed the expression of GSLs with GlcNAcβ1-3 epitope in acute leukemia cell lines and mouse fetal tissues using these mAbs, in which antigens were distributed comparatively. These mAbs are useful for studying the precise distribution of GlcNAcβ1-3Gal-terminating GSL expression in tissues as well as for detecting GSLs carrying terminal GlcNAcβ1-3Gal carbohydrate structure.

Natural killer T (NKT) cells recognize CD1d loaded with glycosphingolipid (GSL) antigens. After being stimulated by GSL antigens, NKT cells immediately secrete cytokines. 2-4)The variety of cytokines release from NKT cells can promote immunoresponses against tumors and microbial infections, and regulate autoimmune responses, 5,6) although a puzzling aspect of these responses is that both Th1 and Th2 cytokines can be secreted in response to the stimulation.The fungal glycolipids are exogenous antigens for NKT cells 7) ; therefore, we focused on acidic glycosphingolipids (AGLs) of edible mushrooms (Hypsizigus marmoreus and Pleurotus eryngii) 8,9) and reported that AGLs induced interferon-g (IFN-g) and interleukin-4 (IL-4) release from murine T cells in a CD11c-positive cell-dependent manner. 10)Invariant natural killer T (iNKT) cells, that are more than 80% of murine NKT cells, express an invariant T cell receptor Va14 chain in combination with certain Vb chains. In mice, CD1d-dependent NKT cells are either CD4CD8-double negative or CD4-positive. The significance of CD4 expression on NKT cells is ambiguous because there is no evidence of any interaction between CD4 and MHC class II molecules on antigen-presenting cells (APCs), although CD4 can directly interacts with CD1d and enhances NKT cell activation.11) Additionally, NKT cells mainly leave the thymus when NK1.1 is negative. 6) In this study, we reported that edible mushroom AGLs activated CD4-positive NK1.1-negative iNKT hybridoma cells that express Va14-Ja18/Vb8.2 12) in a CD1d-dependent manner and intravenous AGL injection elevated the cytokine level. MATERIALS AND METHODS MaterialsPurified AGLs of H. marmoreus and P. eryngii were structurally elucidated according to previous reports, as described briefly.10,13) Saponified and dialyzed extracts were fractionated on a DEAE-Sephadex A-25 column (acetate form) (Amersham Biosciences AB, Uppsala, Sweden), and further purification was performed using a silica gel-6RS-8060 Iatrobeads column (Iatron Laboratories Inc., Tokyo, Japan). The composition of fatty acids and sugars, identification of phosphate bonds, confirmation of inositolphosphate content, determination of linkages among sugars and between sugar and inositol in purified AGLs, and AGL purity was confirmed by treatment with the appropriate reagents and using assays including columns, thin layer chromatography, a microwave oven, gas chromatography, gas-liquid chromatography/mass spectrometry, matrix-assisted laser desorption ionization-time of flight/mass spectrometry and 1 H-NMR. a-Galactosylceramide (a-GalCer) (KRN7000: ALX-306-027) and isoglobotrihexosylceramide (iGb3) (ALX-306-028) were purchased from ALEXIS Biochemicals (Lausanne, Switzerland).Mice and Protocols for in Vivo Experiments AGLs were dissolved in 100% polysorbate-20, diluted with sterilized phosphate buffered saline (PBS) to 0.5% polysorbate-20, and then kept at 4°C until used. Stock solutions were further diluted to 200 and 100 mg/ml AGL or 5 mg/ml a-GalCer in PBS containing 0.025% polyso...

A bovine lipocalin, previously identified as a putative odorant-binding protein in bovine colostrum (bcOBP), was cloned and expressed, and its monoclonal antibody was established. bcOBP was constantly secreted into milk on day of parturition until at least 10 d postpartum at a concentration of 181±39 µg/L. Besides milk, bcOBP occurred in the nasal mucus, saliva, amniotic fluid, vaginal discharge, and blood plasma. Despite its low concentration, the distribution pattern and the finding that bcOBP harbored a characteristic sequence motif, CxxxC, which is conserved among insect and mammal pheromone binding proteins, suggest that bcOBP functions as a pheromone carrier. The presence of bcOBP in the plasma at varied concentrations depending on the lactation period does not exclude the possibility that bcOBP is secreted into milk from the blood. Cross-reactivity of the monoclonal antibody indicated presence of proteins homologous to bcOBP in the colostrum of farm animals of Cetartiodactyla.

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