Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a L-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galβ1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, Dgalactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent D-mannose and D-xylose, respectively), underlying the molecular basis of the FMSinduced IgM antibodies against tumor-specific glycans. mushroom polysaccharide | antitumor activity | anti-Globo H antibody V arious forms of herbal medicine polysaccharides have become valuable as health supplements worldwide (1, 2), suggesting that administration of such polysaccharides may improve innate immunity in vivo. The underlying molecular mechanisms, however, still remain ambiguous. Aberrant terminal fucosylation as well as sialylation in tumor-associated glycans is one of several glycosylation events important in cancer progression (3, 4), and such unusual glycans have recently been used for the development of anticancer vaccines (5-7). As an example, the Globo H-based glycoconjugate vaccines are currently undergoing large-scale clinical trials and have shown promise in therapeutic treatment (8, 9). Studies on the immune response to pathogenic microorganisms (such as Haemophilus influenza type B and Streptococcus pneumonia) have demonstrated that polysaccharides containing repeating antigenic units are generally T cell-independent (TI) (10, 11). Furthermore, recent findings revealed that specific B-cell subsets could establish memory for providing specific Ig synthesis in response to TI-associated polysaccharides (12)(13)(14). In an attempt to understand the biological significance of polysaccharides derived from natural sources, we previously isolated and characterized a crude extract fraction of water-soluble and L-fucose (Fuc)-containing polysaccharides (F3) from Ganoderma lucidum (Reishi) (a mushroom that has been long used as a herb medicine) (15). F3 has since been shown essential for regulation of cytokine network...

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Therefore, developing the early, high-sensitivity diagnostic biomarkers to prevent HCC is urgently needed. Serum a-fetoprotein (AFP), the clinical biomarker in current use, is elevated in only ∼60% of patients with HCC; therefore, identification of additional biomarkers is expected to have a significant impact on public health. In this study, we used glycan microarray analysis to explore the potential diagnostic value of several cancer-associated carbohydrate antigens (CACAs) as biomarkers for HCC. We used glycan microarray analysis with 58 different glycan analogs for quantitative comparison of 593 human serum samples (293 HCC samples; 133 chronic hepatitis B virus (HBV) infection samples, 134 chronic hepatitis C virus (HCV) infection samples, and 33 healthy donor samples) to explore the diagnostic possibility of serum antibody changes as biomarkers for HCC. Serum concentrations of anti-disialosyl galactosyl globoside (DSGG), anti-fucosyl GM1 and anti-Gb2 were significantly higher in patients with HCC than in chronic HBV infection individuals not in chronic HCV infection patients. Overall, in our study population, the biomarker candidates DSGG, fucosyl GM1 and Gb2 of CACAs achieved better predictive sensitivity than AFP. We identified potential biomarkers suitable for early detection of HCC. Glycan microarray analysis provides a powerful tool for high-sensitivity and high-throughput detection of serum antibodies against CACAs, which may be valuable serum biomarkers for the early detection of persons at high risk for HCC.

Sweetening the deal: N. meningitidis serogroup W135 capsular oligosaccharides were synthesized in lengths from disaccharides to decasaccharides. Sera from mice immunized with these oligosaccharide–protein conjugates were examined by a glycan microarray (see picture) and bactericidal assay for antibody specificity and the ability to kill bacteria.

An efficient method for examining the selectivity of inhibitors on two alpha-fucosidases, one from Thermotoga maritima and the other from human, was established. The X-ray crystal structure of the former enzyme makes possible the homology modeling of the human alpha-fucosidase, indicating the major difference between both enzymes in the periphery of the catalytic site. To investigate the difference at the molecular level, a variety of fuconojirimycin (FNJ) derivatives with substitution at C1, C2, C6, or N were rapidly prepared in microplates and screened without purification for the inhibition activities of the two alpha-fucosidases. Among the molecules that were tested, only the substitution at C1 can significantly enhance the inhibitory potency, in contrast to the control (no substitution) and compounds with substitution at other positions. The majority of C1-substituted FNJs were found to be slow tight-binding inhibitors of the Thermotoga enzyme, while acting as the reversible inhibitors of the human fucosidase. The best inhibitor exhibited 13,700-fold difference in affinity between the two enzymes, which was attributed to the dissimilar aglycon binding site. Further investigations were carried out, including site-directed mutagenesis, the comparison of K(i) values among the wild type and mutants, and the intrinsic fluorescence change upon inhibitor titration, all supporting the idea that Tyr64 and Tyr267 of the Thermotoga alpha-fucosidase are critically involved in closely interacting with the aglycon of inhibitors. The increased level of contact thus induced conformational change, leading to the observed slow tight-binding inhibition.