Application of the Antibody-Inducing Activity of Glycosphingolipids to Human Diseases

Glycosphingolipids (GSLs) are composed of a mono-, di-, or oligosaccharide and a ceramide and function as constituents of cell membranes. Various molecular species of GSLs have been identified in mammalian cells due to differences in the structures of oligosaccharides. The oligosaccharide structure can vary depending on cell lineage, differentiation stage, and pathology; this property can be used as a cell identification marker. Furthermore, GSLs are involved in various aspects of the immune response, such as cytokine production, immune signaling, migration of immune cells, and antibody production. GSLs containing certain structures exhibit strong immunogenicity in immunized animals and promote the production of anti-GSL antibodies. By exploiting this property, it is possible to generate antibodies that recognize the fine oligosaccharide structure of specific GSLs or glycoproteins. In our study using artificially synthesized GSLs (artGSLs), we found that several structural features are correlated with the antibody-inducing activity of GSLs. Based on these findings, we designed artGSLs that efficiently induce the production of antibodies accompanied by class switching and developed several antibodies that recognize not only certain glycan structures of GSLs but also those of glycoproteins. This review comprehensively introduces the immune activities of GSLs and their application as pharmaceuticals.

Bifidobacterium infantis Metabolizes 2′Fucosyllactose-Derived and Free Fucose Through a Common Catabolic Pathway Resulting in 1,2-Propanediol Secretion

Human milk oligosaccharides (HMOs) enrich beneficial bifidobacteria in the infant gut microbiome which produce molecules that impact development and physiology. 2′fucosyllactose (2′FL) is a highly abundant fucosylated HMO which is utilized by Bifidobacterium longum subsp. infantis, despite limited scientific understanding of the underlying mechanism. Moreover, there is not a current consensus on whether free fucose could be metabolized when not incorporated in a larger oligosaccharide structure. Based on metabolic and genomic analyses, we hypothesize that B. infantis catabolizes both free fucose and fucosyl oligosaccharide residues to produce 1,2-propanediol (1,2-PD). Accordingly, systems-level approaches including transcriptomics and proteomics support this metabolic path. Co-fermentation of fucose and limiting lactose or glucose was found to promote significantly higher biomass and 1,2-PD concentrations than individual substrates, suggesting a synergistic effect. In addition, and during growth on 2′FL, B. infantis achieves significantly higher biomass corresponding to increased 1,2-PD. These findings support a singular fucose catabolic pathway in B. infantis that is active on both free and HMO-derived fucose and intimately linked with central metabolism. The impact of fucose and 2′FL metabolism on B. infantis physiology provides insight into the role of fucosylated HMOs in influencing host- and microbe-microbe interactions within the infant gut microbiome.

The molecular weight of ulvan affects the in vitro inflammatory response of a murine macrophage

© 2020 Elsevier B.V. Ulvan, a sulfated polysaccharide extracted from the green seaweed genus Ulva, has bioactive properties including an immunomodulating capacity. The immunomodulatory capacity of ulvan from Ulva ohnoi, however, has not been assessed in detail. We depolymerised purified ulvan from U. ohnoi to obtain a range of molecular weight fractions (Mw 7, 9, 13, 21, 209 kDa), which were characterised by constituent sugar analysis, SEC-MALLS, and NMR. Ulvan fractions contained 48.8–54.7 mol% rhamnose, 32.5–35.9 mol% glucuronic acid, 4.5–7.3 mol% iduronic acid, and 3.3–5.6 mol% xylose. 1H and 13C NMR was consistent with hydrolysis occurring at the anomeric centre without further modification to the oligosaccharide structure. The in vitro immunomodulatory effect of ulvan fractions was quantified by measuring levels of inflammatory-mediating signalling molecules released from LPS-stimulated RAW264.7 murine macrophages. All ulvan fractions showed no toxicity on RAW264.7 cells at concentrations below 100 μg mL−1 over 48 h. Secreted interleukin-10 and prostaglandin E2 demonstrated an anti-inflammatory effect by higher molecular weight ulvan fractions at 100 μg mL−1. To a lesser extent, these fractions also enhanced the LPS-induced inflammation through minor increases of IL-1β and IL-6. This study confirms that ulvan from U. ohnoi has a mild in vitro immunomodulatory effect.

The molecular weight of ulvan affects the in vitro inflammatory response of a murine macrophage

© 2020 Elsevier B.V. Ulvan, a sulfated polysaccharide extracted from the green seaweed genus Ulva, has bioactive properties including an immunomodulating capacity. The immunomodulatory capacity of ulvan from Ulva ohnoi, however, has not been assessed in detail. We depolymerised purified ulvan from U. ohnoi to obtain a range of molecular weight fractions (Mw 7, 9, 13, 21, 209 kDa), which were characterised by constituent sugar analysis, SEC-MALLS, and NMR. Ulvan fractions contained 48.8–54.7 mol% rhamnose, 32.5–35.9 mol% glucuronic acid, 4.5–7.3 mol% iduronic acid, and 3.3–5.6 mol% xylose. 1H and 13C NMR was consistent with hydrolysis occurring at the anomeric centre without further modification to the oligosaccharide structure. The in vitro immunomodulatory effect of ulvan fractions was quantified by measuring levels of inflammatory-mediating signalling molecules released from LPS-stimulated RAW264.7 murine macrophages. All ulvan fractions showed no toxicity on RAW264.7 cells at concentrations below 100 μg mL−1 over 48 h. Secreted interleukin-10 and prostaglandin E2 demonstrated an anti-inflammatory effect by higher molecular weight ulvan fractions at 100 μg mL−1. To a lesser extent, these fractions also enhanced the LPS-induced inflammation through minor increases of IL-1β and IL-6. This study confirms that ulvan from U. ohnoi has a mild in vitro immunomodulatory effect.

A Synthetic Glycan Array Containing Cryptococcus Neoformans Glucuronoxylomannan Capsular Polysaccharide Fragments Allows the Mapping of Protective Epitopes

A block synthetic strategy to Cryptococcus neoformans glucuronoxylomannan (GXM) capsular polysaccharide part structures has been developed based on di-, tri-, tetra-, penta- and hexasaccharide thioglycoside building blocks. The approach permitted the synthesis of a library of spacer-containing serotype A and D related GXM oligosaccharide structures, ranging from di- to octadecasaccharides. Ten deprotected GXM compounds (mono- to decasaccharide) were printed onto microarray plates and screened with seventeen mouse monoclonal antibodies (mAbs) to GXM. For the first time a GXM oligosaccharide structure (a serotype A decasaccharide), capable of being recognized by neutralizing forms of these GXMspecific mAbs, has been identified, offering insight into the binding epitopes of a range of protective monoclonal antibodies and furthering our efforts to develop semi-synthetic conjugate vaccine candidates against C. neoformans.<br>

A Synthetic Glycan Array Containing Cryptococcus Neoformans Glucuronoxylomannan Capsular Polysaccharide Fragments Allows the Mapping of Protective Epitopes

A block synthetic strategy to Cryptococcus neoformans glucuronoxylomannan (GXM) capsular polysaccharide part structures has been developed based on di-, tri-, tetra-, penta- and hexasaccharide thioglycoside building blocks. The approach permitted the synthesis of a library of spacer-containing serotype A and D related GXM oligosaccharide structures, ranging from di- to octadecasaccharides. Ten deprotected GXM compounds (mono- to decasaccharide) were printed onto microarray plates and screened with seventeen mouse monoclonal antibodies (mAbs) to GXM. For the first time a GXM oligosaccharide structure (a serotype A decasaccharide), capable of being recognized by neutralizing forms of these GXMspecific mAbs, has been identified, offering insight into the binding epitopes of a range of protective monoclonal antibodies and furthering our efforts to develop semi-synthetic conjugate vaccine candidates against C. neoformans.<br>

Induction of specific adaptive immune responses by immunization with newly designed artificial glycosphingolipids

We previously found that artificial glycosphingolipids (artGSLs) containing very-long-chain fatty acids behave as strong immunogens in mice and promote the production of antibodies recognizing the oligosaccharide portion of artGSLs as the epitope. Here, we report that the oligosaccharide structure of artGSLs influences these immunogenic properties. We evaluated the antibody-inducing activity of artGSLs with different oligosaccharide structures in mice and found strong IgG-inducing activity only with an artGSL containing a core-fucosylated tetraoligosaccharide (Manβ1,4GlcNAcβ1,4[Fucα1,6]GlcNAc). To characterize the immunogenic properties of this artGSL, we analyzed various derivatives and found that the non-reducing terminal mannose structure was critical for the antibody-inducing activity. These artGSLs also exhibited IgG-inducing activity dependent on co-administration of lipid A adjuvant, but no cytokine-inducing activity similar to α-galactosylceramide was detected. Furthermore, repetitive immunization with the artGSL promoted the production of antibodies against a core-fucosylated α-fetoprotein isoform (AFP-L3) known as a hepatocellular carcinoma–specific antigen. These results indicate that the newly designed artGSLs specifically induce adaptive immune responses and promote antibody production by B cells, which can be utilized to develop anti-glycoconjugate antibodies and cancer vaccines targeting tumor-associated carbohydrate antigens.

Role of FSH glycan structure in the regulation of Sertoli cell inhibin production

Variations in follicle-stimulating hormone (FSH) carbohydrate composition and structure are associated with important structural and functional changes in Sertoli cells (SCs) during sexual maturation. The aim of the present study was to investigate the impact of FSH oligosaccharide structure and its interaction with gonadal factors on the regulation of monomeric and dimeric inhibin production at different maturation stages of the SC. Recombinant human FSH (rhFSH) glycosylation variants were isolated according to their sialylation degree (AC and BA) and complexity of oligosaccharides (CO and HY). Native rhFSH stimulated inhibin α-subunit (Pro-αC) but did not show any effect on inhibin B (INHB) production in immature SCs isolated from 8-day-old rats. Activin A stimulated INHB and had a synergistic effect on FSH to stimulate Pro-αC. The less acidic/sialylated rhFSH charge analogues, BA, were the only charge analogue mix that stimulated INHB as well as the most potent stimulus for Pro-αC production. Native rhFSH stimulated both Pro-αC and INHB in SCs at a more advanced maturation stage, isolated from 20-day-old rats. In these cells, all rhFSH glycosylation variants increased INHB and Pro-αC production, even in the presence of growth factors. The BA preparation exerted a more marked stimulatory effect on INHB and Pro-αC than the AC. Glycoforms bearing high mannose and hybrid-type oligosaccharides, HY, stimulated INHB and Pro-αC more effectively than those bearing complex oligosaccharides, CO, even in the presence of gonadal growth factors. These findings demonstrate the modulatory effect of FSH oligosaccharide structure on the regulation of inhibin production in the male gonad.