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.
Low-carbohydrate ketogenic diets (LCKDs) are used for treating obesity and epilepsy; however, the molecular mechanism of LCKDs in tissues has not been fully investigated. In this study, novel ...LCKD-associated molecular targets were explored using gene expression profiling in the liver of mice fed a LCKD. The result showed that the LCKD promoted the expression of glycosyltransferase genes involved in ganglioside synthesis and suppressed the expression of Gm2a, the gene encoding GM2 ganglioside activator protein, a lysosomal protein indispensable for ganglioside degradation. These changes were correlated with increased ganglioside content in the liver and serum. As gangliosides are mainly expressed in central nervous tissues, we also analyzed LCKD effect on cerebral cortex. Although ganglioside levels were unchanged in mice on the LCKD, Gm2a expression was significantly down-regulated. Further analyses suggested that the LCKD altered the expression levels of gangliosides in a limited area of central nervous system tissues susceptible to Gm2a.
We previously generated three types of anti-glycan monoclonal IgM antibodies that react with certain structures on the glycans of glycosphingolipids and glycoproteins. As the nucleotide sequences for ...the variable regions of these IgM antibodies showed homology with those of anti-DNA antibodies deposited in public databases, we analyzed the reactivity of the anti-glycan IgM antibodies to DNA by ELISA. We found that anti-α2,6-sialyl LacNAc IgM in the supernatant of a hybridoma culture cross-reacted with DNA, and after purification of the IgM by zirconia column chromatography, the highly purified IgM showed increased cross-reactivity to DNA. As most of the contaminating bovine serum proteins in the culture supernatant were removed by the purification process, it is likely that a part of the removed components influences antibody reactivity to DNA. Purified anti-DNA antibodies prepared from lupus model NZB/W F1 and MRL/lpr mouse sera and normal human serum were then analyzed, and similar results showing increased reactivity to DNA were obtained. Furthermore, ELISA using these purified antibodies and various carbohydrate antigens showed that the antigen-binding specificity of these antibodies was altered by the purification process from serum-containing antibody preparations. Our results indicate that mammalian serum contains components that strongly influence antibody reactivity to carbohydrate antigens, including DNA.
The data presented herein pertain to a research article entitled “A low-carbohydrate ketogenic diet promotes ganglioside synthesis via the transcriptional regulation of ganglioside metabolism-related ...genes” 1. The present article provides additional structural analysis data for the characterization of hepatic glycoproteins in mice fed a low-carbohydrate ketogenic diet (LCKD). Analysis of hepatic glycoproteins by enzyme-linked assay using the lectins UEA-I, ConA, LCA, and WGA showed that the LCKD decreased mature forms of complex-type glycans but increased immature forms of glycans on glycoproteins. An enzyme-linked immunosorbent assay using an anti–α2,6-sialyl LacNAc antibody also supported this result, indicating that dietary carbohydrate restriction results in aberrant glycosylation of tissue glycoproteins. These structural alterations of hepatic glycoproteins were not correlated with the expression levels of glycosyltransferase genes but were correlated with down-regulated expression of the Gale gene, which encodes a rate-limiting enzyme for the synthesis of sugar nucleotide donors for protein glycosylation in the liver. This property differed from glycosphingolipid metabolism in the liver of LCKD-fed mice.
Conjugation of carrier molecules to oligosaccharides has been used as a method to enhance the immunogenicity of oligosaccharides in host animals. Since bovine serum albumin and keyhole-limpet ...hemocyanin, which are commonly used for peptide antigens, are relatively ineffective for enhancing the immunogenicity of conjugated oligosaccharides, improved methods using streptavidin, non-toxic mutants of the diphtheria toxin and phosphatidylethanolamine have been investigated. Phosphatidylethanolamine can easily be conjugated with the reducing end of oligosaccharides, which is an advantage over other carrier molecules, but the ability to enhance the immunogenicity of the conjugated oligosaccharide has not been fully verified. We found that conjugates of target oligosaccharides and a ceramide analogue, characterized by having a very long-chain fatty acid, could strongly induce the production of antibodies that respond to the target oligosaccharides on glycoproteins and glycolipids in host mice. By using mice immunized with this conjugate, we successfully obtained a monoclonal antibody that recognizes a sialylated oligosaccharide structure found on the major serum glycoproteins, and confirmed the high affinity and strict specificity against its oligosaccharide epitope on the glycoproteins.
The data presented here pertain to a research article entitled “Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation” (Okuda et ...al., 2010). The present article provides additional structural and gene expression data for the characterization of a TNF-α–inducible glycosphingolipid, globotetraosylceramide (Gb4), in vascular endothelial cells. (i) Structural details of Gb4 in lipid raft–enriched cell membranes were determined by MALDI-TOF MS. These analyses identified Gb4 with very-long-chain fatty acids as the major molecular species in this fraction, and the expression levels of whole molecular species of Gb4 with different fatty acid structures in the membrane are uniformly upregulated by TNF-α stimulation. (ii) The expression levels of genes encoding enzymes for synthesis of the ceramide portion of Gb4 were analyzed by real-time PCR. These assays revealed that TNF-α stimulation promotes transcription of the Elovl1 and Cers5 genes, which are involving in the synthesis of Gb4 with very-long-chain fatty acids. Collectively, these results indicate that TNF-α regulates glycosphingolipid synthesis and lipid raft formation in vascular endothelial cells via transcriptional up-regulation of related genes. These data thus provide new insights useful for understanding the molecular basis of inflammation-associated pathology in vascular endothelia.
The data presented in this article are related to the research article entitled “Generation of anti-oligosaccharide antibodies that recognize mammalian glycoproteins by immunization with a novel ...artificial glycosphingolipid” (Okuda and Fukui, 2018) 1. This article describes the immunogenicity of a mammalian glycosphingolipid (globoside) carrying very long-chain fatty acids. Analysis of serum antibody titer by ELISA showed that this globoside had a strong immunogenicity in mice and could immediately induce production of anti-globoside IgGs. Isolated an IgG3 (κ) monoclonal antibody (mAb PA4.2) from the immunized mouse showed high specificity and reactivity against globoside. These data provide a novel antigen design method useful for obtaining IgG antibodies against glycosphingolipids.
By using EDTPA-modified zirconia particles that selectively adsorb immunoglobulins in a column, we developed a chromatography separation system for efficient concentrating and purifying of IgM from ...hybridoma culture supernatants. Hybridoma culture supernatants containing IgMs were diluted 3-fold with 10 mM phosphate buffer (pH 7.0) and passed through the column. During this process, zirconia particles selectively adsorbed these IgMs, and most of the contaminating proteins flowed out into the flow-through. The adsorbed IgMs were easily eluted with a small volume of 400 mM phosphate buffer (pH 8.0), and high-concentration IgM solutions were prepared. Subsequent simple processing using a Capto™ Core 400 cartridge column provided highly purified IgM. The operation is easy, and the activity of IgM is maintained because the purification process is performed using only neutral ranges of phosphate buffers. Here, we showed that anti-globoside and anti-CDw75 IgM purified by this method can be used to stain cervical cancer and Burkitt lymphoma cells that specifically express these respective tumor-associated carbohydrate antigens.
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•We developed a zirconia-based chromatography system for purifying IgM.•IgM in hybridoma culture supernatants was efficiently purified by this system.•Additional Capto™ Core 400 column processing provided highly purified IgM.•This system is useful for purifying IgM against tumor-associated carbohydrate antigens.
Here, we describe porous zirconia particles (PZPs) optimized for the purification of immunoglobulins. PZPs, with a pore size of approximately 10 nm, were designed to specifically interact with ...antibodies via surface modification with a phosphate functional group. A simple PZP purification method based on precipitation enabled efficient purification of mouse anti-glycosphingolipid globoside/Gb4Cer monoclonal IgM (κ-light chains) from hybridoma culture supernatants. Over 99% of contaminating proteins were removed by the PZP purification process, and approximately 50% of the IgM was recovered in the purified fraction after eluting the PZP-adsorbed antibodies with 100 mM phosphate buffer. Other IgG3 and IgM monoclonal antibodies that react with Gb4Cer or α2,6-sialyl LacNAc-modified glycoproteins could also be purified using PZPs and elution buffer at concentrations of 100-500 mM. All of the purified antibodies retained their antigen reactivity and specificity, indicating that PZP purification does not affect antibody function. As PZP purification is also suitable for purification of IgM consisting of λ-light chains and IgG derived from other mammalian species, it is expected to be applied to the purification of a variety of antibodies, including anti-glycoconjugate IgMs.
A low-carbohydrate ketogenic diet (LCKD) promotes the progression of hepatic steatosis in C57BL/6 wild-type mice, but improves the condition in leptin-deficient obese (
) mice. Here, we show a novel ...effect of LCKD associated with the conflicting effects on these mice. Gene expression microarray analyses showed that expression of the
gene, which encodes the very-low-density lipoprotein receptor (VLDLR), was induced in LCKD-fed
mice. Although the VLDLR is not normally expressed in the liver, the LCKD led to VLDLR expression in both
and wild-type mice. To clarify this effect on VLDL dynamics, we analyzed the lipid content of serum lipoproteins and found a marked decrease in VLDL-triglycerides only in LCKD-fed wild-type mice. Further analyses suggested that transport of triglycerides via VLDL from the liver to extrahepatic tissues was inhibited by LCKD-induced hepatic VLDLR expression, but rescued under conditions of leptin deficiency.
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