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Ganglioside GD3/GD2 are over-expressed in various neuroectoderm-derived tumors. Previous studies indicated that GD3 is involved in the enhancement of cancer properties such as rapid growth and increased invasiveness. However, little is known about the functions of GD3/GD2 in glioma cells and glioma micro- environments. To clarify the functions of GD3/GD2 in gliomas, we used a mouse glioma model based on the RCAS/Gtv-a system. At first, we compared the gliomas size between wild-type (WT) and GD3 synthase (GD3S) knockout (KO) mice, showing a less malignant histology and slower tumor growth in GD3S-KO mice than in WT mice. Immunohistochemistry of glioma sections from WT and GD3S-KO mice revealed that reactive microglia/macrophages showed different localization patterns between the two genetic types of mice. CD68+ cells were more frequently stained inside glioma tissues of GD3S-KO mice, while they were stained mainly around glioma tissues in WT mice. The number of CD68+ cells markedly increased in tumor tissues of GD3S-KO mice at 2 weeks after injection of transfectant DF-1 cells. Furthermore, CD68+ cells in GD3S(-/-) glioma tissues expressed higher levels of inducible nitric oxide synthase. We observed higher expression levels of pro-inflammatory cytokine genes in primary-cultured glioma cells of WT mice than in GD3S-KO mice. DNA microarray data also revealed differential expression levels of various cytokines and chemokines in glioma tissues between WT and GD3S-KO mice. These results suggest that expression of GD3S allows glioma cells to promote polarization of microglia/macrophages towards M2-like phenotypes by modulating the expression levels of chemokines and cytokines., This is an Open Access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. To view the details of this license, please visit (http://creativecommons.org/licenses/by-nc-nd/4.0/).

To analyze the binding specificity of a sialic acid–recognizing lectin, sialic acid-binding Ig-like lectin 7 (SIGLEC7), to disialyl gangliosides (GD3s), here we established GD3-expressing cells by introducing GD3 synthase (GD3S or ST8SIA1) cDNA into a colon cancer cell line, DLD-1, that expresses no ligands for the recombinant protein SIGLEC7-Fc. SIGLEC7-Fc did not recognize newly-expressed GD3 on DLD-1 cells, even though GD3 was highly expressed, as detected by an anti-GD3 antibody. Because milk-derived GD3 could be recognized by this fusion protein when incorporated onto the surface of DLD-1 cells, we compared the ceramides in DLD-1–generated and milk-derived GD3s to identify the SIGLEC7-specific GD3 structures on the cell membrane, revealing that SIGLEC7 recognizes only GD3-containing regular ceramides but not phytoceramides. This was confirmed by knockdown/knockout of the sphingolipid delta(4)-desaturase/C4-monooxygenase (DES2) gene, involved in phytoceramide synthesis, disclosing that DES2 inhibition confers SIGLEC7 binding. Furthermore, knocking out fatty acid 2-hydroxylase also resulted in the emergence of SIGLEC7 binding to the cell surface. To analyze the effects of binding between SIGLEC7 and various GD3 species on natural killer function, we investigated cytotoxicity of peripheral blood mononuclear cells from healthy donors toward GD3S-transfected DLD-1 (DLD-1–GD3S) cells and DLD-1–GD3S cells with modified ceramides. We found that cytotoxicity is suppressed in DLD-1–GD3S cells with dehydroxylated GD3s. These results indicate that the ceramide structures in glycosphingolipids affect SIGLEC7 binding and distribution on the cell surface and influence cell sensitivity to killing by SIGLEC7-expressing effector cells.

Acidic glycosphingolipids, gangliosides are highly and consistently expressed in nervous tissues of vertebrates at high levels. Therefore, they have been believed to be largely involved in the development and function of nervous systems. Recent studies with genetic engineering of glycosyltransferase genes have revealed novel aspects of roles of gangliosides in the regulation of nervous tissues with substantial basis. In this review, novel findings on the ganglioside functions and their modes of action elucidated mainly by studies of gene knockout mice have been summarized. In particular, roles of gangliosides in the regulation of lipid rafts to maintain the integrity of nervous systems have been reported with a focus on the roles in the regulation of neuro-inflammation and neurodegeneration via complement systems. In addition, recent advances in the studies of congenital neurodisorders due to the genetic mutaions of ganglioside synthase genes, and also in the techniques for the analysis of ganglioside functions have been introduced.