This article is available online at http://www.jlr.org Glycosphingolipids (GSLs), amphipathic compounds consisting of oligosaccharides and ceramide (Cer) moieties, are ubiquitous in the outer leafl et of the plasma membrane. More than 500 species of GSLs differing in oligosaccharides and Cer moieties have been characterized, some of which represent potential biomarkers for diseases and cell development. For instance, Forssman antigen is specifi cally detected in some gastric, colonic, and lung cancers ( 1 ), and stage-specifi c embryonic antigen (SSEA)-3 and SSEA-4, highly expressed at a stage of embryonic development, are utilized as a marker of embryonic stem cells ( 2 ) and induced pluripotent stem cells ( 3 ).The classifi cation of GSLs, into ganglio-series, globoseries and lacto-series GSLs, is entirely based on the structure of the saccharide moiety. However, the Cer moiety, composed of a sphingoid base and fatty acyl chain linked by an N -acyl linkage, shows heterogeneity in carbon chain length, and saturation and hydroxylation status, etc. The complexity of the Cer moiety prevents the comprehensive analysis of cellular GSLs by MS and/or LC-MS. Removal of the Cer moiety makes the analysis of GSLs much simpler and easier. For this purpose, chemical methods such as ozonolysis ( 4 ) and osmium-catalyzed periodate oxidation ( 5 ) have been developed. These chemical methods, however, require complicated and time-consuming procedures with relatively low yield ( 6 ). Alternatively, enzymes capable of detaching intact oligosaccharides from various GSLs Abstract Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the ␤ -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specifi city were found in the culture fl uid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k cat ) ( k cat ) of the recombinant EGCase I to be 22-and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K m of both enzymes was almost the same for these substrates.

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Background: Endoglycoceramidase-related protein 1 (EGCrP1) is a glucocerebrosidase in Cryptococcus neoformans; however, the functions of its paralogue, EGCrP2, remain unknown. Results: EGCrP2, but not EGCrP1, hydrolyzed steryl-␤-glucosides. Disruption of egcrp2 accumulated ergosteryl-3␤-glucoside in C. neoformans. Conclusion: EGCrP2 degraded steryl-␤-glucosides both in vivo and in vitro.Significance: EGCrP2 is a missing link in steryl-␤-glucoside metabolism in fungi. In addition, EGCrP2 is a potential target for antifungal drugs.

In a previous study, we demonstrated that β1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc 3 Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5 −/− ) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc 3 Cer synthase activity was detected in the tissues of these mice. These results demonstrate that β3GnT5 is the sole enzyme synthesizing Lc 3 Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be upregulated in B3gnt5 −/− B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5 −/− resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5 −/− B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5 −/− B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5 −/− B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.A lmost all organisms possess lipids and proteins to which a broad range of carbohydrate chains are linked. Some carbohydrate structures are known to participate in vital processes, such as the molecules responsible for cell-cell, receptor-ligand, and carbohydrate-carbohydrate interactions. It is known that glycosphingolipids (GSLs) have an important role in biological functions. In mammals, GSLs can be classified into several major classes, such as globo-, isoglobo-, ganglio-, and lacto/neolacto-

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