Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids.

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcß1-Cer, Manß1-4Glcß1-Cer, Fucα1-3Manß1-4Glcß1-Cer, GlcNAcß1-3Manß1-4Glcß1-Cer, GlcNAcα1-2Fucα1-3Manß1-4Glcß1-Cer, GalNAcß1-4GlcNAcß1-3Manß1-4Glcß1-Cer, GalNAcß1-4(Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer (CPS), and GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manß4GlcßCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.

Novel neogala-series glycosphingolipids with terminal mannose and glucose residues from Hirsutella rhossiliensis, an aureobasidin A-resistant ascomycete fungus.

Hirsutella rhossiliensis, a nematophagous fungus belonging to the Ascomycota, is resistant to aureobasidin A (AbA). In this fungus, the biosynthetic pathway leading to mannosylinositolphosphoceramides, which is inhibited by AbA, was not detected. Instead, this fungus contains neutral complex glycosphingolipids (GSLs) and monoglycosylceramides. Except for monoglycosylceramides, neutral GSLs share a neogala-series core structure, Galbeta1-6Galbeta1-Cer. Among the GSLs of H. rhossiliensis, three novel GSLs with terminal Man and Glc residues on the sugar chain were elucidated. We analyzed GSL structure using compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry (MALDI-TOF MS), and (1)H nuclear magnetic resonance spectroscopy (NMR). The following structures were determined: Manalpha1-3Galbeta1-6Galbeta1-6Galbeta1-Cer; Glcalpha1-2Galbeta1-6Galbeta1-6Galbeta1-Cer; and Manalpha1-3Galbeta1-6(Glcalpha1-4)Galbeta1-6Galbeta1-Cer. In the ceramides, the fatty acids were predominantly saturated h24:0-acids and the sphingoids were predominately t18:0- or t18:1-sphingoids. In contrast, the ceramides of Glcbeta1-Cer contained d18:2- and d19:2-sphingoids. These findings indicate the presence of a novel biosynthetic pathway of neogala-series GSLs in fungi.

Invariant Valpha14 natural killer T cell activation by edible mushroom acidic glycosphingolipids.

Invariant natural killer T (iNKT) cells regulate multi-immune response through Th1/Th2 cytokine release triggered by the recognition of CD1d-restricted glycosphingolipid antigens. Here we report that acidic glycosphingolipids (AGLs) of mushroom (Hypsizigus marmoreus and Pleurotus eryngii) presented by murine CD1d-transfected rat basophilic leukocytes induced interleukin-2 (IL-2) release from iNKT hybridoma cells. AGL-1, one of the AGLs, containing mannose at the non-reducing ends, induced CD1d-dependent IL-2 release. Al-though alpha-galactosylceramide (alpha-GalCer) presented by CD11c-positive cells induced both interferon-gamma (IFN-gamma) and IL-4 release, all of AGLs presented by CD11c-positive cells and AGL-1 presented by B cells induced IL-4 release from iNKT hybridoma cells. A single intravenous injection of AGLs into B6 mice induced only a little elevation of IL-4 in serum but repeated intravenous injection of AGLs induced prolonged retention of IL-4 in serum; therefore, these results suggested that edible mushroom AGLs might contribute to the retention of immunohomeostasis through the minimum induction of iNKT cell activation in vivo.

Phosphocholine-containing glycosyl inositol-phosphoceramides from Trichoderma viride induce defense responses in cultured rice cells.

We isolated two major zwitterionic glycosphingolipids (ZGLs) from the phytopathogenic filamentous fungus Trichoderma viride. Structural analyses showed that the ZGLs (designated Tv-ZGL2 and Tv-ZGL3) were the same as the glycosphingolipids ZGL2 and ZGL4 from Acremonium sp., which are described in our previous paper. ZGLs have the following structure: Man(alpha1-6)GlcN(alpha1-2)Ins-P-Cer (Tv-ZGL2) and phosphocholine (PC)-->6Man(alpha1-6)GlcN(alpha1-2)Ins-P-Cer (Tv-ZGL3). To determine whether these ZGLs have functional roles in plant-fungus interaction, we tested to determine whether they would induce defense responses in cultured rice cells. We found that T. viride's ZGLs elicited expression of the PAL and PBZ1 genes, both of which are associated with pathogen resistance. Tv-ZGL2 induced cell death at a moderate rate. Tv-ZGL3, which contains a PC moiety, induced a high level of cell death in rice cells.

Structural characterization of glycosylinositolphospholipids with a blood group type B sugar unit from the edible mushroom, Hypsizygus marmoreus.

Edible fungi, mushrooms, are a popular food in Japan and over 15 cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Manalpha1-2Ins1-[PO(4)]-Cer and extensions of Man, Gal, and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO(4)]-Cer (AGL0), Manalpha1-2Ins1-[PO(4)]-Cer (AGL1), Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL2), Fucalpha1- 2Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL3), Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL4), Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL5), and Galalpha1-2Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (1)H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids. By immunochemical detection, the terminal structure of AGL4, Galalpha1-3(Fucalpha1-2)Galbeta-, was shown to have blood group type B activity. Galalpha1-2 and its repeating sequence in AGL5 and AGL6 are novel structures on the nonreducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.

Mushroom acidic glycosphingolipid induction of cytokine secretion from murine T cells and proliferation of NK1.1 alpha/beta TCR-double positive cells in vitro.

Interferon (IFN)-gamma and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- gamma and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of alpha-galactosylceramide (alpha-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by alpha-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 alpha/beta TCR-double positive cells in splenocytes. Administration of a mixture of alpha-GalCer and AGLs affected the stimulation of alpha-GalCer and generally induced a subtle Th1 bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.

Synthetic studies on glycosphingolipids from Protostomia phyla: syntheses and biological activities of amphoteric glycolipids containing a phosphocholine residue from the earthworm Pheretima hilgendorfi.

Two types of amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi, PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (1) and PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (2), and their derivatives (4, 5) were synthesized. These were examined for their ability to enhance production of interleukin-8 (IL-8), a potent inflammatory cytokine involved in neutrophil chemotaxis, in a TNFalpha-stimulated granulocytic HL-60 cells. Compounds 1 and 2 were found to be potent enhancers of IL-8 production.

Structural analysis of neutral glycosphingolipids from the silkworm Bombyx mori and the difference in ceramide composition between larvae and pupae.

Glycosphingolipids (GSLs) from the silkworm Bombyx mori were identified and GSL expression patterns between larvae and pupae were compared. The structural analysis of neutral GSLs from dried pupae revealed the following predominant species: Glcß1Cer, Manß4Glcß1Cer, GlcNAcß3Manß4Glcß1Cer, Galß3Manß4Glcß1Cer, GalNAcα4Galß3Manß4Glcß1Cer, GlcNAcß3Galß3Manß4Glcß1Cer, Galα4Galß3Manß4Glcß1Cer and (GalNAcα4)1-4 GalNAcα4Galß3Manß4Glcß1Cer. Lin-ear elongation of α4-GalNAc was observed at the non-reducing end of Galß3Manß4Glcß1Cer with up to five GalNAc repeats. The arthro-series GSL GlcNAcß3Manß4Glcß1Cer, a characteristic GSL-glycan sequence of other Arthropoda, was detected in silkworms. The main ceramide species in each purified GSL fraction were h20:0-d14:1 and h22:0-d14:1. GSL expression patterns in larvae and pupae were compared using thin-layer chromatography, which demonstrated differences among acidic, polar and neutral GSL fractions, while the zwitterionic fraction showed no difference. Neutral GSLs such as ceramides di-, tri- and tetrasaccharides in larvae showed less abundant than those in pupae. MALDI-TOF MS analysis revealed that larval GSLs contained four types of ceramide species, whereas pupal GSLs contained only two types. The structural analysis of neutral GSLs from silkworms revealed a novel series of GSLs. The comparison of GSL expression patterns between larvae and pupae demonstrated differences in several fractions. Alterations in GSL ceramide composition between larvae and pupae were observed by MALDI-TOF MS analysis.

Identification and characterization of carbohydrate molecules in mammalian cells recognized by dengue virus type 2.

The interaction between cell surface receptors and the envelope glycoprotein (EGP) on the viral membrane surface is the initial step of Dengue virus infection. To understand the host range, tissue tropism, and virulence of this pathogen, it is critical to elucidate the molecular mechanisms of the interaction of EGP with receptor molecules. Here, using a TLC/virus-binding assay, we isolated and characterized a carbohydrate molecule on mammalian cell surfaces that is recognized by dengue virus type 2 (DEN2). Structural determination by immunochemical methods showed that the carbohydrate structure of the purified glycosphingolipid was neolactotetraosylceramide (nLc4Cer). This glycosphingolipid was expressed on the cell surface of susceptible cells, such as human erythroleukemia K562 and baby hamster kidney BHK-21. All serotypes of DEN viruses, DEN1 to DEN4, reacted with nLc4Cer, and the non-reducing terminal disaccharide residue Galbeta1-4GlcNAcbeta1- was found to be a critical determinant for the binding of DEN2. Chemically synthesized derivatives carrying multiple carbohydrate residues of nLc4, but not nLc4 oligosaccharide, inhibited DEN2 infection of BHK-21 cells. These findings strongly suggested that multivalent nLc4 oligosaccharide could act as a competitive inhibitor against the binding of DEN2 to the host cells.

Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells.

Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi ( Acremonium sp.) isolated from soil. Five ZGLs (ZGL1-ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization-time-of-flight MS, (1)H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(alpha1-2)Ins1-P-1Cer (ZGL1), Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL2), Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL3), PC-->6Man(alpha1-6)GlcN(alpha1-2)Ins1- P -1Cer (ZGL4), and PC-->6Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(alpha1-2)Ins1- P, is rather different from those found in other fungi, such as Man(alpha1-2)Ins1- P and Man(alpha1-6)Ins1- P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(alpha1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells.

Synthetic studies on glycosphingolipids from the Protostomia phyla: syntheses of arthro-series glycosphingolipids.

Glycosphingolipids isolated from larvae of the green-bottle fly, Lucilia caesar, have quite unique structures containing GlcNAcbeta-(1 --> 3)-Man and GalNAcbeta-(1 --> 4)-GlcNAcbeta-(1 --> 3)-Man. We have synthesized two glycosphingolipids, beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer and beta-D-GalNAcp-(1 --> 4)-beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer. A key reaction in the synthetic sequence is the application of the intramolecular aglycon delivery (IAD) approach for the synthesis of the beta-mannopyranosidic linkages.

Structural elucidation of the neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides from the marine crab Erimacrus isenbeckii.

The neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides (GL-1, GL-2, GL-3, GL-4a and GL-4b), were identified from whole tissues of the marine crab Erimacrus isenbeckii by successive column chromatography with ion exchange Sephadex (QAE-Sephadex), magnesium silicate (Florisil) and silicic acid (Iatrobeads) resins. Through component analysis, sugar analysis, methylation studies, exoglycosidase cleavage, and various chromatographic and spectrometric techniques, their structures were proposed to be as follows: GL-1, Glcß1-1Cer; GL-2, Manß1-4Glcß1-1Cer; GL-3, Galß1-3Manß1-4Glcß1-1Cer; and GL-4a and GL-4b, Gal3Meα1-4Galß1-3Manß1-4Glcß1-1Cer. The main molecular species of the aliphatic moiety in each purified glycolipid were 18:0, 22:0, 22:1-d14:1 (fatty acid-sphingoid) and 18:0-d16:1 for GL-1; 18:0-d16:1 and 22:1-d14:1, d16:1 for GL-2; 22:1, 24:1-d16:1 for GL-3; 22:1, 24:1-d16:1 for GL-4a; and h22:1, h24:1-d16:1 for GL-4b, respectively. By immunological detection, an arthro-series glycosphingolipid (At3Cer; GlcNAcß1-3Manß1-4Glcß1-1Cer) was also detected as a minor component. The characteristic arthro-series glycosphingolipid has been observed in most animals belonging to the phylum Arthropoda.

Biochemical studies on sphingolipid of Artemia franciscana (I) isolation and characterization of sphingomyelin.

Sphingomyelin was isolated from cysts of the brine shrimp Artemia franciscana using QAE-Sephadex A25, Florisil and Iatrobeads column chromatographies. The chemical structure was identified using thin-layer chromatography, gas-liquid chromatography, infrared spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ceramide moiety of sphingomyelin consisted of stearic, arachidic, and behenic acids as fatty acids, and hexadeca-4- and heptadeca-4-sphingenines as sphingoids. By comparative analysis, the ceramide component of Artemia sphingomyelin appears unique in invertebrates and vertebrates. Biological functions of sphingomyelin have largely been investigated using mammalian-derived sphingomyelin. In mammals, a wide variety of molecular species of sphingomyelins have been reported, especially derived from nerve tissue, while the lower animal Artemia contains this unusual sphingomyelin perhaps because of having a much simpler nervous system. The purified unusual sphingomyelin derived from Artemia franciscana might be a very useful tool in elucidating the functions and mechanisms of action of this mediator.

Sphingomyelins in four ascidians, Ciona intestinalis, Halocynthia roretzi, Halocynthia aurantium, and Styela clava.

Sphingomyelin is rarely found in lower animals, while sphingophospholipid is a characteristic of higher animals. In this study, sphingomyelin was first isolated and characterized from ascidian Ciona intestinalis. Ascidian sphingomyelin was prepared using ion exchange (QAE-Sphadex-A25) and silicic acid (Florisil and Iatrobeads) column chromatographies. The chemical structure was characterized by fatty acid analysis, sphingoid analysis, hydrogen fluoride degradation, acid hydrolysis, enzymatic hydrolysis, infrared analysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ceramide moieties of C. intestinalis sphingomyelin consisted primarily of C16:0, C18:0, and C18:1 fatty acids and d18:2 sphingadiene. Furthermore, sphingomyelins were isolated and characterized from 3 other ascidians, Halocynthia roretzi, Halocynthia aurantium, and Styela clava using the same methods. Comparative analysis of the sphingomyelin structures in 4 ascidian species-C. intestinalis (Enterogona) and H. roretzi, H. aurantium, and S. clava (Pleurogona)-revealed that the major fatty acid composition of the ceramides was similar, and that they differed in minor components.

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi.

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.

A Novel sulfatide, GlcCer-I6 sulfate, from the ascidian Ciona intestinalis.

A novel sulfated glycosphingolipid, SGL-1, was isolated from the ascidian Ciona intestinalis, prepared from chloroform/methanol extracts and fractionated successively on DEAE Sephadex-A25, Florisil and Iatrobeads column chromatographies. Chemical structural analysis was performed using methylation analysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-NMR spectroscopy. This chemical structure is presented as GlcCer I(6)-Sulfate. The ceramide moiety was specified by t16:0, t17:0, br,t17:0, t18:0 and br,t18:0 as sphingoids, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids.

Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of amphoteric glycolipid analogues from the porcine nematode, Ascaris suum.

A novel amphoteric glycosphingolipid, cholinephosphoryl-(-->6)-beta-D-GlcpNAc-(1-->3)-beta-D-Manp-(1-->4)-beta-D-Glcp-(1-->)-Cer, isolated from the porcine parasitic nematode, Ascaris suum, may be expected to be involved in host-parasite interactions. This glycosphingolipid analogue containing octyl residue in place of ceramide was synthesized as follows: The key reaction of this synthetic procedure is the formation of a intramolecular aglycon delivery (IAD) approach for beta-selective mannosylation. Then, a coupling of phosphocholine group at the position C-6'' of 16 was attempted using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by reaction of the resulting cyclic phosphate intermediate with anhydrous trimethylamine to give 17. Subsequent debenzylation and debenzylidenation afforded target compound (2).

Microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids.

For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH(3)OH for 2 min followed by 1 M HCl/CH(3)OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure. Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.

Newly discovered neutral glycosphingolipids in aureobasidin A-resistant zygomycetes: Identification of a novel family of Gala-series glycolipids with core Gal alpha 1-6Gal beta 1-6Gal beta sequences.

We found for the first time that Zygomycetes species showed resistance to Aureobasidin A, an antifungal agent. A novel family of neutral glycosphingolipids (GSLs) was found in these fungi and isolated from Mucor hiemalis, which is a typical Zygomycetes species. Their structures were completely determined by compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry, and (1)H NMR spectroscopy. They were as follows: Gal beta 1-6Gal beta 1-1Cer (CDS), Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTS), Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTeS), and Gal alpha 1-6Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CPS). The ceramide moieties of these GSLs consist of 24:0, 25:0, and 26:0 2-hydroxy acids as major fatty acids and 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid. However, the glycosylinositolphosphoceramide families that are the major GSLs components in fungi were not detected in Zygomycetes at all. This seems to be the reason that Aureobasidin A is not effective for Zygomycetes as an antifungal agent. Our results indicate that the biosynthetic pathway for GSLs in Zygomycetes is significantly different from those in other fungi and suggest that any inhibitor of this pathway may be effective for mucormycosis, which is a serious pathogenic disease for humans.