The isolation and partial characterization of the glycolipids of BP8/C3H ascites-sarcoma cells

1. The total lipid was extracted from BP8/C3H ascites-sarcoma cells with acetone, light petroleum, pyridine and chloroform–methanol successively. Each extract was treated with mild alkali. The alkali-stable lipids from the pyridine and chloroform–methanol extracts, which included the glycolipids, were fractionated on silicic acid and silica gel G columns. 2. The total yield of glycolipid was about 60 mg./100 g. dry wt. of tumour cells, about 0·4% of the total lipid. Four classes of glycolipid were isolated and characterized as ceramide monohexoside (G1), ceramide dihexoside (G2), ceramide trihexoside (G3) and ceramide hexosaminyltrihexoside (G4). 3. G1, G2, G3 and G4 constituted 55, 21, 9 and 15% of the total glycolipid respectively. 4. G1 was a mixture of ceramide glucoside (70%) and ceramide galactoside. 5. The general structures of the oligosaccharide moieties of G2, G3 and G4 were elucidated by partial acid hydrolysis of the glycolipids with water-soluble polystyrenesulphonic acid. G2 was mostly ceramidelactoside with about 10% of ceramide galactosylgalactoside. G3 and G4 were probably a ceramide digalactosylglucoside and a ceramide N-acetylgalactosaminylgalactosylgalactosylglucoside respectively. 6. The fatty acid compositions of the glycolipids were very similar; lignoceric acid and nervonic acid were the major components and all contained monohydroxy acids in proportions varying from 10 to 25% of the total acids.     

Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins of Saccharomyces cerevisiae exist with two types of lipid moiety--diacylglycerol or ceramide--both of which contain 26:0 fatty acids. To understand at which stage of biosynthesis these long-chain fatty acids become incorporated into diacylglycerol anchors, we compared the phosphatidylinositol moieties isolated from myo-[2-(3)H]inositol-labelled protein anchors and from GPI intermediates. There is no evidence for the presence of long-chain fatty acids in any intermediate of GPI biosynthesis. However, GPI-anchored proteins contain either the phosphatidylinositol moiety characteristic of the precursor lipids or a version with a long-chain fatty acid in the sn-2 position of glycerol. The introduction of long-chain fatty acids into sn-2 occurs in the endoplasmic reticulum (ER) and is independent of the sn-2-specific acyltransferase SLC1. Analysis of ceramide anchors revealed the presence of two types of ceramide, one added in the ER and another more polar molecule which is found only on proteins which have reached the mid Golgi. In summary, the lipid of GPI-anchored proteins can be exchanged by at least three different remodelling pathways: (i) remodelling from diacylglycerol to ceramide in the ER as proposed previously; (ii) remodelling from diacylglycerol to a more hydrophobic diacylglycerol with a long-chain fatty acid in sn-2 in the ER; and (iii) remodelling to a more polar ceramide in the Golgi.     

A new 6-hydroxy-4-sphingenine-containing ceramide in human skin.

A new ceramide consisting of 6-hydroxysphingosine linked to a non-hydroxyacid was found in human epidermal lipid. This ceramide was sought because its fatty acid and sphingoid moieties are present in other combinations in human epidermal ceramides. To isolate the new ceramide, the mixture of ceramides in human epidermal lipid was first separated into fractions by thin-layer chromatography (TLC), and then each fraction was further purified by TLC after acetylation of all hydroxyl groups. TLC after acetylation revealed that one of the fractions isolated in the first TLC step contained two components, namely, the ceramide consisting of sphingosine linked to an alpha-hydroxyacid and an unknown ceramide. The new ceramide constituted about 9% of the total ceramides, and was shown by NMR spectroscopy to be N-acyl-6-hydroxysphingosine.     

Two different types of lipid moieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomyces cerevisiae.

Numerous glycoproteins of Saccharomyces cerevisiae are anchored in the lipid bilayer by a glycophosphatidylinositol (GPI) anchor. Mild alkaline hydrolysis reveals that the lipid components of these anchors are heterogeneous in that both base-sensitive and base-resistant lipid moieties can be found on most proteins. The relative abundance of base-resistant lipid moieties is different for different proteins. Strong alkaline or acid hydrolysis of the mild base-resistant lipid component liberates C18-phytosphingosine indicating the presence of a ceramide. Two lines of evidence suggest that proteins are first attached to a base-sensitive GPI anchor, the lipid moiety of which subsequently gets exchanged for a base-resistant ceramide: (i) an early glycolipid intermediate of GPI biosynthesis only contains base-sensitive lipid moieties; (ii) after a pulse with [3H]myo-inositol the relative abundance of base-sensitive GPI anchors decreases significantly during chase. This decrease does not take place if GPI-anchored proteins are retained in the ER.     

Stage-associated expression of ceramide structures in glycosphingolipids from the human trematode parasite Schistosoma mansoni

Glycosphingolipids of Schistosoma mansoni adults, cercariae and eggs comprise ceramide monohexosides (CMH) with glucose or galactose and ceramide dihexosides (CDH) with the schistosome-specific structure GalNAc(beta1-4)Glc(1-1)ceramide. Ceramide analysis revealed C18- and C20-phytosphingosines in egg CMH, C18-sphinganine as well as C18-, C19- and C20-phytosphingosines in cercarial CMH, and C18- and C20-phytosphingosines as well as C18-sphingosine and C18-sphinganine in adult CMH. For all three life cycle stages, the predominant fatty acid was C16h:0. As a characteristic feature, a range of saturated, unsaturated and hydroxylated long-chain fatty acids with 24-28 carbon atoms were additionally found in minor cercarial CMH species. The corresponding ceramides represented major constituents in cercarial CDH, while adult and egg CDH were dominated by ceramides with short fatty acid chains. The resultant ceramide patterns could be correlated with the differential expression of carbohydrate antigens on schistosomal glycolipids at various stages. A possible impact of ceramide structure on the biosynthesis of the carbohydrate moieties is discussed.     

Hydroxylation state of fatty acid and long-chain base moieties of sphingolipid determine the sensitivity to growth inhibition due to AUR1 repression in Saccharomyces cerevisiae

The structures of ceramide found in the yeast Saccharomyces cerevisiae are classified into five groups according to the hydroxylation states of the long-chain base and fatty acid moieties. This diversity is created through the action of enzymes encoded by SUR2, SCS7, and as yet unidentified hydroxylation enzyme(s). Aur1p is an enzyme catalyzing the formation of inositol phosphorylceramide in the yeast, and the defect leads to strong growth inhibition due to accumulation of ceramide and reductions in complex sphingolipid levels. In this study, we found that the deletion of SCS7 results in the enhancement of growth inhibition due to repression of AUR1 expression under the control of a tetracycline-regulatable promoter, whereas the deletion of SUR2 attenuates the growth inhibition. Under AUR1-repressive conditions, SCS7 and SUR2 mutants showed reductions in the complex sphingolipid levels and the accumulation of ceramide, like wild-type cells. On the other hand, the deletion of SCS7 had no effect on the growth inhibition through reductions in the complex sphingolipid levels caused by repression of LIP1 encoding a ceramide synthase subunit. Furthermore, the deletion of SUR2 did not suppress the growth inhibition under LIP1-repressive conditions. Therefore, it is suggested that the deletion of sphingolipid hydroxylases changes the toxicity of ceramide under AUR1-repressive conditions.     

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)‐anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI‐anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A₂ Per1p and O‐acyltransferase Gup1p play essential roles in diacylglycerol‐type lipid remodelling of GPI‐anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphorylceramide by Cwh43p. In this report, we constructed double‐mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI‐anchored proteins. Based on our analyses of single‐ and double‐mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso‐phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag‐tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.     

A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.

Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingosine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease. This study reports a new assay method to detect acid ceramidase activity in vitro using Bodipy or lissamine rhodamine-conjugated ceramide (C12 ceramide; dodecanoylsphingosine). Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[(14)C]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substrate provided data very similar to those of the radioactive substrate, but under the experimental conditions tested, it was significantly more sensitive. Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy dodecanoic acid, could be detected, providing an accurate measure of acid ceramidase activity as low as 0.1 pmol/mg protein/h. Acid ceramidase activities in skin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, confirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatographic technique is a reliable, rapid, and highly sensitive method to determine acid ceramidase activity, and that it could be useful wherever the in vitro detection of acid ceramidase activity is of importance.     

Proton nuclear magnetic resonance of neutral and acidic glycosphingolipids

A number of intact neutral glycosphingolipids (globo, asialoganglio, neolacto, and gala series), gangliosides, and sulfatide were analyzed by proton nuclear magnetic resonance (NMR) using dimethyl-d6 sulfoxide as a solvent at different conditions of measurement. The chemical shifts of amide proton of ceramide, N-acetylhexosamine and sialic acid moieties were positioned with regularity, thus providing their molar composition. The chemical shifts of amide proton in ceramide moiety differed with respect to constituent fatty acids; delta 7.45 to 7.52 ppm at 25 degrees C for the nonhydroxy acids and 7.32 to 7.42 ppm for the hydroxy acids. The chemical shifts of methyl proton in N-acetyl group were distinguished between N-acetylhexosamine and N-acetylneuraminic acid, and those in N-acetylgalactosamine were discriminated between neutral glycolipids and gangliosides. In the presence or absence of D2O in dimethyl sulfoxide at 110 degrees C, the anomeric protons resonated with regularity characteristic of respective monosaccharide linkages, and the anomeric protons of N-acetylgalactosamine in neutral glycolipids and gangliosides were clearly distinguished. The present study thus demonstrates the general applicability of NMR procedure to glycosphingolipids, providing the determination of chemical composition of both the lipophilic and carbohydrate moieties and the structural elucidation.     

Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms

Complex glycoinositolphosphoryl ceramides (GIPCs) have been purified from a pathogenic encapsulated wild-type (WT) strain of Cryptococcus neoformans var. neoformans and from an acapsular mutant (Cap67). The structures of the GIPCs were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance spectroscopy, methylation analysis, gas chromatography-mass spectrometry, and chemical degradation. The main GIPC from the WT strain had the structure Manp(alpha1-3)[Xylp(beta1-2)] Manp(alpha1-4)Galp(beta1-6)Manp(alpha1-2)Ins-1-phosphoryl ceramide (GIPC A), whereas the compounds from the acapsular mutant were more heterogeneous in their glycan chains, and variants with Manp(alpha1-6) (GIPC B), Manp(alpha1-6) Manp(alpha1-6) (GIPC C), and Manp(alpha1-2)Manp(alpha1-6)Manp(alpha1-6) (GIPC D) substituents linked to the nonreducing terminal mannose residue found in the WT GIPC A were abundant. The ceramide moieties of C. neoformans GIPCs were composed of a C(18) phytosphingosine long-chain base mainly N-acylated with 2-hydroxy-tetracosanoic acid in the WT GIPC while in the acapsular Cap67 mutant GIPCs, as well as 2-hydroxy-tetracosanoic acid, the unusual 2,3-dihydroxy-tetracosanoic acid was characterized. In addition, structural analysis revealed that the amount of GIPC in the WT cells was fourfold less of that in the acapsular mutant.     

Structures and fatty acid compositions of neutral glycosphingolipids of human plasma

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.     

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.     

Characterization of neutral sphingolipids from chicken erythrocytes

The neutral sphingolipids from chicken erythrocytes were characterized. The total concentration of neutral sphingolipids was found to be 480 nmol/g of dry stroma. They were isolated and purified by droplet counter-current chromatography, Iatrobeads column chromatography, and preparative thin-layer chromatography. The major neutral sphingolipids were free ceramide, ceramide monohexoside, ceramide dihexoside, and ceramide pentahexoside, which represented 43%, 23.5%, 10.0%, and 3.6% of the long chain bases, respectively. Thus, free ceramide was the most abundant neutral sphingolipid in chicken erythrocytes. Ceramide monohexoside was composed of more galactosylceramide than glucosylceramide. Galabiosylceramide was found in the ceramide dihexoside fraction together with lactosylceramide. Ceramide pentahexoside was a Forssman glycolipid. There were two groups of neutral sphingolipids; one had mainly C16 fatty acid and the other had C22 and C24 fatty acids. In both groups sphingosine (d18:1) was predominant as a long chain base. 2-Hydroxy-C16 fatty acid was a major component of one of the ceramide monohexosides.     

Kinetic Changes in Free Ceramide and Cerebroside during Germination of Black Gram

Free ceramide and cerebroside were isolated from black gram sprouts of all germinating stages. Free ceramide and cerebroside were found to increase during germination.

The major sphingosine bases of free ceramide were 4-hydroxysphingenine and 4-hydroxy-sphinganine (trihydroxy type) while that of cerebroside was sphinga-4,8-dienine (dihydroxy type). A change in the component sphingosine base was that 4-hydroxysphingenine in free ceramide and cerebroside increased slightly after germination.

The major fatty acid of free ceramide was α-hydroxylignoceric acid while that of cerebroside was α-hydroxypalmitic acid. Changes in component fatty acid were that α-hydroxylignoceric acid in both sphingolipids increased after germination.     

2n-fatty acids from phosphatidylcholine label sphingolipids--a novel role of phospholipase A2?

In order to find out whether there is a phospholipase A2 (PLA2)-mediated link between glycerophospholipids and sphingolipids, L929 cells were labeled with 1n-palmitoyl-2n-[1-14C]palmitoyl phosphatidylcholine for 16-18 h or 90 min. After labeling for 16-18 h, 14C-sphingomyelin (SM), 14C-ceramide and 14C-sphingosine were demonstrated on autoradiograms of thin layer chromatograms of untreated or mildly hydrolyzed lipid extracts in different chromatographic systems. Strong hydrolysis of labeled SM proved that both possible moieties of SM, sphingosine and acyl moiety, had been labeled. The identity of SM and its enzymatic degradation product, ceramide, was verified by mass spectrometry. The label in SM-derived ceramide was demonstrated on an autoradiogram after thin layer chromatography. The inhibitor of (dihydro)ceramide synthase fumonisin B1 suppressed the label in sphingolipids significantly during 16-18 h (ceramide and SM), as well as during 90-min labeling (SM). The presence of inhibitors of PLA2 (bromoenol lactone, aristolochic acid and quinacrine dihydrochloride) diminished the label in SM significantly during the 90-min labeling. These results demonstrate a close metabolic relationship between glycerophospholipids and sphingolipids and give evidence for a novel role of PLA2.     

Structural analysis of sphingophospholipids derived from Sphingobacterium spiritivorum, the type species of genus Sphingobacterium

The unique feature of the genus Sphingobacterium is the presence of sphingophospholipids and ceramides, besides diacylglycerophospholipids. As major cellular lipid components, five kinds of sphingophospholipids were purified from Sphingobacterium spiritivorum ATCC 33861(T), the type species of genus Sphingobacterium. They were identified as ceramide phosphorylethanolamines (CerPE-1 and CerPE-2), ceramide phosphoryl-myo-inositols (CerPI-1 and CerPI-2), and ceramide phosphorylmannose (CerPM-1). The ceramide of CerPE-1, CerPI-1, and CerPM-1 was composed of 15-methylhexadecasphinganine (isoheptadeca sphinganine, iso-C17:0) and 13-methyltetradecanoic acid (isopentadecanoic acid, iso-C15:0), whereas that of CerPE-2 and CerPI-2 was composed of isoheptadeca sphinganine and 2-hydroxy-13-methyltetradecanoic acid (2-hydroxy isopentadecanoic acid, 2-OH iso-C15:0). These sphingophospholipids were also found in cellular lipids of Sphingobacterium multivorum ATCC 33613(T), Sphingobacterium mizutaii ATCC 33299(T), Sphingobacterium faecium IFO 15299(T), Sphingobacterium thalpophilum ATCC 43320(T), and Sphingobacterium antarcticum ATCC 51969(T). To our knowledge, the existence of CerPM-1 is a novel sphingophospholipid through eukaryotic and prokaryotic cells.     

Polar glycosphingolipids in annelida. A novel series of glycosphingolipids containing choline phosphate from the earthworm, Pheretima hilgendorf.

A novel series of glycosphingolipids containing choline phosphate has been demonstrated in whole tissues of the earthworm, Pheretima hilgendorfi. The thin layer chromatographic pattern of the total polar glycolipids revealed the presence of more than three components with positive reactions toward orcinol-sulfuric acid (sugar), molybdate (phosphate), and Dragendorff's (choline) spray reagents. Two of these polar glycolipids (PGL1 and PGL2) were purified by the use of successive column chromatography on QAE-Sephadex A-25 and silicic acid (Iatrobeads) and detected during elution by the presence of galactose-bound choline phosphate. The structural elucidation of the oligosaccharide moieties was performed by compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and methylation analysis. Thus, the structures of PGL1 and PGL2 were deduced to be as follows: cholinephosphoryl-->6Gal beta 1-1Cer and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-1Cer. Although the oligosaccharide structures of both PGL1 and PGL2 have previously been found in other organisms, the presence of a choline phosphate group as an oligosaccharide substituent is the first finding in nature. The main molecular species of the ceramide moieties were composed of beheninyl- and lignocerinyloctadecasphingenines and their nonadecasphingenine homologues.     

Mass spectra of permethyl derivatives of glycosphingolipids

Mass spectra of the permethyl derivatives of a series of glycosphingolipids have been recorded. Fragments containing one, two and in some cases three sugar moieties were detected for the polyglycosyl compounds, several of these being of high intensity. The utility of such ions in establishing the position of hexosamine in an oligosaccharide chain was demonstrated. Low molecular weight gangliosides gave peaks corresponding to permethylated sialic acid under certain experimental conditions, permitting identification of N-acetyl- and N-glycoclylneuraminic acids. Glass sample containers appeared to catalyze destruction of these units. Peaks corresponding to the entire ceramide unit were detected but only in a few instances did these reflect the true ceramide composition. Specific fragments derived from the fatty acids and long-chain bases, respectively, were shown to be useful for qualitative identification of these components.     

Characterization of glycoinositolphosphoryl ceramide structure mutant strains of Cryptococcus neoformans

In fungi, glycoinositolphosphoryl ceramide (GIPC) biosynthetic pathway produces essential molecules for growth, viability, and virulence. In previous studies, we demonstrated that the opportunistic fungus Cryptococcus neoformans synthesizes a complex family of xylose-(Xyl) branched GIPCs, all of which have not been previously reported in fungi. As an effort to understand the biosynthesis of these sphingolipids, we have now characterized the structures of GIPCs from C. neoformans wild-type (KN99alpha) and mutant strains that lack UDP-Xyl, by disruption of either UDP-glucose dehydrogenase (NE321) or UDP-glucuronic acid decarboxylase (NE178). The structures of GIPCs were determined by a combination of nuclear magnetic resonance (NMR) spectroscopy, tandem mass spectrometry (MS), and gas chromatography-MS. The main and largest GIPC from wild-type strain was identified as an alpha-Manp(1 --> 6)alpha-Manp(1 --> 3)alpha-Manp[beta-Xylp(1 --> 2)]alpha-Manp(1 --> 4)beta-Galp(1 --> 6)alpha-Manp(1 --> 2) Ins-1-P-Ceramide, whereas the most abundant GIPC from both mutant strains was found to be an alpha-Manp(1 --> 3)alpha-Manp(1 --> 4)beta-Galp(1 --> 6)alpha-Manp(1 --> 2)Ins-1-P-Ceramide. The ceramide moieties of C. neoformans wild-type and mutant strains were composed of a C(18) phytosphingosine, which was N-acylated with 2-hydroxy tetra-, or hexacosanoic acid, and 2,3-dihydroxy-tetracosanoic acid. Our structural analysis results indicate that the C. neoformans mutant strains are unable to complete the assembly of the GIPC-oligosaccharide moiety due the absence of Xyl side chain.