Glycosphingolipid Transfer Research Articles

Multimodal regulation of encystation in Giardia duodenalis revealed by deep proteomics

Cyst formation in the parasitic protist Giardia duodenalis is critical to its transmission. Existing proteomic data quantifies only 17% of coding genes transcribed during encystation and does not cover the complete process from trophozoite to mature cyst. Using high-resolution mass spectrometry, we have quantified proteomic changes across encystation and compared this with published transcriptomic data. We reproducibly identified 3863 (64.5% of Giardia proteins) and quantified 3382 proteins (56.5% of Giardia proteins) over standard trophozoite growth (TY), during low-bile encystation priming (LB), 16 h into encystation (EC), and at cyst maturation (C). This work provides the first known expanded observation of encystation at the proteomic level and triples the coverage of previous encystation proteomes. One-third (1169 proteins) of the quantified proteome is differentially expressed in the mature cyst relative to the trophozoite, including proteasomal machinery, metabolic pathways, and secretory proteins. Changes in lipid metabolism indicated a shift in lipid species dependency during encystation. Consistent with this, we identified the first, putative lipid transporters in this species, representing the steroidogenic acute regulatory protein–related lipid transfer (StARkin), oxysterol binding protein related protein (ORP/Osh) and glycosphingolipid transfer protein (GLTP) families, and follow their differential expression over cyst formation. Lastly, we undertook correlation analyses of the transcriptome and proteome of trophozoites and cysts, and found evidence of post-transcriptional regulation of key protein classes (RNA binding proteins) and stage-specific genes (encystation markers) implicating translation-repression in encystation. We provide the most extensive proteomic analysis of encystation in Giardia to date and the first known exploration across its complete duration. This work identifies encystation as highly coordinated, involving major changes in proteostasis, metabolism and membrane dynamics, and indicates a potential role for post-transcriptional regulation, mediated through RNA-binding proteins. Together our work provides a valuable resource for Giardia research and the development of transmission-blocking anti-giardials.

Bio-recognition and functional lipidomics by glycosphingolipid transfer technology.

Through glycosphingolipid biochemical research, we developed two types of transcription technologies. One is a biochemical transfer of glycosphingolipids to peptides. The other is a physicochemical transfer of glycosphingolipids in silica gel to the surface of a plastic membrane. Using the first technology, we could prepare peptides which mimic the shapes of glycosphingolipid molecules by biopanning with a phage-displayed peptide library and anti-glycosphingolipid antibodies as templates. The peptides thus obtained showed biological properties and functions similar to those of the original glycosphingolipids, such as lectin binding, glycosidase modulation, inhibition of tumor metastasis and immune response against the original antigen glycosphingolipid, and we named them glyco-replica peptides. The results showed that the newly prepared peptides could be used effectively as a bio-recognition system and suggest that the glyco-replica peptides can be widely applied to therapeutic fields. Using the second technology, we could establish a functional lipidomics with a thin-layer chromatography-blot/matrix-assisted laser desorption ionization-time of flight mass spectrometry (TLC-Blot/MALDI-TOF MS) system. By transferring glycosphingolipids on a plastic membrane surface from a TLC plate, innovative biochemical approaches such as simple purification of individual glycosphingolipids, binding studies, and enzyme reactions could be developed. The combinations of these biochemical approaches and MALDI-TOF MS on the plastic membrane could provide new strategies for glycosphingolipid science and the field of lipidomics. In this review, typical applications of these two transfer technologies are introduced.

Enhanced Selectivity for Sulfatide by Engineered Human Glycolipid Transfer Protein

Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D‖D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of "portal entrance" residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homodimerization. "Door-opening" conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of "engineered GLTPs" with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.

Identification of a glycosphingolipid transfer protein GLTP1 in Arabidopsis thaliana

Arabidopsis thaliana At2g33470 encodes a glycolipid transfer protein (GLTP) that enhances the intervesicular trafficking of glycosphingolipids in vitro. GLTPs have previously been identified in animals and fungi but not in plants. Thus, At2g33470 is the first identified plant GLTP and we have designated it AtGTLP1. AtGLTP1 transferred BODIPY-glucosylceramide at a rate of 0.7 pmol x s(-1), but BODIPY-galactosylceramide and BODIPY-lactosylceramide were transferred slowly, with rates below 0.1 pmol x s(-1). AtGLTP1 did not transfer BODIPY-sphingomyelin, monogalactosyldiacylglycerol or digalactosyldiacylglycerol. The human GLTP transfers BODIPY-glucosylceramide, BODIPY-galactosylceramide and BODIPY-lactosylceramide with rates greater than 0.8 pmol.s(-1). Structural models showed that the residues that are most critical for glycosphingolipid binding in human GLTP are conserved in AtGLTP1, but some of the sugar-binding residues are unique, and this provides an explanation for the distinctly different transfer preferences of AtGLTP1 and human GLTP. The AtGLTP1 variant Arg59Lys/Asn95Leu showed low BODIPY-glucosylceramide transfer activity, indicating that Arg59 and/or Asn95 are important for the specific binding of glucosylceramide to AtGLTP1. We also show that, in A. thaliana, AtGLTP1 together with At1g21360 and At3g21260 constitute a small gene family orthologous to the mammalian GLTPs. However, At1g21360 and At3g21260 did not transfer any of the tested lipids in vitro.

Effect of Sterol Carrier Protein‐2 Expression on Sphingolipid Distribution in Plasma Membrane Lipid Rafts/Caveolae

Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.

Characterization of the Arabidopsis thaliana glycosphingolipid transfer protein GLTP1

Characterization of the Arabidopsis thaliana glycosphingolipid transfer protein GLTP1

Identification and functional analysis of plant glycosphingolipid transfer proteins

Identification and functional analysis of plant glycosphingolipid transfer proteins

The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells

The Niemann-Pick type C2 (NPC2) protein is a small, soluble, lysosomal protein important for cholesterol and sphingolipid transport in the lysosome. The immunological phenotype of NPC2-deficient mice was limited to an impaired thymic selection of Vα14 natural killer T cells (NKT cells) and a subsequent reduction of NKT cells in the periphery. The remaining NKT cells failed to produce measurable quantities of interferon-γ in vivo and in vitro after activation with α-galactosylceramide. In addition, thymocytes and splenocytes from NPC2-deficient mice were poor presenters of endogenous and exogenous lipids to CD1d-restricted Vα14 hybridoma cells.Importantly, we determined that similar to saposins, recombinant NPC2 was able to unload lipids from and load lipids into CD1d. This transfer activity was associated with a dimeric form of NPC2, suggesting a unique mechanism of glycosphingolipid transfer by NPC2. Similar to saposin B, NPC2 dimers were able to load isoglobotrihexosylceramide (iGb3), the natural selecting ligand of NKT cells in the thymus, into CD1d. These observations strongly suggested that the phenotype observed in NPC2-deficient animals was directly linked to the efficiency of the loading of iGb3 into CD1d molecules expressed by thymocytes. This conclusion was supported by the rescue of endogenous and exogenous iGb3 presentation by recombinant NPC2. Thus, the loading of endogenous and exogenous lipids and glycolipids onto CD1d is dependent on various small, soluble lipid transfer proteins present in the lysosome.

Direct Visualization of Saposin Remodelling of Lipid Bilayers

Saposins A, B, C and D are soluble, non-enzymatic proteins that interact with lysosomal membranes to activate the breakdown and transfer of glycosphingolipids. The mechanisms of hydrolase activation and lipid transfer by saposins remain unknown. We have used in situ atomic force microscopy (AFM) with simultaneous confocal fluorescence microscopy to investigate the interactions of saposins with lipid membranes. AFM images of the effect of saposins A, B and C on supported lipid bilayers showed a time and concentration-dependent nucleated spread of membrane transformation. Saposin B produced deep gaps that ultimately filled with granular material, while saposins A and C lead to localized areas of membrane that were reduced in height by approximately 1.5 nm. Fluorescence-labeled saposin C co-localized with the transformed areas of the bilayer, indicating stable binding to the membrane. Fluorescence resonance energy transfer confirmed a direct interaction between saposin C and lipid. Under certain conditions of membrane lipid composition and saposin concentration, extensive bilayer lipid removal was observed. We propose a multi-step mechanism that integrates the structural features and amphipathic properties of the saposin proteins.

Glycolipid Transfer Protein Mediated Transfer of Glycosphingolipids between Membranes

Glycolipid Transfer Protein Mediated Transfer of Glycosphingolipids between Membranes

Glycolipid Transfer Protein Mediated Transfer of Glycosphingolipids between Membranes: A Model for Action Based on Kinetic and Thermodynamic Analyses

Glycolipid transfer protein (GLTP) catalyzes the intermembrane transfer of lipids that have sugars beta-linked to either diacylglycerol or ceramide backbones, including simple glycosphingolipids (GSLs) and gangliosides. The present study provides a quantitative understanding of GLTP action involving bilayer vesicles that have high and low curvature stress, i.e., small and large unilamellar vesicles (SUVs and LUVs). When the GSL intervesicular transfer was monitored in real time using an established fluorescence resonance energy approach, the initial GSL transfer rates (v(0)) and net transfer equilibrium (K(eq)) were determined for GLTP-mediated transfer from SUVs and LUVs over the temperature range of 30-44 degrees C. v(0) exhibited a linear dependence with respect to varying GLTP concentrations (0-143 nM range) in SUVs and LUVs, suggesting a first order dependence on the GLTP bulk concentration. Thermodynamic parameters associated with the GLTP-GSL transition-state complex and GSL net transfer were determined from linear Arrhenius and van't Hoff plots, respectively. Although initial transfer rates were lower for LUVs than for SUVs, the activation energy barriers were higher for LUVs, while the Gibbs's free energy of the transition states were similar. The formation of a transition-state complex was predominantly enthalpy driven, whereas the net transfer of GSLs was mainly entropy driven. The rate-limiting step for GLTP action appeared to be the surface processes leading to the GLTP-GSL complex formation and release associated with a shuttle/carrier mode of action. Because surface processes leading to the GLTP-GSL complex formation were limiting for GLTP action with SUVs and LUVs, it was concluded that GLTP is likely to be a valuable tool to probe and manipulate GSL environments in membranes.

Structural basis for glycosphingolipid transfer specificity.

Lipid transfer proteins are important in membrane vesicle biogenesis and trafficking, signal transduction and immunological presentation processes. The conserved and ubiquitous mammalian glycolipid transfer proteins (GLTPs) serve as potential regulators of cell processes mediated by glycosphingolipids, ranging from differentiation and proliferation to invasive adhesion, neurodegeneration and apoptosis. Here we report crystal structures of apo-GLTP (1.65 A resolution) and lactosylceramide-bound (1.95 A) GLTP, in which the bound glycosphingolipid is sandwiched, after adaptive recognition, within a previously unknown two-layer all-alpha-helical topology. Glycosphingolipid binding specificity is achieved through recognition and anchoring of the sugar-amide headgroup to the GLTP recognition centre by hydrogen bond networks and hydrophobic contacts, and encapsulation of both lipid chains, in a precisely oriented manner within a 'moulded-to-fit' hydrophobic tunnel. A cleft-like conformational gating mechanism, involving two interhelical loops and one alpha-helix of GLTP, could enable the glycolipid chains to enter and leave the tunnel in the membrane-associated state. Mutation and functional analyses of residues in the glycolipid recognition centre and within the hydrophobic tunnel support a framework for understanding how GLTPs acquire and release glycosphingolipids during lipid intermembrane transfer and presentation processes.

Glycolipid intermembrane transfer is accelerated by HET-C2, a filamentous fungus gene product involved in the cell-cell incompatibility response.

Among filamentous fungi capable of mycelial growth, het genes play crucial roles by regulating heterokaryon formation between different individuals. When fusion occurs between fungal mycelia that differ genetically at their het loci, the resulting heterokaryotic cells are quickly destroyed. It is unclear how het gene products of Podospora anserina trigger heterokaryon incompatibility. One unexplored possibility is that glycosphingolipids play a role because the het-c2 gene encodes a protein that displays 32% sequence identity and an additional 30% similarity to the mammalian glycolipid transfer protein. Here, P. anserina protoplasts containing wild-type het-c2 genes were shown to have greater glycosphingolipid transfer activity than protoplasts with disrupted het-c2 genes, a condition previously linked to altered cell compatibility following hyphal fusion. The observed glycolipid transfer activity could not be accounted for by nonspecific lipid transfer protein activity. Direct assessment showed that purified, recombinant HET-C2 accelerates the intermembrane transfer of glycolipid in vitro, but that the HET-C2 activity is mitigated much less by negatively charged membranes than the mammalian glycolipid transfer protein. The findings are discussed within the context of HET-C2 being a member of an emerging family of ancestral sphingolipid transfer proteins that play important roles in cell proliferation and accelerated death.

Charged membrane surfaces impede the protein-mediated transfer of glycosphingolipids between phospholipid bilayers.

A lipid transfer protein that facilitates the transfer of glycolipids between donor and acceptor membranes has been investigated using a fluorescence resonance energy transfer assay. The glycolipid transfer protein (23-24 kDa, pI 9.0) catalyzes the high specificity transfer of lipids that have sugars beta-linked to either a ceramide or a diacylglycerol backbone, such as simple glycolipids and gangliosides, but not the transfer of phospholipids, cholesterol, or cholesterol esters. In this study, we examined the effect of different charged lipids on the rate of transfer of anthrylvinyl-labeled galactosylceramide (1 mol %) from a donor to acceptor vesicle population at neutral pH. Compared to neutral donor vesicle membranes, introduction of negatively charged lipid at 5 or 10 mol % into the donor vesicles significantly decreased the transfer rate. Introduction of the same amount of negative charge into the acceptor vesicle membrane did not impede the transfer rate as effectively. Also, positive charge in the donor vesicle membrane was not as effective at slowing the transfer rate as was negative charge in the donor vesicle. Increasing the ionic strength of the buffer with NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (pI congruent with 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that the transfer process follows first-order kinetics and that the off-rate of the transfer protein from the donor vesicle surface is the rate-limiting step in the transfer process.

Glycosphingolipid specifity of the human sulfatide activator protein

The interaction of the sulfatide activator protein with different glycosphingolipids have been studied in detail. The following findings were made. 1. The sulfatide activator protein forms water-soluble complexes with sulfatides [Fischer, G. and Jatzkewitz, H. (1977) Hoppe-Seyler's Z. Physiol. Chem. 356, 6588-6591] and various other glycospingolipids. 2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein, transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GM2, GM1 and GD1a were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules. 3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein. 4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by beta-galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long-chain fatty acids in their hydrophobic ceramide anchor.

46] Glycolipid transfer protein from pig brain

Publisher Summary The membrane-free cytosol of pig brain contains glycolipid transfer protein (GL-TP) that stimulates the transfer of various glycosphingolipids and glyceroglycolipids among membranes in vitro. The protein has been purified to homogeneity from this source. The GL-TP from pig brain functions as a specific carrier of glycolipids most likely by forming a 1 : 1 molecular complex. Under conditions where one of the membranes lacks glycolipids, GL-TP has been found to catalyze a net transfer reaction. The chapter describes the purification of GL-TP from pig brain. GL-TP has also been partially purified from rat brain and several cell lines of rat, mouse, and human origin. Antisera raised against pig GL-TP in rabbits are cross-reactive with rat, mouse, and human GL-TP.

Nonspecific lipid transfer protein in the assay of a membrane-bound enzyme CMP-N-acetyl-neuraminate:lactosylceramide sialyltransferase.

CMP-N-acetylneuraminate:lactosylceramide alpha-2,3-sialyltransferase is tightly associated with the luminal side of the Golgi membrane as is its lipid substrate, lactosylceramide. In order to understand the kinetics, properties, and regulation of this enzyme, it is necessary to alter the amount and type of substrate in the membrane while minimizing changes in the membrane environment or in the conformation of the enzyme. Therefore, nonspecific lipid transfer protein, which accelerates the transfer of phospholipids, cholesterol, and glycosphingolipids between membranes was used to study the properties and kinetics of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase. These results are compared to those obtained in parallel experiments using detergent-solubilized substrate. Enzyme activity was increased four- to fivefold by transfer protein and was consistently higher than the activity measured in the presence of detergents. In contrast to the results obtained with detergents, the enzyme activity increased linearly with both Golgi protein and with incubation time for up to 60 min. The Km values for the water-soluble substrate, CMP-neuraminic acid, were virtually identical when determined in the presence of transfer protein (0.23 mM) or detergents (0.27 mM). On the other hand, the apparent Km values for the lipophilic substrate, lactosylceramide, were markedly different when determined in the presence of transfer protein (47.9 microM) or in the presence of detergents (1.2 microM). These observations suggest that transfer protein is a useful tool to study the properties and kinetics of membrane-bound enzymes when both the enzyme and substrate are components of the same membrane.

Isolation of two glycolipid transfer proteins from bovine brain: reactivity toward gangliosides and neutral glycosphingolipids.

Two glycolipid transfer proteins that catalyze the transfer of gangliosides and neutral glycosphingolipids from phosphatidylcholine vesicles to erythrocyte ghosts have been isolated from calf brain. Purification procedures included differential centrifugation, precipitation at pH 5.1, ammonium sulfate precipitation, and gel filtration on Sephadex G-50 and G-75. The final stage employed fast protein liquid chromatography (Mono S), producing two peaks of activity. Apparent purity of the major peak (TP I) was approximately 85-90%, as judged by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis. That of the minor fraction (TP II) was less. The major band of both fractions had a molecular mass of approximately 20,000 daltons. Both proteins catalyzed the transfer of ganglioside GM1 as well as asialo-GM1, but transfer protein I was more effective with di- and trisialogangliosides. Transfer protein II appeared to be somewhat more specific for neutral glycolipids in that GA1 was transferred more rapidly than any of the gangliosides; however, lactosylceramide transfer was relatively slow. Neither protein catalyzed transfer of phosphatidylcholine.

Evidence for the presence of a glycosphingolipid-transfer protein in rat brain cytosol.

Proteins in the postmicrosomal supernatant fraction of rat brain catalyzed the transfer of bovine brain galactocerebroside, sulfatide, and ganglioside GM1 from unilamellar liposomes to the rat erythrocytes or ghosts. The vesicles were made with egg yolk lecithin, cholesterol, 3H-labelled glycolipid, and a trace of [14C]triolein as a nonexchangeable marker. The routine assay of the glycosphingolipid transfer consisted of incubation of the donor liposomes with erythrocytes in the presence or absence of supernatant protein in physiological buffer at 37 degrees C for various time intervals. After the incubation, the erythrocytes were separated from the vesicles by centrifugation and the extent of protein-catalyzed transfer of labelled glycolipid in the membrane-bound total lipid fraction was determined by scintillation spectrometry. The fraction of [3H]glycosphingolipid transferred is represented by a change in the 3H/14C ratios at initial and subsequent time intervals. The glycosphingolipid transfer catalyzed by the supernatant protein was found to be logarithmic, whereas the protein-independent transfer was linear over a period of 3-4 h. The rate constant (K) and half time (t1/2) of the protein-catalyzed transfer reaction of cerebrosides and sulfatides were almost the same, while the transfer of ganglioside GM1 occurred at a slightly faster rate, probably owing to the greater aqueous solubility of this lipid. The transfer activity was also increased in a manner dependent on the amount of supernatant protein added up to 10 mg. The catalytic activity of the protein was lost when heated at 70 degrees C for 5 min. The pH optimum of the activity was around 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycolipid transfer protein from pig brain transfers glycolipids with β-linked sugars but not with α-linked sugars at the sugar-lipid linkage

The glycolipid transfer protein purified from pig brain facilitates the transfer of various glycosphingolipids and glyceroglycolipids (Yamada K., Abe A. and Sasaki T. (1985) J. Biol. Chem. 260, 4615–4621). In this paper, the transfer of Man β1 → 4Glc β1-Cer and Man α1 → 4Man β1 → 4Glc β1-Cer isolated from a bivalve, Corbicula japonica, the transfer of 3-[ Glcα1-]- sn-1,2-diacylglycerol and 3-[ Glcα1 → 2 Glcα1-]- sn-1,2-diacylglycerol prepared from Streptococcus lactis, and the transfer of 3-[Glcβ1-]-rac-1,2-dipalmitylglycerol have been investigated. The transfer of these lipids from liposomes to mitochondria was assayed by the decrease of these lipids in the donor liposomes. These lipids were determined by chromatographic isolation of the lipids, acid hydrolysis of the isolated lipids, and subsequent determination of glucose in the hydrolysate. The glycolipid transfer protein facilitated the transfer of ManGlcCer and ManManGlcCer. The transfer protein did not facilitate the transfer of Glcα-diacylglycerol or GlcαGlcα-diacylglycerol. However, the transfer of Glcβ-dipalmitylglycerol was facilitated by the protein. These results strongly suggest that the glycolipid transfer protein has the specificity to the presence of β-linked glucose or galactose directly linked to either ceramide or diacylglycerol.