Dynamic modulation of the glycosphingolipid content in supported lipid bilayers by glycolipid transfer protein

Supported lipid bilayers (SLBs) are popular models of cell membranes. Owing to the importance of glycosphingolipids (GSLs) in modulating structure and function of membranes and membrane proteins, methods to tune the GSL content in SLBs would be desirable. Glycolipid transfer protein (GLTP) can selectively transfer GSLs between membrane compartments. Using the ganglioside GM1 as a model GSL, and two mass-sensitive and label-free characterization techniques—quartz crystal microbalance with dissipation monitoring and ellipsometry—we demonstrate that GLTP is an efficient and robust biochemical tool to dynamically modulate the GSL content of SLBs up to 10 mol % GM1, and to quantitatively control the GSL content in the bulk-facing SLB leaflet. By exploiting what we believe to be a novel tool, we provide evidence that GM1 distributes highly asymmetrically in silica-supported lipid bilayers, with ∼85% of the ganglioside being present in the bulk-facing membrane leaflet. We report also that the pentameric B-subunit of cholera toxin binds with close-to-maximal stoichiometry to GM1 in SLBs over a large range of GM1 concentrations. Furthermore, we quantify the liganding affinity of GLTP for GM1 in an SLB context to be 1.5 μM.     

Preparation of homogenous oligosaccharide chains from glycosphingolipids

After the discovery of glycosphingolipid (GSL) glycan detaching enzymes, Rhodococcal endoglycoceramidase (EGCase) and leech ceramide glycanase (CGase), the method for enzymatically releasing glycans from GSLs has become the method of choice for preparing intact ceramide-free oligosaccharide chains from GSLs. This paper describes (1) the preparation of the intact oligosaccharides from GM1 (II³NeuAcGgOse₄Cer) and GbOse₄Cer as examples to show the use of CGase to prepare intact glycan chains from GSLs, and (2) the specificity and detergent requirements of Rhodococcal EGCases for the release of glycan chains from different GSLs.     

Oxidation and base-catalyzed elimination of the saccharide portion of GSLs having very different polarities

Glycosphingolipids (GSLs), present in cell membranes, participate in a variety of biological functions. Although their exact role(s) may not be understood, it has been shown that 1) embryos lacking glucosylceramide synthase activity do not develop normally, 2) GSLs can affect neuritogenesis, and 3) they can function as receptors for some pathogens. To study the role of the saccharide portion of a GSL in any of these functions, it is necessary to either isolate it from the intact GSL or synthesize it. Because syntheses are more complex, modifications were made to the oxidation/elimination procedure previously described for the isolation of the saccharide portion of GM1 and GD1a to enable it to be used with GSLs of varying polarity. The key is to use a mixture of GSLs that differ in polarity. This appears to eliminate problems encountered when purified GSLs such as sulfatide or GT1b are used.     

Traveling for the glycosphingolipid path

Our studies on glycosphingolipids (GSLs) were initiated through isolation and structural characterization of lacto-series type 1 and 2 GSLs, and globo-series GSLs. Lacto-series structures included histo-blood group ABH and I/i antigens. Our subsequent studies were focused on GSL changes associated with: (i) ontogenic development and differentiation; (ii) oncogenic transformation and tumor progression. Various novel types of GSLs such as extended globo-series, sialyl-Le^x (SLe^x), sialyl-dimeric-Le^x (SLe^x-Le^x), dimeric-Le^x (Le^x-Le^x), Le^y-on-Le^x, dimeric-Le^a (Le^a-Le^a), Le^b-on-Le^a, etc. were identified as tumor-associated antigens. These studies provide an essential basis for up- or down-regulation of key glycosyltransferase genes controlling development, differentiation, and oncogenesis. GSL structures established in our laboratory are summarized in Table 1, and structural changes of GSLs associated with ontogenesis and oncogenesis are summarized in Sections 2 and 3.Based on these results, we endeavored to find out the cell biological significance of GSL changes, focused on (i) cell adhesion, e.g., the compaction process of preimplantation embryo in which Le^x-to-Le^x, Gb4-to-GalGb4 or -nLc₄ play major roles; and (ii) modulation of signal transduction through interaction of growth factor receptor tyrosine kinase with ganglioside, e.g., EGF receptor tyrosine kinase with GM3. Recent trends of studies on i and ii lead to the concept that GSL clusters (microdomains) are organized with various signal transducer molecules to form glycosignaling domains (GSD). GSL-dependent adhesion occurs through clustered GSLs, and is coupled with activation of signal transducers (cSrc, Src family kinase, Rho A, etc.). Clustered GSLs involved in cell adhesion are recognized by GSLs on counterpart cells (carbohydrate-to-carbohydrate interaction), or by lectins (e.g., siglecs, selectins).Our major effort in utilization of GSLs in medical science has been for: (i) cancer diagnosis and treatment (vaccine development) based on tumor-associated GSLs and glycoepitopes; (ii) genetically defined phenotype for susceptibility to E. coli infection; (iii) clear identification of physiological E-selectin epitope (myeloglycan) expressed on neutrophils and myelocytes; (iv) characterization of sialyl poly-LacNAc epitopes recognized as male-specific antigens. Utilization of these GSLs or glycoepitopes in development of anti-adhesion approach to prevent tumor metastasis, infection, inflammation, or fertilization (i.e., contraceptive) is discussed. For each approach, development of mimetics of key GSLs or glycoepitopes is an important subject of future study.     

Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice

The apolipoprotein E gene knockout (apoE-/-) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE-/- mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE-/- and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAcbeta1-4Galbeta1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE-/- mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE-/- mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE-/- mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by N-glycolyl GM2 and the former comprised of NeuNAcalpha2-3Galbeta1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE-/- mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE-/- mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.     

Physiological relevance of sphingolipid activator proteins in cultured human fibroblasts

The physiological degradation of several membrane-bound glycosphingolipids (GSLs) by water-soluble lysosomal exohydrolases requires the assistance of sphingolipid activator proteins (SAPs). Four of these SAPs are synthesized from a single precursor protein (prosaposin). Inherited deficiency of this precursor results in a rare disease in humans with an accumulation of ceramide (Cer) and glycolipids such as glucosylceramide and lactosylceramide (LacCer). In a previous study, we have shown that human SAP-D stimulates the lysosomal degradation of Cer in precursor deficient cells. In order to study the role of SAPs (or saposins) A-D in cellular GSL catabolism, we recently investigated the catabolism of exogenously added [(3)H]labeled ganglioside GM1, Forssman lipid, and endogenously [(14)C]labeled GSLs in SAP-precursor deficient human fibroblasts after the addition of recombinant SAP-A, -B, -C and -D. We found that activator protein deficient cells are still able to slowly degrade gangliosides GM1 and GM3, Forssman lipid and globotriaosylceramide to a significant extent, while LacCer catabolism critically depends on the presence of SAPs. The addition of either of the SAPs, SAP-A, SAP-B or SAP-C, resulted in an efficient hydrolysis of LacCer.     

Novel class of glycosphingolipids involved in male fertility

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1-/- mice are infertile, male Siat9-/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.     

High-performance liquid chromatographic analysis of neutral glycosphingolipids as their per-O-benzoyl derivatives

Previous reports from this laboratory (1–4) described the perbenzoylation of neutral glycosphingolipids (GSL)¹ with benzoyl chloride in pyridine and analysis of the perbenzoylated derivatives by high performance liquid chromatography (hplc). A disadvantage of this procedure is that N-benzoylation occurs as well as the desired O-benzoylation. This does not permit recovery of the parent GSL after mild alkaline hydrolysis due to formation of a mixture of N-acylated and N-benzoylated GSLs(1). It has also been demonstrated that the benzoylation with benzoic anhydride in pyridine does not lead to the formation of N-benzoylated products. However, the anhydride reaction is sluggish and the benzoyl chloride method has been the preferred procedure.Gupta et al. (5) used N,N-dimethyl-4 amino pyridine (DMAP) as a catalyst in the acylation of phospholipids by the anhydrides of fatty acids. F. B. Jungalwala (private communication) has shown that this catalyst greatly accelerates the reaction of benzoic anhydride with sulfatides.In this communication we report the preparation and hplc analysis of per-O-benzoyl derivatives of GSLs by reaction with benzoic acid anhydride in the presence of DMAP as a catalyst. Reaction with these reagents avoids amide acylation, forms single products with satisfactory chromatographic properties and parent GSLs can be regenerated by mild alkaline hydrolysis.     

Immunohistological and flow cytometric analysis of glycosphingolipid expression in mouse lymphoid tissues.

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylceramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb(4)Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg(4)Cer, and Gb(4)Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.     

High expression of lactotriaosylceramide, a differentiation-associated glycosphingolipid, in the bone marrow of acute myeloid leukemia patients

Glycosphingolipids (GSLs) are information-bearing biomolecules that play critical roles in embryonic development, signal transduction and carcinogenesis. Previous studies indicate that certain GSLs are associated with differentiation in acute myeloid leukemia (AML) cells. In this study, we collected bone marrow samples from healthy donors and AML patients and analyzed the GSL expression profiles comprehensively using electrospray ionization linear ion-trap mass spectrometry. The results showed that AML patients had higher expression of the GSL lactotriaosylceramide (Lc3), GM3 and neolactotetraosylceramide (nLc4) in their bone marrow than did the healthy donors (P < 0.05), especially the M1 subtype of AML. To further explore the molecular mechanisms of Lc3, we examined the expression of the Lc3 synthase β1,3-N-acetylglucosaminyltransferase5 (β3Gn-T5) and found that the bone marrow samples of AML patients had 16-fold higher expression of β3Gn-T5 than those of healthy donors (P < 0.05). Our results suggest that AML-associated GSLs Lc3, GM3 and nLc4 are possibly involved in initiation and differentiation of AML.     

Selective caveolin-1-dependent endocytosis of glycosphingolipids

We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80-90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.     

Glucosinolate structures in evolution

By 2000, around 106 natural glucosinolates (GSLs) were probably documented. In the past decade, 26 additional natural GSL structures have been elucidated and documented. Hence, the total number of documented GSLs from nature by 2011 can be estimated to around 132. A considerable number of additional suggested structures are concluded not to be sufficiently documented. In many cases, NMR spectroscopy would have provided the missing structural information. Of the GSLs documented in the past decade, several are of previously unexpected structures and occur at considerable levels. Most originate from just four species: Barbarea vulgaris, Arabidopsis thaliana, Eruca sativa and Isatis tinctoria. Acyl derivatives of known GSLs comprised 15 of the 26 newly documented structures, while the remaining exhibited new substitution patterns or chain length, or contained a mercapto group or related thio-functionality.GSL identification methods are reviewed, and the importance of using authentic references and structure-sensitive detection methods such as MS and NMR is stressed, especially when species with relatively unknown chemistry are analyzed. An example of qualitative GSL analysis is presented with experimental details (group separation and HPLC of both intact and desulfated GSLs, detection and structure determination by UV, MS, NMR and susceptibility to myrosinase) with emphasis on the use of NMR for structure elucidation of even minor GSLs and GSL hydrolysis products. The example includes identification of a novel GSL, (R)-2-hydroxy-2-(3-hydroxyphenyl)ethylglucosinolate.Recent investigations of GSL evolution, based on investigations of species with well established phylogeny, are reviewed. From the relatively few such investigations, it is already clear that GSL profiles are regularly subject to evolution. This result is compatible with natural selection for specific GSL side chains. The probable existence of structure-specific GSL catabolism in intact plants suggests that biochemical evolution of GSLs has more complex implications than the mere liberation of a different hydrolysis product upon tissue disruption.     

Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice

Glycosphingolipids (GSLs) have been implicated as potential atherogenic lipids. Studies in apolipoprotein E-null (apoE(-/-)) mice indicate that exacerbated tissue GSL accumulation resulting from alpha-galactosidase deficiency promotes atherosclerosis, whereas the serine palmitoyl transferase inhibitor myriocin (which reduces plasma and tissue levels of several sphingolipids, including sphingomyelin, ceramide, sphingosine-1-phosphate, and GSLs) inhibits atherosclerosis. It is not clear whether GSL synthesis inhibition per se has an impact on atherosclerosis. To address this issue, apoE(-/-) mice maintained on a high-fat diet were treated with a potent glucosylceramide synthesis inhibitor, d-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4), 10 mg/kg/day for 94 days, and lesion development was compared in mice that were treated with vehicle only. EtDO-P4 reduced plasma GSL concentration by approximately 50% but did not affect cholesterol or triglyceride levels. Assessment of atherosclerotic lesions at four different sites indicated that EtDO-P4 had no significant impact on lesion area. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, and the in vitro evidence implying that GSLs may be pro-atherogenic, our current data indicate that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo.     

Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis

The cancer-preventive activity of cruciferous vegetables is commonly attributed to isothiocyanates resulting from the breakdown of the natural products glucosinolates (GSLs). Sulforaphane, the isothiocyanate derived from 4-methylsulfinylbutyl GSL, is thought to be the major agent conferring cancer-preventive properties, whereas the isothiocyanate of 4-methylthiobutyl GSL does not have the same activity. We report the identification of an Arabidopsis flavin-monooxygenase (FMO) enzyme, FMO(GS-OX1), which catalyzes the conversion of methylthioalkyl GSLs into methylsulfinylalkyl GSLs. This is evidenced by biochemical characterization of the recombinant protein, and analyses of the GSL content in FMO(GS-OX1) overexpression lines and an FMO(GS-OX1) knock-out mutant of Arabidopsis. The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs, with an approximately fivefold increase in 4-methylsulfinylbutyl GSL in seeds. Identification of FMO(GS-OX1) provides a molecular tool for breeding of Brassica vegetable crops with increased levels of this important GSL, which has implications for production of functional foods enriched with the cancer-preventive sulforaphane.     

Novel bioresources for studies of Brassica oleracea: identification of a kale MYB transcription factor responsible for glucosinolate production

Plants belonging to the Brassicaceae family exhibit species‐specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health–promoting properties. Among them, glucoraphanin (aliphatic 4‐methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full‐length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild‐type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale.     

Expression of glycosphingolipids in lymph nodes of mice lacking TNF receptor 1: biochemical and flow cytometry analysis

The expression of gangliosides and neutral glycosphingolipids (GSLs) in the lymph nodes of mice lacking the gene for the tumour necrosis factor-alpha receptor p55 (TNFR1) has been investigated. GSL expression in the tissues of mice homozygous (TNFR1-/-) or heterozygous (TNFR1+/-) for the gene deletion was analysed by flow cytometry and high-performance thin-layer chromatography (HPTLC) followed by immunostaining with specific antibodies. HPTLC immunostaining revealed that lymph nodes from TNFR1-/- mice had reduced expression of ganglioside GM1b and GalNAc-GM1b, neolacto-series gangliosides, as well as the globo- (Gb3, Gb4 and Gb5) and ganglio-series (Gg3 and Gg4) neutral GSLs. Flow cytometry of freshly isolated lymph node cells showed no significant differences in GSL expression, except for the GalNAc-GM1b ganglioside, which was less abundant on T lymphocytes from TNFR1-/- lymph nodes. In TNFR1-/- mice, GalNAc-GM1b+/CD4+ T cells were twofold less abundant (3.8% vs 7.6% in the control mice), whereas GalNAc-GM1b+/CD8+ T cells were fourfold less abundant (5.0% vs 20.2% in the control mice). This study provides in vivo evidence that TNF signalling via the TNFR1 is important for the activation of GM1b-type ganglioside biosynthetic pathway in CD8 T lymphocytes, suggesting its possible role in the effector T lymphocyte function.     

A rapid preparative method for isolation of neutral and acidic glycosphingolipids by radial thin-layer chromatography

An efficient method to separate neutral and acidic glycosphingolipids (GSLs) from their mixtures within a short period (45-60 min) and with low consumption of solvents (chloroform-methanol-water, 60/35/8 (v/v/v); 250-500 ml) has been developed. This method utilizes a centrifugal thin-layer chromatograph (Chromatotron) and the GSL mixtures (30-400 mg) are applied to glass plates coated with a 1-mm layer of silica gel 60 PF-254. The method (radial thin-layer chromatography) is rapid and simple and the recovery of glycosphingolipids is high (70-80%).     

Glycosphingolipid composition of a new immortalized human cerebromicrovascular endothelial cell line

Previous studies have demonstrated the involvement of glycosphingolipid (GSL) antigens in the pathogenesis of immune-mediated neurological disorders such as peripheral neuropathies and multiple sclerosis. To study the role of the blood-brain barrier (BBB) in these disorders, we used a new human cerebromicrovascular endothelial cell (HCEC) line that has been immortalized through transfection with the plasmid pSV3-neo encoding for the SV40 large T-antigen and the neomycin gene. The immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates but maintained phenotypic properties of early-passage control cells. Therefore, this human cell line may serve as a useful in vitro model for studying the properties of the human BBB. We first investigated the GSL composition of cultured SV-HCECs. The major gangliosides were GM3 (62% of total gangliosides), GM2 (18%), GM1 (3%), and GD1a (15%). The major neutral GSLs were glucosylceramide (15% of the total neutral glycolipids), lactosylceramide (36%), globotriaosylceramide (3%), and globoside (43%). Trace amounts of paragloboside, lactosaminyl paragloboside, and sulfoglucuronyl paragloboside could also be detected by TLC-immunostaining. These results provide the basis for further investigations of the expression of these cell surface antigens in cultured SV-HCECs on activation with inflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma, which have been implicated as playing an important role in the pathogenesis of many nervous system disorders.     

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.