Accumulation of glycosphingolipids in human atherosclerotic plaque and unaffected aorta tissues

We have measured the levels of glycosphingolipids and the activityof glycosphingolipid glycosyltransferases in human aortic intimaand media from patients who died of atherosclerosis. The effectsof lactosylceramide (LacCer) and glucosylceramide (GlcCer) fromplaque intima on smooth muscle cell proliferation were assessed.When the GIcCer data was expressed as (pg GlcCer/mg cholesteroland/mg total phospholipid, a 28-fold and 7-told increase inplaque intima compared to normal intima was observed. Similarly,the level of LacCer was elevated 5-fold and 4-fold, respectively,compared to unaffected intima. The activity of UDPGicCer: ceramideß1     

Glycosphingolipids of human aorta

The structures of the main gangliosides of human aorta (intima and media) were elucidated. The main component (67%) was identified as N-acetylneuraminosyl-lactosylceramide (ganglioside GM3). The aorta tissue contained also gangliosides GM1, GD3, GD1a, and GT1. All sialic acid residues in gangliosides were present as N-acetyl-neuraminosyl derivatives. Among neutral glycosphingolipids of human aorta, the main components were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The preliminary data suggest that the composition of the investigated glycosphingolipids in tissue might vary upon atherosclerosis lesions of aorta.     

The human blood fluke Schistosoma mansoni synthesizes a novel type of glycosphingolipid.

Previous studies have shown that the glycoprotein oligosaccharides synthesized by adult Schistosoma mansoni, the organism responsible for human schistosomiasis, are unusual in that they contain terminal beta-GalNAc residues and lack sialic acid. These observations and other studies indicating that schistosome glycoproteins and glycolipids are antigenic in infected animals led us to investigate the structures of the glycosphingolipids synthesized by these organisms and to determine whether they are structurally related to those synthesized by their vertebrate hosts. For our studies, adult schistosomes were metabolically radiolabeled with either [3H]galactose or [3H]glucosamine, and the newly synthesized glycosphingolipids were isolated and characterized. The major glycosphingolipids synthesized by adult schistosomes were found to be galactosylceramide and glucosylceramide. The adult worms synthesized no lactosylceramide (Gal beta 1-4Glc-ceramide), a common constituent of vertebrate cells; however, another disaccharide-containing glycosphingolipid cleavable by ceramide glycanase was found. The results of compositional and methylation analyses and exoglycosidase treatments demonstrated that this ceramide-disaccharide has the structure GalNAc beta 1-4Glc-ceramide. We also found that extracts of adult schistosomes are unable to transfer Gal from UDP-Gal to glucosylceramide, whereas extracts of Chinese hamster ovary cells, as a control, are able to do so, confirming that schistosomes are unable to synthesize lactosylceramide. Low levels of higher molecular weight glycosphingolipids were also found to be synthesized by adult schistosomes, and although their levels were too small to allow definitive characterization, compositional analyses indicated that they also contained GalNAc. We have tentatively designated the new disaccharide structure GalNAc beta 1, 4Glc- the "schistocore", which may represent a new type of glycosphingolipid core series.     

An improved technique for separation of neutral glycosphingolipids by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) procedure for separation of O-acetyl-N-p-nitrobenzoyl derivatives of six neutral glycosphingolipids: glucosylceramide, lactosylceramide, globotriaosylceramide, lactotriaosylceramide, globotetraosylceramide, and neolactotetraosylceramide. The recoveries of glucosylceramide and globotetraosylceramide for the derivatization procedure and HPLC analysis were approximately 75%, and one nanomole of glycolipid could be detected. The procedure was used for analysis of human erythrocyte neutral glycolipids.     

The glycosphingolipid composition and glycosyltransferase activities of the small intestinal mucosa of testosterone-treated rats

Prior studies have demonstrated that sex hormones can influence the glycosphingolipid composition of different organs, including small intestine. However, to date, the effects of testosterone on glycosphingolipids of rat small intestinal mucosa have not been examined. Experiments were conducted to examine the effect of subcutaneous administration of synthetic testosterone (500 micrograms/100 g body wt.) on the gangliosides and neutral glycosphingolipids of rat small intestinal mucosa. Their results demonstrated that testosterone administrations: (i) increased the ganglioside content including hematoside (GM3); (ii) increased the total content of neutral glycosphingolipids, which was due to the increases in glucosylceramide and globotriaosylceramide; (iii) increased the activities of cytidine 5'-monophosphate-N-acetylneuraminic acid: lactosylceramide sialyltransferase, and UDPgalactose: lactosylceramide galactosyltransferase; (iv) increased the percentage of the long chain base phytosphingosine in hematoside, glucosyl-, and globotriaosylceramide; and (v) significantly altered the fatty acid composition of each of these glycosphingolipids. These results demonstrate that administration of testosterone induces alterations in glycosphingolipid composition and glycosyltransferases activities in rat small intestinal mucosa.     

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.     

Glycosphingolipids from bovine milk and milk fat globule membranes: a comparative study. Adhesion to enterotoxigenic Escherichia coli strains

Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.     

Localization of neutral glycosphingolipids in human plasma.

1. The localization of the neutral glycosphingolipids glucosylceramide, lactosylceramide, trihexosylceramide and globoside in human plasma was investigated. Glycosphingolipids were isolated and analysed by gas-liquid chromatography. 2. After Sephadex gel chromatography of human plasma, about 75% of the glycosphingolipids were found in the fraction containing most of the lipoproteins. 3. After fractionation of the lipoproteins by ionic precipitation, 15-25% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction, 30-45% in the low density lipoprotein fraction and 40-50% in the high density lipoprotein fraction. 4. After fractionation of lipoproteins by density-gradient ultracentrifugation, 15% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction and 85% in the low density and high density lipoprotein fractions. No glycosphingolipids could be detected in the ultracentrifugal residue which contains the bulk of the albumin.     

Hydrazinolysis of glycosphingolipids. A new method for preparation of N-deacylated (lyso) glycosphingolipids

A useful method for N-deacylation of the ceramide moiety of glycosphingolipids has been developed. Galactosylceramide, glucosylceramide, lactosylceramide, and galactosyllactosylceramide were effectively deacylated by heating with anhydrous hydrazine at 150 degrees C for 15-25 h. The lyso-derivative as the deacylated product of the ceramide moiety of each glycosphingolipid was isolated by preparative silica gel thin layer chromatography with a 70-85% yield from the starting glycolipids. Hydrazine sulfate was an effective catalyst for the deacylation of the ceramide moiety. No dissociation of oligosaccharide moieties of the glycolipids on hydrazinolysis was confirmed by gas chromatographic analysis and N-acylation of these lysoderivatives. The free amino groups of the lysoglycosphingolipids can be combined with various kinds of probes giving useful derivatives for biochemical and immunological studies on glycosphingolipids.     

Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.     

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.     

Gangliosides and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung

Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor-associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).     

Neutral glycosphingolipids and gangliosides of human lung and lung tumours.

In order to help determine whether alterations of the profiles of glycosphingolipids occur consistently in human tumours, the neutral glycosphingolipids and gangliosides of nine lung tumours (one adenocarcinoma, four squamous cell, two mixed adeno-squamous cell, one large cell and one oat-cell carcinomata) were analysed. The control tissue consisted of adjacent lung; it contained neutral glycosphingolipids corresponding in properties to glucosyl-, lactosyl-, globotriaosyl- and globotetraosyl-ceramides. All of the tumours also contained these four neutral glycosphingolipids. However, in addition, five of the tumours (two of the squamous, the large cell and the two mixed adeno-squamous cell carcinomata) contained neutral glycosphingolipids corresponding in properties to lactotriaosyl- and neolactotetraosyl-ceramides; these same tumours also exhibited higher amounts of lactosylceramide than the other tumours analysed. Both of the two former neutral glycosphingolipids and very substantial amounts of the latter neutral glycosphingolipid were detected in pneumonic lung and in polymorphonuclear leucocytes; it thus appears possible that these particular compounds were derived from these latter cells rather than from the tumour cells. The ganglioside patterns of the tumours were almost equivalent in complexity to that exhibited by the control lung tissue. This study shows that the profiles of two major classes of glycosphingolipids (neutral glycosphingolipids and gangliosides) occurring in lung tumours are almost as complex as those of the parent tissue, a finding in contrast with the notably simplified patterns of these lipids found in many cancer cells grown in vitro. It also suggests that when lactotriaosyl- and neolactotetraosyl-ceramides and high amounts of lactosylceramide are detected in human tumours, the possibility must be considered that these compounds are derived from polymorphonuclear leucocytes.     

Neutral glycosphingolipids of serum lipoproteins in Fabry's disease.

The neutral glycosphingolipid compositions of lipoprotein fractions of serum from eight healthy male volunteers and three patients with Fabry's disease were determined. Four fractions were studied: very low density lipoprotein (VLDL, d less than 1.006); low density lipoprotein (LDL, d 1.006-1.063); high density lipoprotein (HDL, d 1.063-1.21); and ultracentrifugal residue (Residue, d less than 1.21). All lipoprotein fractions contained the four major neutral glycosphingolipids (glucosylceramide, lactosylceramide, galactosylgalactosylglucosylceramide and N-acetylgalactosaminylgalactosylgalactosylglucosylceramide). The LDL and HDL, however, accounted for most of the total glycosphingolipid (69 and 20%, respectively); only small amounts were demonstrated in the VLDL and Residue. The relative distributions of the glycosphingolipids within the LDL and HDL fractions were similar to the distribution in unfractionated serum. Galactosylgalactosylglucosylceramide levels were 3-5 times normal in all three patients with Fabry's disease, and in two the distribution of the excess lipid among the major lipoprotein fractions was similar to that in the control group. In the third patient, who exhibited the presence of "sinking pre-beta lipoprotein", the amount of glycosphingolipid isolated with the HDL was greater than that in the LDL.     

P blood group regulation of glycosphingolipid levels in human erythrocytes.

The neutral glycosphingolipid content of normal human erythrocytes was analyzed by a new method which utilizes high performance liquid chromatography. This rapid and accurate technique permits the quantitation of each of the major neutral glycolipids from individual blood samples. A correlation between the P blood group and the relative quantities of neutral glycosphingolipids is demonstrated. Erythrocytes from P1 individuals are shown to contain more globotriaosylceramide and less lactosylceramide than do erythrocytes from P2 individuals. The results of these experiments suggest the existence of a new phenotype in the P blood group system, and have further implications regarding the biosynthesis of the P blood group glycosphingolipids.     

Human gastric glycosphingolipids recognized by Helicobacter pylori vacuolating cytotoxin VacA

Many bacterial toxins utilize cell surface glycoconjugate receptors for attachment to target cells. In the present study the potential carbohydrate binding of Helicobacter pylori vacuolating cytotoxin VacA was investigated by binding to human gastric glycosphingolipids on thin-layer chromatograms. Thereby a distinct binding of the toxin to two compounds in the non-acid glycosphingolipid fraction was detected. The VacA-binding glycosphingolipids were isolated and characterized by mass spectrometry and proton NMR as galactosylceramide (Galbeta1Cer) and galabiosylceramide (Galalpha4Galbeta1Cer). Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources showed an additional recognition of glucosylceramide (Glcbeta1Cer), lactosylceramide (Galbeta4Glcbeta1Cer) and globotriaosylceramide (Galalpha4Galbeta4Glcbeta1Cer). No binding to the glycosphingolipids recognized by the VacA holotoxin was obtained with a mutant toxin with deletion of the 37 kDa fragment of VacA (P58 molecule). Collectively our data show that the VacA cytotoxin is a glycosphingolipid binding protein, where the 37 kDa moiety is required for carbohydrate recognition. The ability to bind to short chain glycosphingolipids will position the toxin close to the cell membrane, which may facilitate toxin internalization.     

Neutral Glycosphingolipids and Ceramide Composition of Ethylnitrosourea-Induced Rat Neural Tumors: Accumulation of Ceramide in Tumors

Abstract: Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20 mg/kg body weight, in the tail vein of the mother. The neutral glycosphingolipid, sulfatide, and ceramide composition of the tumors and the normal tissues from which the tumors originated is described. The content of nonhydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) containing ceramide in all the neural tumors so far examined was significantly increased compared with the corresponding normal neural tissue. Some 8 to 18 mol% of total neutral glycolipids was as ceramide in neurinomas, oligodendrogliomas, and menin-giomas. Lactosylceramide in normal neural tissues was about 1 mol% of the total neutral glycosphingolipids. In various neural tumors lactosylceramide increased up to 8 mol%. NFA- and HFA-containing cerebrosides constitute 94–100% of the neutral glycosphingolipids in normal neural tissues. In various neural tumors the mol percent of cerebrosides was significantly reduced. A high performance liquid chromatographic method was modified to analyze simultaneously ceramides, cerebrosides, and higher neutral glycosphingolipids.     

Glycosphingolipid patterns of fetal and adult small intestinal mucosa in the sheep

The ganglioside and neutral glycosphingolipid composition of fetal and adult sheep small intestinal mucosa were characterized and compared. Mono- and tetrahexosylceramide were the major neutral glycolipids of both fetal and adult tissue. Fetal mucosa, however, possessed a higher content of monohexosylceramide than its adult counterpart. Similarly, G_D1a, G_M3 and G_M2 were found to be the principal gangliosides in fetal and adult tissue. Adult intestinal mucosa possessed significant amounts of G_T1a not present in fetal tissue. Analysis of the hydroxy and nonhydroxy fatty acids as well as the long-chain bases of the major glycosphingolipids revealed differences between these lipophilic components of glycolipids in fetal and adult intestinal mucosa. The results, therefore, indicate that both quantitative and qualitative differences in glycosphingolipid composition exist between fetal and adult sheep small intestinal mucosa.     

Immunogenic properties of mannose-containing ceramide disaccharide and immunochemical detection of its hapten in the two kinds of crustacean, Euphausia superba and Macrobrachium nipponense.

Antiserum against Man beta 1-4Glc beta 1-1Ceramide (MIOse2Cer), a mannolipid isolated from spermatozoa of the fresh-water bivalve, Hyriopsis schlegellii, has been elicited in rabbits by repeated injection of a mixture of hapten-bovine serum albumin (1:1, mg/ml) with Freund's adjuvant. The specificity of the affinity-purified antibody (immunoglobulin G type) obtained from the serum was examined, using other glycosphingolipids and glyco-proteins structurally related to MIOse2Cer, by means of ELISA and TLC-immunostaining. The purified antibody was highly specific to MIOse2Cer and lacked reactivity with other glycolipids and glycoproteins including glucosylceramide, lactosylceramide, dimannosylglucosylceramide (MIOse3Cer), glucosaminylmannosylglucosylceramide (ArOse3Cer), thyroglobulin and alpha 1-acid glycoprotein. The antibody was found to bind, although less efficiently, to certain other compounds containing the group Man beta 1-4Glc and/or Man beta 1-4GlcNAc at their termini, such as MIOse2-sphingosine and Man beta 1-4GlcNAc beta 1-p-aminobenzoic acid ethylester derivatives. The present antibody was applied to the detection of the natural hapten in crustacean glycolipids. The purified antibody reacted with a neutral glycosphingolipid present in the two kinds of crustacean, Euphausia superba (antarctic krill) and Macrobrachium nipponense (fresh-water shrimp) as shown by TLC-immunostaining. The crustacean glycolipid antigen was isolated and characterized to be the Man beta 1-4Glc-Cer. This is the first report on the presence of a mannose-containing glycosphingolipid in the crustacean.