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Glycolipids were isolated from primary human lung carcinoma tissue of various histologic types: adenocarcinoma, squamous cell carcinoma, and undifferentiated small cell carcinoma. Each type of carcinoma had a characteristic glycolipid pattern. The major glycolipids isolated were ceramide monohexosides, ceramide dihexosides, ceramide trihexosides, globoside, and hematoside. Squamous cell carcinoma and undifferentiated small cell carcinoma showed marked increases of ceramide monohexosides and dihexosides. Adenocarcinoma had a much higher level of the sulfatide (ceramide 3-sulfate-galactoside) as compared to squamous cell carcinoma, undifferentiated small cell carcinoma, or normal lung tissue. Embryonic tissue had more significant levels of sulfatide than did the other carcinomas. Adenocarcinoma had significantly lower levels of glycolipids due mainly to a decrease in the amount of ceramide monohexosides and dihexosides and hematoside.

Infection with verotoxin producing Escherichia coli has been strongly implicated in the etiology of the hemolytic uremic syndrome (HUS). We have previously shown that this toxin specifically binds to a glycolipid receptor-globotriosyl ceramide (Gb3). We have therefore quantitated the level of this glycolipid by HPLC in human renal cortex and medulla as a function of age. We have also measured the binding of verotoxin to Gb3 isolated from each renal tissue sample. Gb3 was a major component of the glycolipid fraction of all renal samples analyzed. The levels were found to be higher in the cortex than medulla, correlating with the clinical incidence of renal lesions in HUS, but reduced in the kidneys of infants as compared to adults. Verotoxin binding was directly proportional to the renal Gb3 content. Thus, human renal tissue is a rich source of the verotoxin receptor glycolipid. However, changes in receptor concentration cannot explain the age-related incidence of HUS.

T cells treated with cancer sera frequently suppressed immunoglobulin production by autologous lymphocytes stimulated with pokeweed mitogen. Sera from healthy individuals did not induce suppressor activity. This suppression is not caused by TG cells (T cells bearing receptors for the Fc portion of immunoglobulin G [IgG]) interacting with immune complexes, because we used T cells depleted of TG cells. The suppressor-inducing factors were separated into an IgG-containing fraction and a fraction with a smaller molecular weight. IgG fractions from all sera that were positive for anti-globoside antibody induced suppressor T cells, and elimination of the anti-globoside antibody from these IgG fractions reduced the ability to induce suppressor T cells. T cells treated with rabbit anti-globoside antiserum also activated suppressor T cells. These observations indicate that the antibodies directed to the globoside antigen on suppressor T cells stimulate the suppressor T cells, and that anti-globoside antibody in cancer sera may play a role in causing immunodeficiency in cancer patients.

The antigenic structures associated with the functional alleles of the human ABO, H, Lewis and P blood group genetic systems are carbohydrate and hence are secondary products of the blood group genes. The carbohydrate structures that confer A, B, H and Lewis specificities are part of glycosphingolipid molecules on cell surfaces and of glycoprotein molecules in secretions. The carbohydrate chains of the glycolipids or glycoproteins are built up by the sequential action of a series of glycosyltransferase enzymes determined by genes at several different loci. The genes at the ABO, H and Lewis loci are concerned only with the final stages of the biosynthesis of the carbohydrate structures; the functional alleles each control the formation of a single glycosyltransferase. The enzymic product of the H gene is an α-2-L-fucosyltransferase that conveys the sugar L-fucose to terminal β-D-galactosyl residues. The H-active structures thus formed constitute acceptors for the sugar N -acetyl-D-galactosamine, added by the A -gene specified enzyme or D-galactose, added by the B -gene specified enzyme. The regulatory gene Se controls the activity of the H gene in cells synthesizing the secreted glycoproteins and other regulatory genes modify the expression of the A, B and H genes in the cells synthesizing the glycolipids. The functional allele at the Lewis locus, Le , controls the formation of an α-4-L-fucosyltransferase, that conveys L-fucose to subterminal N -acetylglucosamine residues. If the adjacent terminal β-galactosyl residue is unsubstituted the structure formed has Le a specificity; if the β-galactosyl unit is substituted with α-2-linked fucose the structure has Le b specificity. The genetics of the P blood group system are less well understood than those of the ABO and Lewis systems. A glycoprotein isolated from hydatid cyst fluid provided a convenient source of P 1ˉ and P k -active substance for immunochemical studies and allowed the structure of the P 1 determinant to be established. P 1 , P k and P antigens have now been isolated from red cells and found to be glycosphingolipids. The antigen, P k , ceramide trihexoside, is the precursor of the P antigen, globoside, but the precise genetic and biosynthetic relation of the P 1 specific structure to P and P k determinants has still to be clarified.

We demonstrate that human fibroblasts of the rare Pk phenotype lack globoside, which was identified as the blood group P antigen, and that p cells possess neither globoside nor trihexosyl ceramide, which was identified as Pk antigen. Our investigations indicate also that these glycosphingolipid patterns are most likely caused by inherited preferential biosynthetic pathways in the abnormal phenotypes rather than by excess catabolism of the antigens. Evidence is presented that the fibroblasts of Pk phenotype lack beta-N-acetylgalactosaminyltransferase (globoside synthetase; UDP-N-acetylgalactosamine:trihexosylceramide beta-N-acetylgalactosaminyltransferase; EC 2.4.1.79) activity, and those of p are deficient in alpha-galactosyltransferase (trihexosylceramide synthetase; UDP galactose:lactosylceramide alpha-galactosyltransferase) and possibly also in globoside synthetase. The diminished globoside synthetase activity in p cells, however, is not caused by the defect in the gene coding for this enzyme. It appears, rather, to be caused by a failure in gene expression because one-third of Pk X p hybrids became able to express P antigenicity with a time lag of 3-4 days after cell fusion [Fellous, M., Gerbal, A., Nobillot, G. & Weils, J. (1977) Vox Sang. 32, 262-268].

Twenty-four years after the first discovery in 1900 of the blood group antigens on erythrocytes by Landsteiner and others (1–3) several researchers successfully extracted small amounts of ABH substances from red cell membranes using 95% ethanol (4–7) as an elutrient. These findings that ethanol could extract some ABH substance were confirmed by others in subsequent years leading to the idea that lipids carried the ABH antigens on erythrocytes. The main proponents of this idea were Masamune and his students Hirata, Tokura, and Satoh (8–10) in the 1950’s. Yamakawa, a pioneer in this area, also isolated at the same time a “globoside” fraction containing ABH activity from erythrocyte stroma using methanol-ether (11, 12). Modern knowledge suggests that these substances were probably the shorter, 5–20 sugar lactosaminyl ceramides having terminal ABH groups. Masamune’s student, Hakomori, has performed the most careful and detailed work on the latter class of lipids and has developed methods for their purification and characterization (13).

Uropathogenic Escherichia coli frequently express globoside-specific adhesins, shown to mediate binding to uroepithelial cells. For one gene cluster pap, it recently has been demonstrated that globoside binding is not dependent on expression of the pilus subunit gene papA. Instead, two other pap genes papF and papG are specifically required for globoside binding (F. P. Lindberg et al., EMBO J. 3:1167-1173, 1984). By restriction enzyme mapping, DNA hybridization, DNA sequencing, and protein expression in minicells, we show that three gene clusters encoding globoside binding have a very similar structure and gene organization, although they were cloned from different E. coli isolates. Major differences between the adhesin clones were restricted to the central part of the pilin gene (papA) and to one of the two adhesin gene (papG). The three functional units required for biogenesis of globoside-binding pili, i.e., pilin synthesis, pilin export, and pilin assembly, as well as expression of adhesion function, were all trans complementable among the gene clusters.

A glycolipid was isolated from normal lung and lung carcinoma tissues. This glycolipid was identified as the Forssman antigen of pentaglycosyl ceramide by means of chemical and immunological methods. The presence of this antigenic glycolipid was observed in all the tissues examined of adult and embryo lungs, and of lung tumors irrespective of histological type. The extracts of human lung and lung tumors were capable of catalyzing the synthesis of Forssman antigen from globoside.

Equine renal glycopshingolipids were composed of galactocerebroside, glucocerbroside, ceramide dihexoside, ceramide trihexoside, sulfatide, globoside I, Forssman globoside, and hematoside. Free ceramide and sphingomyelin were also found in equine kidney. Their long chain bases consisted of sphingosine, dihydrosphingosine, C18-phytosphingosine, and C20-phytosphingosine, whereas the fatty acids were separated into two groups: nonhydroxy and hydroxy fatty acids. Ceramide monohexoside was separated into five spots by TLC on borax-impregnated plates. The major component of ceramide monohexoside was glucocerbroside which accounted for 46.6% of the total ceramide monohexoside and contained a ceramide consisting of phytosphingosines and hydroxy fatty acids. The long chain bases of hematoside and sulfatide contained dihydroxy and trihydroxy bases in nearly equal ratios. On the other hand, the other glycosphingolipids contained mainly dihydroxy bases, though with significant amounts of trihydroxy bases. Free ceramides were separated into four groups by silicic acid column chromatography and the major ceramides were of two kinds, consisting of dihydroxy bases and nonhydroxy fatty acids (49.9% of the total ceramide) and of trihydroxy bases and nonhydroxy fatty acids (38.5% of the total ceramide). The minor ceramides contained predominantly hydroxy fatty acids. Neither trihydroxy bases nor hydroxy fatty acids were detected in spingomyelin.

UDP-N-acetylgalactosamine:globoside alpha-N-acetylgalactosaminyltransferase (EC 2.4.1.-) synthesizing Forssman hapten was solubilized from dog spleen microsomes by a combination of Triton X-100 treatment and sonication. The solubilized enzyme was partially purified by calcium phosphate gel, ammonium sulfate fractionation and then DEAE-cellulose column chromatography. The enzymatic activity of the purified preparation was stimulated by exogenously added phosphatidylserine, as found in the particulate enzyme. When the properties of the purified enzyme were examined in the presence of exogenous phosphatidylserine, the enzyme had an absolute requirement for Mn2+; this was not substituted by Ca2+ or Mg2+. Apparent Km values for UDP-N-acetylgalactosamine and globoside were 1-10(-5) and 5-10(-4) M, respectively. It had a pH optimum of 6.55 regardless of the presence or absence of exogenous lipids. Since the partially purified enzyme was completely free of uridine diphosphatase which was found in the particulate preparaton, the effect of UDP on the transferase activity could be studied. Thus, UDP inhibited 85% of the activity at a concentration of 1.5 mM. p-Cholormercuribenzoate inhibited over 90% of the activity at 2 mM, indicating the transferase to be SH-enzyme.

Summary A healthy 22-year-old woman was noted to have erythrocytes of the Pk phenotype: a strong Pk antigen, no detectable P antigen and anti-P antibody in her serum. Her erythrocytes contained four to six times as much Pk glycolipid (globotriaosylceramide or CTH) and approximately half as much P glycolipid (globotertraosylceramide or globoside) as normal red cells. The structures of CTH and globoside were characterized by analysis of permethylated sugars and complement fixation in addition to chromaographic mobility and sugar composition. Inasmuch as the erythrocytes of two Pk individuals that were analysed previously (Marcus et al., 1976) contained no detectable globoside, these abnormalities appear o represent a new phenotype in the P blood group system.

The carbohydrate portion of mammalian glycosphingolipids varies in the number of glycose residues, their composition, and the sequence, linkages and anomeric configuration between the sugars. This structural diversity makes possible a very large number of substances; this and also evidence for the occurrence of closely related isomers with different immunological specificities suggests an urgent need for additional fractionation procedures and methods of structural analysis. Such techniques as high performance liquid chromatography, and field desorption and chemical ionization mass spectrometry are reviewed and the results of studies using fuco-pentaose I and glucosylceramide as model compounds are presented. The 90.5 MHz, proton decoupled, natural abundance carbon-13 nuclear magnetic resonance spectra have been obtained for glucosylceramide, lactosylceramide, globotriglycosylceramide and globoside. Single lines were observed for most of the sugar, sphingosine and fatty acyl carbon components. A comparison of their resonances with those of simpler model compounds and other glycolipids allowed the determination of the sugar anomeric configuration, the intersugar linkage position and the configuration of the sugar ring structure. These studies also permitted the estimation of fatty acyl composition in terms of degree of unsaturation and chain length from chemical shift assignments and integration values of specific nuclei. Preliminary results of studies involving the uptake and conversion of 1-13C-mannose into virally transformed hamster (NIL-HSV) cell metabolites are also discussed.

The glycosphingolipids (GSLs) of two sublines of Madin-Darby canine kidney (MDCK) cells, an epithelial cell line, were characterized by t.l.c., antibody overlay and mass spectrometry. The major characteristic which distinguishes the two MDCK cell strains is their trans-epithelial electrical resistance which is typically of the order of 3000 ohm.cm2 for strain I and 100 ohm.cm2 for strain II cells. Strain I and II cells were equally rich in glycolipids, the cellular GSL/phospholipid ratio being 0.04. However, while the phospholipid patterns were identical, the GSLs showed striking differences, and each cell strain expressed appreciable amounts of GSLs that were not found in the other strain. Both cell types possessed neutral GSLs with one, two or three carbohydrate moieties. The monoglycosylceramide accounted for 50% of the total GSLs in each strain. However, while in strain I cells over 90% of this monoglycosylceramide was monoglucosylceramide, in strain II cells over 90% consisted of monogalactosylceramide. In addition, MDCK strain II cells selectively expressed GSLs belonging to the globo series (26% of its neutral GSLs), including globoside and Forssman antigen, a globoside derivative. MDCK strain I cells, on the other hand, expressed another series of GSLs with 4-7 carbohydrate moieties characterized by the common sequence Hex-HexNAc-Hex-Hex-Cer. The presence of two fucosylated GSLs in these series was established. Both MDCK strain I and II cells contained negatively charged GSLs, the major component of which was the ganglioside GM3. MDCK strain II cells in addition expressed sulfatide, the sulfated derivative of galactosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)

Several novel glycosphingolipids have been isolated from the human teratocarcinoma cell line HT-E (833K). These cells contain two neutral glycolipids which metabolically incorporate radio-labelled fucose. In addition, there are three new gangliosides present, which are all members of the globoside series of glycolipids. One of the fucose containing glycolipids forms globoside when treated with α-fucosidase, and one of the gangliosides forms globoside when treated with neuraminidase. On the basis of chromatographic behavior, exoglycosidase treatment and antibody reactivity, the tentative structures of these new glycolipids are: Fuc (α1−?) GalNAc (βl−3) Gal (α1−4) Gal (β1−4) Glc (β1- Cer ) and NeuAc (α2−3) GalNAc (β1−3) Gal (α1−4) Gal (β7−4) Gle (β1− Cer )

Two Escherichia coli cytotoxins (verotoxins 1 and 2) have been previously implicated in the cytopathology of the Hemolytic Uremic Syndrome. We have examined the glycolipid binding specificity of verotoxin (VT)2. This toxin specifically binds to globotriosyl ceramide (galactose alpha 1-4 galactose beta 1-4 glucosyl ceramide). Removal, or substitution of the terminal a galactose residue with N-acetyl galactosamine in beta 1-3 linkage, deletes binding activity. The toxin does not recognize similar terminal a galactose residues on a glycoglycerolipid. Thus the binding specificity of VT2 is the same as previously reported for VT1. Liposomes containing globotriosyl ceramide are able to specifically remove VT1 and VT2 cytotoxicity and cell lines selected in vitro for resistance to VT1 are cross resistant to VT2.

The mechanism for the synthesis of Forssman glycolipid (GalNAc-alpha-GalNAc-beta-Gal-Gal-Glc-Cer) from trihexosylceramide (Gal-Gal-Glc-Cer, Hex3Cer) and radioactively labeled UDP-N-acetylgalactosamine catalyzed by particulate beta- and alpha-N-acetylgalactosaminyl-transferase of hamster NIL-2K cells was studied. The radioactivity incorporated into the Forssman glycolipid using a mixture of Hex3Cer and globoside (GalNAc-beta-Gal-Gal-Glc-Cer) as the substrates was greater than that from each substrate singly. When the radioactive Forssman glycolipid from the mixed substrates was hydrolyzed with alpha-N-acetylgalactosaminidase, the ratio of 14C-labeled globoside to the liberated N-acetyl[14C]galactosamine was 0.38, indicating that Hex3Cer could serve as a substrate. From these results, the following hypothesis (baton pass model) is proposed. There exists an enzyme complex consisting of beta- and alpha-transferases in membranes. In this complex, it is not possible to saturate alpha-transferase with exogenous globoside; however, it can utilize a transient globoside synthesized from Hex3Cer. It thus appears that Forssman glycolipid is in part formed from Hex3Cer through this enzyme complex without the accumulation of the intermediate globoside. The abbreviations used are: GalNAc, N-acetylgalactosamine; Cer, ceramide.

Summary Liposomes prepared from total lipid extracts of human and rabbit erythrocyte ghosts were tested for agglutinability with lectins. Soya bean lectin aggregated liposomes prepared with human or rabbit lipids in keeping with the fact that galactose is the predominant sugar in glycolipids. Wheat germ lectin agglutinates liposomes prepared from rabbit lipids but not human lipids. The internal position of acetylglucosamine in the major globoside of rabbit erythrocytes would indicate that wheat germ lectin can interact with a non-terminal sugar. Concanavalin A did not agglutinate either liposomes from human or rabbit lipids. We conclude that glycolipids form an additional class of lectin receptors on membranes.

The human erythrocyte P blood group system consists of three known antigens, P1, P and Pk. We have identified the P antigen as the glycosphingo-lipid globoside, βGalNAc(1→3)αGal(1→4)βGal(1→4)Glc-cer, and the Pk antigen as ceramide trihexoside, αGal(1→4)βGal(1→4)Glc-cer. These data suggest, in contrast to previous hypotheses, that the Pk antigen is a biosynthetic precursor of P, and that neither P nor Pk is a precursor of P1. These findings also provide an explanation for the apparent recessive inheritance of the Pk antigen, and for the nature of the biochemical abnormality in individuals of the rare Pk and p phenotypes.

The activities of the two enzymes (UDP-acetylgalactosamine-globoside alpha-N-acetylgalactosaminyltransferase and alpha-N-acetyl-D-galactosaminidase) involved in the synthesis and degradation of Forssman antigen were studied in uninvolved and neoplastic human lungs. The Forssman synthetic enzyme activities of 17 of 18 squamous cell carcinomas were higher than those of the uninvolved lung tissues of the subjects studied, and the degradation enzyme activities of 16 of 18 squamous cell carcinomas were higher than those of the uninvolved portions. No consistent abnormalities in both enzyme activities were seen in 28 adenocarcinomas, whereas the mean activities of the two enzymes were elevated in these neoplasms. These differences in enzyme activities between those samples may indicate that the synthesis and degradation of Forssman antigen in adenocarcinoma of the lung are expressed or repressed according to the individual, whereas in squamous cell carcinoma, these activities are expressed unrelated to the individual.

THE nervous system has a unique distribution of glycolipids1, which changes markedly during growth of the animals2 and in various inherited metabolic disorders3. No physiological roles for glycolipids in neural function are known, however. But, in nonneural cells glycolipids of the cell membrane are essential for cell recognition and interaction4. For example, some gangliosides act as receptors for various bacterial toxins5 and globoside has been identified as the human P blood type antigen6. When administered in the medium, ganglioside can be incorporated into cell membranes and function as toxin receptors5,7. Ganglioside added to the medium competes with membrane receptors by binding to the toxins5. We reported previously8,9 that the effects of some drugs and sera on the formation of neuromuscular junction in culture could be evaluated by recording the endplate potential (e.p.p.) from muscle cells combined with the spinal cord. We describe here the effect of glycolipids on the development of neuromuscular junction in culture of skeletal muscle and the spinal cord. Globoside had biphasic effects, an inhibitory effect at the higher concentration and a facilitatory one at the lower concentration or on brief exposure at the beginning of the cultivation. Our evidence suggests that glycolipids are involved in formation of the neuromuscular junction.

In a previous paper, we reported the presence of globoside as a major neutral glycolipid in PC12 pheochromocytoma cells [Ariga, T., Macala, L. J., Saito, M., Margolis, R. K., Greene, L. A., Margolis, R. U., & Yu, R. K. (1988) Biochemistry 27, 52-58]. Recently, we found that subcloned PC12h cells accumulated another unusual neutral glycolipid. In order to characterize this glycolipid, PC12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated two major neutral glycolipids, which were purified by Iatrobeads column and preparative thin-layer chromatographies. One of the glycolipids was found to be globoside, and the other had a globotriaosyl structure with an additional terminal Gal alpha 1-3 residue. Its structure was determined by fast atom bombardment mass spectrometry, two-dimensional proton nuclear magnetic resonance spectrometry (2D NMR), permethylation study, sequential degradation with exoglycosidase, and mild acid hydrolysis to be Gal(alpha 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1')Cer.

The plasma membranes from ejaculated human spermatozoa were removed by nitrogen cavitation (600 PSI for 10 min) and isolated by centrifugation followed by a discontinuous sucrose density gradient centrifugation. Glycolipid analysis of the plasma membrane revealed a three-fold enrichment in gangliosides: GM3 and GD1a/GD1b and neutral glycolipids: globoside and sulfatide as compared to that of whole human sperm. Two dimensional electrophoresis of human sperm plasma membranes revealed about 75 polypeptides. Several of these polypeptides were similar in migration and in display of shape and color to that found in boar sperm plasma membranes.

Specific immune damage to liposomes containing Forssman or globoside glycolipid was inhibited when the liposomes also contained ganglioside. The activity of a human monoclonal Waldenstrom macroglobulin antibody to Forssman glycolipid was inhibited by each of three gangliosides tested, G M3 , G D1a and G D1b . Inhibition of the monoclonal antibody was dependent on the amount of ganglioside in the liposomes, and was diminished by reducing the relative amount of ganglioside. Inhibition also correlated positively with the number of ganglioside sialic acid groups, with inhibition by G T1b > G D1a > G M3 . Naturally occurring human antibodies to globoside glycolipid were detected in 18% (9 out of 50) of normal human sera tested. Immune damage to liposomes induced by each of the three highest-reacting human anti-globoside sera was blocked by liposomal G M3 . We conclude that gangliosides can strongly influence immune damage to membranes induced by antibody interactions with adjacent neutral glycolipids.

The type 2 fimbrial lectin of Actinomyces naeslundii WVU45 mediated the binding of this bacterium to glycosphingolipids chromatographed on thin-layer silica gel plates. Radioiodinated bacteria attached to GM1, GD1b, and globoside. After chromatograms were treated with sialidase, the bacteria also bound to GD1a and GT1b. The actinomyces lectin apparently recognized the Gal beta 3GalNAc termini of these gangliosides and the GalNAc beta 3Gal terminus of globoside, suggesting that glycolipids containing these sequences may serve as receptors for A. naeslundii on mammalian cells.

The analysis of two‐dimensional proton shift‐correlated 500 MHz NMR spectra of several peracetylated glycosphingolipids confirmed that peracetylated oligosaccharides are particularly well suited for the identification of the constituent sugar residues and the elucidation of the sites of glycosidic linkage. Although a number of partial sequences could be determined by inter‐residue nuclear Overhauser effect or long‐range scalar coupling across the glycosidic bonds, the full sequence determination of larger oligosaccharides has several limitations owing to experimental difficulties and possible ambiguities in assignments due to signal overlap. The following peracetylated glycosphingolipids were investigated by two‐dimensional methods at 500 MHz: Galα(1→4)Galβ(1→4)Glcβ(1→1)Cer (Cer = ceramide), GalNAcβ(1→3)Galα(1→4)Galβ(1→4)Glcβ(1→1) Cer (globoside) and Galα(1→3)Galβ(1→4)GlcNAcβ(1→3)Galα(1→3)Galβ(1→4)GlcNAcβ(1→6)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glcβ(1→1)Cer. To compare the potential of one‐ and two‐dimensional methods, the spectra of peracetylated Galβ(1→4)Glcβ(1→1)Cer, Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glcβ(1→1)Cer. To compare the potential of one‐ and two‐dimentional methods, the spectra of peracetylted Galβ(1→4) Glcβ(1→1)Cer were assigned with the aid of spin‐decoupling difference spectroscopy at 360 MHz.

To further define the molecules that may mediate spontaneous abortion due to maternal-fetal blood group incompatibility within the P blood group system, we have examined the fine specificities of maternal antibodies and the glycolipid antigens from the placenta of a P infant born to a Pk1 mother. Maternal antibodies obtained during therapeutic plasmapheresis were analyzed to determine their reactivities with placental glycolipid extracts on thin-layer plates. Second antibodies specific for IgM, IgG, and IgA revealed immunoglobulins of all of these classes strongly reactive with one major placental glycolipid that comigrates with globoside. GC/MS analysis confirmed that the major P-active pentaglycosylceramide of placenta has the same structure as that previously shown for the P antigen of red blood cells: GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. Serum antibodies partially purified by affinity chromatography on globoside-octyl-Sepharose specifically recognize glycolipids that contain terminal GalNAc beta 1-3Gal . . . residues and also recognize the same sequence as an internal determinant in some, but not all, glycolipids with extended globoside core regions. Thus, in the blood group P incompatible fetus, the major P antigen present in placenta has the same carbohydrate structure as the P antigen present in fetal and adult erythrocytes and might be a target for the maternal immune system.

In his review on sphingolipidoses (1) which appeared in the New England Journal of Medicine in 1966, R.O. Brady predicted that a sphingolipodystrophy remains to be discovered where abnormal quantities of the entire globoside molecule might accumulate because of attenuation of a hexosaminidase that catalyses the hydrolysis of the terminal N-acetylgalactosamine.

The chemical characterization of certain phospholipids and glycolipids of the pig erythrocyte is reported. The main phospholipids under investigation were phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and sphingomyelin, with lesser amounts of phosphatidyl inositol. Perhaps the most unique characteristics of the phosphoglycerides were the lack of any vinyl ether or glyceryl ether groups and the occurrence of significant concentrations of phosphatidyl serine (containing only C18 fatty acids). The major glycolipids present in these erythrocytes were of the globoside (“tetraglycosyl” ceramide) type, with smaller amounts of other glycosyl ceramides and traces of gangliosides. Two major forms of the globosides, labeled glycolipid C and glycolipid D, were isolated and identified. Both C and D were of the same basic structure, i.e.N-acetyl-galactosaminyl(→)galactosyl(→)galactosyl(→)glucosyl(→)ceramide, with the major difference being that C contained normal straight chain fatty acids whereas D contained only monohydroxy fatty acids. The composition of these fatty acids from these two glycolipids, as related to chain length and unsaturation, were comparable and contained mainly 22:0, 24:0 and 24:1 types. On the basis of periodate-permanganate cleavage results, it was concluded that the double bond in the 24h:1 acid was located at the 8:9 position (from carboxyl end). Unequivocal proof is offered that the hydroxy function was located at the 2 position of the fatty acids. Other glycolipids containing both hydroxy and normal fatty acids were detected in these erythrocyte lipids.

A glycolipid hapten of the Forssman antigen of horse spleen was found to be a ceramide pentasaccharide, which contained 2 moles of N-acetylgalactosamine, 2 moles of galactose, and 1 mole of glucose per ceramide. An α-N-acetylgalactosaminidase of hog liver (Weissman, B., and Hinrichsen, D. F., Biochemistry, 8, 2034 (1969)) hydrolyzed the terminal α-N-acetylgalactosaminosyl residue with a simultaneous loss of the Forssman hapten activity. The resulting compound was identical with globoside (Yamakawa, T., Nishimura, S., and Kamimura, H., Jap. J. Exp. Med., 35, 201 (1965); Hakomori, S., Siddiqui, B., Li, Y. T., Li, S. C., and Hellerqvist, C. G., J. Biol. Chem. 246, 2271 (1971)) and gave a specific precipitin reaction with anti-globoside antiserum. The oligosaccharide released from the Forssman hapten gave an intense Morgan-Elson reaction and 1 of the 2 moles of galactosamine was periodate resistant. With these results and those of methylation studies, the structure of Forssman hapten was proposed as N-acetylgalactosaminosyl-α-(1 → 3) N-acetylgalactosaminosyl-β-(1 → 3)galactopyranosyl-α-(1 → 4)galactopyranosyl-β-(1 → 4)glucopyranosyl-(1 → 1)ceramide.

The injection of phenylhydrazine, zymosan, and human erythrocyte stroma caused an increase in the specific activities of various hydrolytic enzymes in rat spleen and liver tissues. The activity of spleen glucocerebrosidase and sphingomyelinase was particularly augmented by the injection of phenylhydrazine whereas the increases obtained with zymosan were not statistically significant. The injection of red-cell stroma brought about a significant increase in the activity of these enzymes; the injection of lipid-extracted stroma had no effect. The injection of partially purified stromal sphingolipids consisting mainly of globoside and sphingomyelin caused an increase in the activity of glucocerebrosidase and sphingomyelinase in liver but not in spleen.

Globoside added to culture medium was taken up by NIL cells and accumulated as a component of plasma membrane. This was evidenced by the recovery of 3 H-labelled globoside from plasma membrane fractions and by the higher chemical quantity of globoside found in NIL cells cultured in medium containing globoside. Concomitantly the following changes in growth behavior were manifested: a reduction in growth rate due to an extended prereplicative phase and a reduced saturation density which may result from changed adhesive properties of cells.

Thin-layer chromatographic methods are described for the separation of the following monosaccharides: glucose, galactose, glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine and neuraminic acids. Neutral solvent systems and unaltered silica gel G plates were used. The monosaccharides are separated sharply, which allows these methods to be used for the determination of these compounds. The preparation and use is discussed of seven facile spray reagents that afford further specificity for detection and evaluation of the sugars at the microgram level. The feasibility of these procedures was demonstrated with a hydrolyzate of human erythrocyte globoside.

The galactose oxidase-sodium borohydride method was used to specifically label the terminal N-acetylgalactosamine of three glycosphingolipids, Gm2-ganglioside, asialo-Gm2-ganglioside, and globoside. All of the compounds showed a minimum of 95% radiopurity, and generally more than 90% of the total radioactivity was located in the terminal galactosamine moiety. Globoside and asialo-Gm2-ganglioside were labeled to high specific activities comparable with those of the sphingolipids with a terminal galactose moiety, labeled with the same procedure. These labeled compounds were well suited as substrates for the study of specific sphingolipid N-acetylgalactosaminidase. Gm2-ganglioside, however, was a poor substrate for galactose oxidase, and its specific activity was only a small percentage of the others. Furthermore, because of the low specific activity of the galactosamine moiety, it was necessary to pretreat Gm2-ganglioside with unlabeled sodium borohydride to reduce the nonspecific labeling of other portions of the molecule. The use of labeled sodium borohydride of a very high specific activity may yield specifically labeled Gm2-ganglioside suitable for metabolic studies. Thus, the method is useful for labeling not only terminal galactose but also terminal N-acetylgalactosamine of glycosphingolipids.

By a double-diffusion precipitation-in-gel technique, isolated cholera toxin as well as its natural toxoid were shown to be fixed and precipitated by the ganglioside G M1 but not by any of the related glycolipids G M3 , G M2 , G M1 -GlcNAc, G D1a , G D1b , G T1 , globoside, G A1 , and tetrahexoside-GlcNAc. Twenty-five nanograms of G M1 was enough to give a precipitation line with 1.2 μg of toxin, whereas about 50 ng was required with this amount of toxoid. G M1 also inactivated the toxin in the ileal loop as well as in the intradermal models in rabbits. A 1: 1 molar ratio of ganglioside to toxin was found limiting, e.g., 100 pg of G M1 could inactivate 5 ng (about 50 blueing doses) of isolated toxin. G M1 inactivated crude toxin (culture fil rate) with the same efficiency as isolated toxin, and the inactivating capacity of G M1 was unaffected by mixing with other gangliosides, indicating the specificity in the reaction between G M1 and toxin. The other glycolipids tested did not inactivate toxin except G D1a and G A1 which did so with approximately 1,000 times less efficiency than G M1 . This identified the portion Gal → GalNAc [Formula: see text] as the critical region in G M1 for toxin fixation, and it is postulated that this may be the tissue receptor structure for the cholera toxin.

The lipid and mucopolysaccharide storage diseases were first detected by characterizing the material which accumulated in liver or brain. In the case of the glycosphingolipidoses, these abnormal accumulations have been shown to be the result of specific inherited lysosomal hydrolase deficiencies. Normal human liver contains five major glycosphingolipid components, glucosylceramide (GL-1a), lactosylceramide (GL-2a), trihexosylceramide (GL-3), globoside (GL-4) and hematoside (GM3) (4,15) together with smaller amounts of disialohematoside (GD3) and galactosylceramide (GL-lb) (Fig. 1). Human liver also contains trace amounts of sulfatide (GL-lbS), gangliosides such as GM1, and fucoglycosphingolipids (blood group substances). Further, liver has been shown to contain most of the lysosomal glycosyl hydrolases associated with the catabolism of glycosphingolipids. Therefore, the determination of glycosphingolipid levels in this organ (which can be readily biopsied) should be useful in understanding the biochemical defects in a wide range of storage diseases.

Glycosphingolipids were isolated from the liver of four patients with globoid cell leukodystrophy and compared to those from normal liver. The ratio of glucosylceramide to galactosylceramide was 0.8, 1.2, 1.3 and 7.5 in the four cases studied, compared to a value of greater than 10 in liver from control patients of the same age and sex. In addition to this accumulation of galactosylceramide, all four livers showed an elevated level of sulfatide, and in one case trihexosylceramide and globoside were also markedly elevated.

Ceramide trihexosides and globosides were isolated from mouse Nakahara-Fukuoka sarcoma, porcine spleen, and rat kidney. The oligosaccharide portions were liberated from the glycolipids by ozonolysis and successive alkali fragmentation by Wiegandt's method. The oligosaccharides were purified by charcoal chromatography, followed by reduction with sodium borohydride. Reduced oligosaccharides were subjected to 220 MHz p.m.r. spectroscopy. The spectra of the oligosaccharides showed clearly resolved doublets for the anomeric protons, confirming the previous finding that the anomeric configuration of the terminal galactose in the ceramide trihexoside is indeed an α-configuration. Further, the structure of the globosides was studied by the above technique and with specific N-acetyl-β-hexosaminidase. From the results, the structure of rat renal and porcine splenic (cytolipin K) globosides were shown to be N-acetylgalactosaminyl (β1→3) galactosyl (α1→4) galactosyl (β1→4) glucosyl ceramide.

Antibody-complement dependent damage to liposomal model membranes has been previously investigated by measuring the release of low molecular weight markers such as glucose. To determine whether larger solutes are also released under these conditions, experiments have been performed using immunologically sensitive liposomes that contained not only trapped glucose, but also enzymes (hexokinase, glucose-6-phosphate dehydrogenase, β-galactosidase) as macromolecular markers. The largest of these enzymes (β-galactosidase) has dimensions which closely approximate the diameter of the lesions detected by negative staining in natural membranes after immune lysis. Liposomes prepared with lecithin, and either actively sensitized with globoside or passively sensitized with alkali-treated lipopolysaccharide, released the enzymes in parallel with glucose upon incubation with the appropriate antiserum and native guinea pig serum as source of complement. Immune damage to sphingomyelin liposomes was characterized by a significantly lower loss of the enzymes in comparison to the percentage of glucose released; a comparable response was manifested by liposomes prepared from sheep erythrocyte lipids. Electron microscopic examination of negatively stained lecithin liposomes, which had released the macromolecular markers, failed to reveal the characteristics lesions; these findings are consistent with evidence obtained by other laboratories suggesting that the lesions may not correspond to functional holes. Lesions were, however, consistently observed in liposome preparations that had been treated with the polyene antibiotics, filipin; this antibiotic causes appreciable loss of both glucose and enzymes from either lecithin or sphingomyelin liposomes.

Sandhoff’s disease results from the deficient activity of both N-Acetyl-β-hexosaminidase A and B (14,17,19–22) and is characterized by the neural and visceral deposition of Gm2 ganglioside, its asialo derivative (NAcgal-gal-glc-cer) and globoside (NAcgal-gal-gal-glc-cer) in affected individuals (Figure 1) (3,6,9,10,14,19–22,24,25).

The glycolipid content of human non-seminomatous germ cell tumour cell lines correlates with their differentiation lineage. To analyse whether this reflects the situation in primary tumours, we studied five embryonal carcinomas, five yolk sac tumours and nine (mixed) non-seminomas, using thin-layer chromatography and carbohydrate immunostaining. We also analysed the glycolipid content of 19 seminomas to reveal their relationship with non-seminomas. Lactosylceramide (CDH) was detected in all embryonal carcinomas, but in fewer than half of the seminomas. Seminomas and embryonal carcinomas contained globoseries glycolipids, including globotriosylceramide (Gb3), globoside (Gb4), galactosy globoside (Gb5) and sialy1 galactosyl globoside (GL7). The lacto-series glycolipid Le(x) was found in all embryonal carcinomas, but only in one seminoma. Gangliosides GD3 and GT3 were detected in many seminomas, but rarely in embryonal carcinomas. Yolk sac tumours displayed a heterogeneous glycolipid profile. Compared with seminomas and pure embryonal carcinomas, differentiated non-seminomas had reduced levels of globo-series glycolipids, especially Gb3 and Gb5, whereas CDH, Le(x), GD3 and GT3 were found in the majority of cases. Thus, the glycolipid content of non-seminoma cell lines reflects the situation in primary tumours. Globo-series glycolipids are similarly expressed in seminomas and embryonal carcinomas. The expression of Gb3 and Gb5 is reduced in non-seminomas upon differentiation. Le(x) expression in non-seminomas, including embryonal carcinomas, allows discrimination from seminomas. Expression of gangliosides in seminomas might indicate their maturation from ganglioside-negative precursor cells. Reprogramming of these precursors would result in the formation of Le(x)-expressing embryonal carcinomas. Images Figure 1 Figure 2

A UDP-N-acetylgalactosamine:globoside alpha-3-N-acetylgalactosaminyltransferase has been purified over 3500-fold in 4% yield from a Triton X-100 extract of canine spleen microsomes by affinity chromatography on globoside acid-agarose. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed two major bands with molecular weights of 66,000 and 56,000. Judging from the molecular weight of 120,000 estimated by Sephadex gel filtration in Triton X-100 and the above electrophoretic result, the enzyme presumably exists normally as a dimer. It required Mn2+ for its activity and had a pH optimum at 6.7-6.9. The enzyme catalyzes the transfer of N-acetylgalactosamine in alpha 1 leads to 3 linkage to globoside. Neither Fuc alpha 1 leads to 2Gal beta 1 leads to 4Glc nor H blood group substance nor deglycosylated mucin were acceptors. This indicates that the enzyme was distinct from both the N-acetylgalactosaminyltransferases converting the H to the A blood group substance and catalyzing synthesis of the Ser(Thr)-GalNAc linkage. Studies on substrate specificities indicate that the preferred substrates have the general structure GalNAc beta 1 leads to 3Gal-OR in which the nature of the R moiety has relatively little effect on activity. Kinetic analysis indicates UDP is a competitive inhibitor with respect to UDP-N-acetylgalactosamine and a noncompetitive inhibitor with respect to globoside. These studies demonstrate the first report of the properties of a purified enzyme catalyzing the transfer of sugar residues to glycolipids.

The glycosphingolipid (GSL) globoside (Gb4) is essential for parvovirus B19 (B19V) infection. Historically considered the cellular receptor of B19V, the role of Gb4 and its interaction with B19V are controversial. In this study, we applied artificial viral particles, genetically modified cells, and specific competitors to address the interplay between the virus and the GSL. Our findings demonstrate that Gb4 is not involved in the binding or internalization process of the virus into permissive erythroid cells, a function that corresponds to the VP1u cognate receptor. However, Gb4 is essential at a post-internalization step before the delivery of the single-stranded viral DNA into the nucleus. In susceptible erythroid Gb4 knockout cells, incoming viruses were arrested in the endosomal compartment, showing no cytoplasmic spreading of capsids as observed in Gb4-expressing cells. Hemagglutination and binding assays revealed that pH acts as a switch to modulate the affinity between the virus and the GSL. Capsids interact with Gb4 exclusively under acidic conditions and dissociate at neutral pH. Inducing a specific Gb4-mediated attachment to permissive erythroid cells by acidification of the extracellular environment led to a non-infectious uptake of the virus, indicating that low pH-mediated binding to the GSL initiates active membrane processes resulting in vesicle formation. In summary, this study provides mechanistic insight into the interaction of B19V with Gb4. The strict pH-dependent binding to the ubiquitously expressed GSL prevents the redirection of the virus to nonpermissive tissues while promoting the interaction in acidic intracellular compartments as an essential step in infectious endocytic trafficking.

In this study we compared the specificity for the globoseries of glycolipids of Escherichia coli expressing the O-negative, A-positive (ONAP) adhesin and clones transformed with the pap-like (prs or pap-2) gene cluster. Receptor-active glycolipids were identified by the ability of radiolabeled bacteria to bind to the glycolipids on thin-layer chromatogram plates. The ONAP adhesin and pap-like clones bound with high affinity to the globo-A and Forssman glycolipids. The ONAP strains did not recognize other glycolipids of the globoseries. In contrast, the pap-like clones also showed weak binding to globotriaosylceramide and reacted weakly with Gal alpha 1----4 Gal beta-latex beads. We suggest that the pap-like and ONAP adhesins recognize an epitope shared by the globo-A and Forssman structures, e.g., terminal GalNAc alpha 1----3 bound to Gal alpha 1----4Gal beta-containing glycolipids.

The exposure of several major red-cell glycolipids to galactose oxidase was studied by oxidizing the cells with the enzyme and reducing them with NaB2H4. After isolation, the deuterium label was detected by mass fragmentography. 60-70% globoside in human and porcine erythrocytes was exposed as measured by this method. In contrast, asialo-GM2 in guinea-pig erythrocytes and Forssman glycolipid in sheep erythrocytes were mainly in a cryptic state. Neuraminidase treatment increased the incorporation of deuterium label to asialo-GM2 4-8-fold. A similar effect was seen in Forssman glycolipid when sheep red cells were labeled with the neuraminidase/galactose oxidase/NaB3H4 method. In contrast, the increase in labeling was only about 10-40% in porcine and human globosides, which were efficiently exposed to galactose oxidase already in native red cells.

Plasma membrane glycolipids are localized at the outer leaflet of the lipid bilayer, and their carbohydrate portions are exposed to the environment. The efficiency of exposure has, however, not been known. We have been able to determine the availability of the major red cell glycolipid, globoside, to externally added galactose oxidase. Red cells were extensively treated with the enzyme and the oxidized cells reduced with NaBD4. After isolation the extent of exposed globoside was estimated by mass spectrometry. The results show that the exposure of globoside varies in red cells of different individuals from 37-66%. The fatty acid composition of externally available globoside was the same as that of non-oxidized globoside. The exposure was not influenced by protease treatment of intact cells and no correlation was found with different ABO blood groups.

A genetic neurodegenerative disorder with lysosomal storage of Gm2 ganglioside, its asialo derivative, and globoside, and β-hexosaminidase deficiency has been described in domestic cats (Cork et al, Science, 1977, in press). To strengthen the apparent analogy with human Gm2 gangliosidosis type II, feline β-hexosaminidase has been studied. Two major forms of this lysosomal enzyme, Hex A and Hex B, are present in organs and body fluids from normal animals, with biochemical and kinetic properties remarkably similar to those of the corresponding human enzymes. These data, and the deficiency of both components in affected cats, support the analogy between feline and human diseases. Thus it is possible to utilize this animal model for enzyme therapy experiments. Structural differences between human and feline β-hexosaminidase, however, may result in different cellular uptake, endocellular activity and stability, limiting the relevance of replacement experiments with feline enzyme. Thus, plasma clearance and organ disposition of human placental Hex A and Hex B has been studied in normal cats, utilizing the apparent absence of interspecific immunologic cross-reactivity. The results so far indicate a rapid, preferential hepatic uptake, possibly via a saturable receptor mechanism, with recovery of the exogenous enzymes in a lysosomal fraction. Further studies using human enzymes should result in enzyme replacement strategies, to be tested in diseased cats, relevant to therapeutic attempts in human Gm2 gangliosidosis.