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A 62-year-old woman gave clinical manifestation of liver cirrhosis. Urinary protein was false positive, no uremia was found and renal changes were entirely overlooked. Deposition of abundant lipids (globoside and ceramide trihexoside) was found in the kidneys; essentially degenerative changes of the tubular epithelia were noted. These renal changes were compared with those in Fabry's disease.
The expression patterns of glycolipid from prostatic hyperplasia, prostatic cancer and normal prostate tissue were observed. A further analysis of antigen recognized by mouse monoclonal antibody APG1, which was gained by immunizing glycolipids extracted from human prostate cancer, was also performed. In cancer tissue, both of the lactosyl and globoside series glycolipids were found to be generally reduced, although in the ganglioside series, GM3 and GD3 were not reduced and only the glycolipids with longer chains than GD2 were found to be reduced. These results indicated that the inhibition of sugar chain elongation, but not sialylation, was the main synthetic change occurring with carcinogenesis of the human prostate. APG1 reacted with only two bands near GM2 and GD2 of the ganglioside fraction on a thin-layer chromatography plate, but it did not react with any of the known gangliosides of the ganglioside series including GM2 and GD2. Histochemically, APG1 showed intense reaction only in frozen tissue sections of human prostate, and the reactivity decreased with the increasing grade of cancer. Therefore, this antigen was considered to be a prostate-specific and differentiated antigen reacting with nonganglioseries gangliosides.
We recently showed that human skin fibroblasts internalize fluorescent analogues of the glycosphingolipids lactosylceramide and globoside almost exclusively by a clathrin-independent mechanism involving caveolae. In contrast, a sphingomyelin analogue is internalized approximately equally via clathrin-dependent and caveolar routes. Here, we further characterized the caveolar pathway for glycosphingolipids, showing that Golgi targeting of sphingolipids internalized via caveolae required microtubules and phosphoinositol 3-kinases and was inhibited in cells expressing dominant-negative Rab7 and Rab9 constructs. In addition, overexpression of wild-type Rab7 or Rab9 (but not Rab11) in Niemann-Pick type C (NP-C) lipid storage disease fibroblasts resulted in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM(1) ganglioside, and a dramatic reduction in intracellular cholesterol stores. Our results demonstrate a role for Rab7 and Rab9 in the Golgi targeting of glycosphingolipids and suggest a new therapeutic approach for restoring normal lipid trafficking in NP-C cells.
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.
Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.
Glycosphingolipids (GSLs), together with glycopeptides, are typical constitutents of various cell membranes in a wide variety of organisms. In particular, it is known that GSLs have numerous physiological functions due to variations in the sugar chain, in spite of the very small quantity of constituents. Those are classified into cerebrosides, sulfatides, ceramide oligohexosides, globosides, and gangliosides based on the constituent sugars. Gangliosides, sialic acid-containing GSLs, are especially enriched in the brain and nervous tissues and are involved in the regulation of many cellular events. Recently, a number of GSLs have been isolated from marine invertebrates such as echinoderms, poriferans, and mollusks. We have also been researching biologically active GSLs from echinoderms to elucidate the structure-function relationships of GSLs and to develop novel medicinal resources. This review summarizes the structures and biological activities of GSLs from sea cucumbers. This study showed that the characteristics of GSLs and structure-activity relationships had neuritogenic activity toward the rat pheochromocytoma cell line PC12. That is, most of the cerebroside constituents of the sea cucumber are same glucocerebrosides as in other animals, except for some constituents, while the ganglioside constituents were unique in that a sialic acid directly binds to the glucose of cerebroside, they are mutually connected in tandem, and some are located in the internal parts of the sugar chain. It also became apparent that sialic acid is indispensable for the neuritogenic activities.
The pore-forming activity of CEL-III, a Gal/GalNAc specific lectin from the Holothuroidea Cucumaria echinata, was examined using artificial lipid membranes as a model system of erythrocyte membrane. The carboxyfluorescein (CF)-leakage studies clearly indicated that CEL-III induced the formation of pores in the dipalmitoyl phosphatidyl choline (DPPC)-lactosyl ceramide (LacCer) liposomes effectively but not in the DPPC-glucosyl ceramide (GlcCer) liposomes or DPPC liposomes. Such a leakage of CF was strongly inhibited by lactose, a potent inhibitor of CEL-III, suggesting that the leakage is mediated through the specific binding of CEL-III to the carbohydrate chains on the surface of the liposomes. The leakage of CF from the DPPC-lactosyl ceramide liposomes was pH-dependent, and it increased with increasing pH. The immunoblotting analysis and circular dichroism data indicated that upon interaction with liposomes, CEL-III associated to form an oligomer concomitantly with a marked conformational change. Furthermore, channel measurements showed that CEL-III has an ability to form small ion channels in the planar lipid bilayers consisting of diphytanoylphosphatidylcholine and human globoside (Gb4Cer)/LacCer.
In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.
Syrian hamster embryo cells were treated with galactose oxidase, followed by reduction with tritiated sodium borohydride at pH 7.4. The labeling patterns of galactosyl and N-acetyl galactosaminyl residues on the cell surface were altered in comparing scraped vs. unscraped and buffer vs. media-soaked cells treated with galactose oxidase. From these preliminary studies, the procedure to be used in most of the asbestos treatment studies was to treat cells in situ, in buffer with galactose oxidase, and then to label treated scraped cells with NaB(3)H(4). After 20 hr interaction between chrysotile asbestos and Syrian hamster cell cultures, an alteration in surface labeling of glycolipids and glycoproteins was observed. Tritiated disialogangliosides (G(Dla)) and the higher molecular weight labeled glycoproteins were significantly reduced by asbestos treatment. Similar chrysotile asbestos-treated cultures were grown in monolayers in MEM (Eagles) with 10% fetal bovine serum for 2, 24, 48, and 72 hr and then surface-labeled with galactose oxidase-. NaB(3)H(4) in phosphate buffer. Little or no difference was observed between surface-labeled lipid or protein distribution in untreated cells and those treated with asbestos for 2 hr. Asbestos-induced polar and neutral glycolipid pattern changes were observed at 24, 48, and 72 hr. Disialo- and trisialogangliosides (the more complex gangliosides) were decreased 85%, whereas globoside GL-4 was decreased by 60% at 72 hr. An overall decrease of labeled glycoproteins was observed at 24-48 hr. By 72 hr there was a complete loss of labeled protein bands with 80,000 dalton molecular mass. Since the changes in glycoproteins and glycolipids occur only after extended exposure of the cells to asbestos, the present studies support the concept that a metabolic rather than immediate masking effect is involved. Comparisons of treatment of Syrian hamster embryo cells with various asbestos fibers for 48 hr in the order of decreasing reduction in complex gangliosides were crocidolite>chrysotile (intermediate)>amosite. Effects of the above fibers on high molecular weight glycoproteins labeling followed the same order. The labeling pattern is reminiscent of the increased simplification of glycolipids and glycoproteins found in transformed cells. In the case of asbestos which appears to have no independent mutagenic capability, it is more likely that the membrane changes induced by asbestos serve to allow other mutagens to pass into the cell so as to act on the nuclear structure.
The carbohydrate composition of the stereociliary glycocalyx of the utricle was analysed quantitatively. The utricular sensory epithelium was collected from adult albino guinea pigs, and its apical surface structure was blotted onto a PVDF membrane, which was then subjected to acid hydrolysis. The hydrolysate was treated with 1-phenyl-3-methyl-5-pyrazolone for labelling and analysed by reversed-phase HPLC coupled with detection of UV absorbance at 245 nm. Man, GlcN, GaIN, Glc, Gal and Fuc were detected and estimated to be 211, 219, 46, 1,270, 266 and 36 pmoles per 10 utricular maculae, respectively. The presence of Man indicates the presence of N-linked glycoconjugates, and the considerable amounts of GLcN and Gal suggest the presence of complex-type N-glycosides, poly-lactosamine and/or keratan sulfate. The relatively low GaIN content indicates that O-glycosides, chondroitin sulfates and GaINAc-containing glycosphingolipids, i.e. gangliosides and globosides, are minor components. Electronmicroscopic and confocal laser scanning microscopic observations revealed that the blotted apical surface structure constituted mostly the ciliary bundle. Consequently, the observed carbohydrate composition is probably that of the stereociliary glycocalyx of the utricular sensory epithelium.
A new genetic model of the P blood group system is presented. The system is controlled by two chromosomal loci. The first locus has three allelic genes. The pk gene codes for an alpha galactosyl transferase that converts ceramide dihexoside to ceramide trihexoside (or the pk antigen). The second allele, the pk gene, codes for an alpha galactosyl transferase that converts both ceramide dihexoside to ceramide trihexoside (or the pk antigen) and paragloboside to the P1 antigen. The third allele does not produce an active codes for a beta N-acetyl galactosaminyl transferase that converts ceramide trihexoside to globoside (or the P antigen). The second allele does not produce an active product. The predictions of the model are in agreement with family studies and fibroblast fusion studies. The current model and previous genetic models, however, predict different possible phenotypes from rare 2 x p or p2k x p matings or fibroblast fusions.
Although changes in surface carbohydrate expression of abnormally expanded MRL/Mp-lpr/lpr (MRL/lpr) lymph node (LN) cells have previously been described, the composition and function of glycolipids present on these cells as well as the spectrum of specificity of anti-carbohydrate antibodies reactive with these cells remains obscure. Analysis of antibodies to a panel of 22 carbohydrate structures using a liposome immune lysis assay (LILA) showed that, except for anti-asialo GM2 (GA2) antibodies, marked reduction of antiglycolipid antibody levels was observed in sera from 4-mo-old MRL/lpr mice compared with these from MRL/Mp(-)+/+ (MRL/+) mice. Absorption experiments revealed that both anti-asialo GM1 (GA1) and globoside antibodies had binding capacity to MRL/lpr LN cells. To elucidate the glycolipid profiles of MRL/lpr LN cells, glycolipids were extracted from LN cells of both MRL/lpr and MRL/+ mice and analysed. A 30-fold elevation of GM1 was found in MRL/lpr LN cells compared with MRL/+ LN cells. From the results of LILA using GA1/GM1 mixed liposomes, aberrantly expressed GM1 inhibited the classical complement pathway but did not interfere with the binding of anti-GA1 antibodies to liposomal GA1. These findings suggest that a drastic GM1 increase on MRL/lpr LN cells would inhibit the action of anti-GA1 antibodies and complement on the cell surface. This may explain the escape of these cells from an activated self directed immune response.
The author engaged himself in the studies of ABO blood group system for the last three decades, and reviewed the progresses in this period, which were classified into following 5 items. 1. H-, A- and B-active oligosaccharides were isolated from the globoside fractions from human erythrocytes by ozonolysis. One of the H-active oligosaccharide with short carbohydrate chain is a pentasaccharide: Fuc(alpha 1-->2)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc, and the other with long carbohydrate chain is a heptasaccharide: Fuc(alpha 1-->2)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc. Hexa- or octasaccharides with blood group A- or B-activity have an additional alpha-N-acetylgalactosaminyl residue or alpha-galactosyl residue, which joints with alpha 1-->3 linkage to subterminal beta-galactose of the both of H-active oligosaccharides, respectively. 2. A blood group A-gene specified alpha-N-acetyl-galactosaminyltransferase (A-enzyme) catalyzes the transfer of N-acetylgalactosamine from the UDP-sugar to the subterminal beta-galactosyl residue of blood group H-active carbohydrate chain, and a blood group B-gene specified alpha-galactosyltransferase (B-enzyme) catalyzes the transfer of galactose from the UDP-sugar to the subterminal beta-galactosyl residue of blood group H-active carbohydrate chain, respectively. Either the A- or B-enzyme can not transfer the substrate sugar to the carbohydrate chain lacking alpha-fucosyl residue of H-determinant, and it is the reason why the synthesis of blood group A- or B-antigenic structure in inhibited in the tissues of Bombay phenotype and in the secretory glands of the nonsecretor. 3. Specific antibody either to the A- or B-enzyme can be introduced in the serum of the rabbit which was immunized with the A- or B-enzyme preparation, respectively. And immunological cross reaction is also present between the A- and B-enzyme, but the immunologically cross reactive material can not be found in the blood group O individual. The absence of immunologically cross reactive material in the blood group O individual is supported by a fact that the cross reactive antibody similar to the antibody in rabbit serum was present in the serum of the chronic myeloid leukemia patient, who was belonged to blood group B and treated with blood group incompatible bone marrow transplantation from blood group O donor, because it is acceptable to speculate that the grafted lymphocytes react to the B-enzyme in the recipient and produce the anti-enzyme antibody. 4. The immunological profiles described above are compatible with the cDNA structures of human blood group ABO alleles presented by Yamamoto F. et al. Their gene model is that the cDNAs of blood group ABO alleles are highly homologous, but the cDNA of common O allele is non-functional due to a single nucleotide deletion close to the 5'end of the coding sequence, which causes a frame shift of the codon, and results in truncated peptide. 5. Transcription of the human blood group ABO gene can be enhanced by a CBF/NF-Y which binds the minisatellite on the 5'-upstream sequence of the gene.
Two brothers with oligosymptomatic mucopolysaccharidosis VII were observed from age 11 8/12 to 16 years, and 15 1/2 to 19 years, respectively. Asymptomatic thoracic kyphosis and mild scoliosis were the prominent clinical features. Herniae, hepatosplenomegaly, corneal clouding and shortness of stature were absent. Both had Alder type granulations in polymorphonuclear leukocytes and to a lesser degree in monocytes. Ultrastructural analysis of blood leukocytes revealed polymorphous inclusions of probably more than one class of organic substances. Radiological signs were mild, confined to the spine and consisted of irregularities of upper and lower vertebral plates, of vertebral flattening and some osteophytic changes. Both patients excreted excessive amounts of acid mucopolysaccharides in urine and also globoside. Cultured skin fibroblasts of both patients contained metachromatic granules, had only approx. 10% of normal beta-glucuronidase activity and degraded sulfated mucopolysaccharides at a slower than normal rate. Sera of the patients had none or minimal beta-glucuronidase activity, the mother's serum had subnormal and the father's serum low-normal activity. The older brother is the oldest known case of mucopolysaccharidosis VII. As this hereditary disorder may take a remarkably mild clinical course, beta-glucuronidase-deficient juveniles may exist undetected in the general population.
A crude soluble preparation of human hepatic N-acetyl-beta-D-hexosaminidase was examined for its activities toward three natural glycosphingolipid substrates after fractionation by the isoelectric focusing procedure. Profiles of activities toward N-acetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM2-ganglioside) and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide (globoside) were always identical with that of nonspecific N-acetyl-beta-D-hexosaminidase as determined with artificial substrates. The Component A of the enzyme had the activity peak at an isoelectric point of 5.0 to 5.1. In contrast, hydrolytic activities toward N-acetylgalactosaminyl-[N-acetylneuraminyl]galactosyl- glucosylceramide (GM2-ganglioside) were associated with only the most acidic subfraction of the hexosaminidase A component. The activity to hydrolyze GM2-ganglioside had its peak at an isoelectric point of 4.8 to 4.9. These findings might provide an explanation for the GM2-ganglioside accumulation in juvenile GM2-gangliosidosis (partial deficiency of hexosaminidase A) and in the so-called AB variant of GM2-gangliosidosis (apparently normal hexosaminidase A and B activity).
A patient with myoclonus epilepsy as a main clinical symptom was histopathologically diagnosed as a generalized sphingolipidosis. It was found that both sphingomyelin and globoside I fairly increased in kidney, heart, lung and liver. While, only sphingomyelin was found to increase in cerebral gray and white matters and cerebellum, but other lipids were within the normal range. Sphingomyelin accounted for 22% of the total phospholipids especially in cerebellum. No cholesterol ester and ganglioside GM2 or asialo GM2 were in particular found in the brain. Fatty acid compositions of phospholipids, glycosphingolipids and gangliosides were found to be normal. Judging from the sphingomyelin storage not only in visceral organs but also in brain tissues, it was proposed that this disease might be a variant in Niemann-Pick disease Type C, although an enzymatic assay of sphingomyelinase still remains.
Parvo B19 (Fifth disease) is an erythrotropic virus which attaches through the 'P' globoside receptor on the surface of human red blood cells and precursors. This typically benign viral infection can cause a transient aplastic anemia in patients with underlying red cell disorders. In this case, a two-year-old child presents with severe aplastic anemia without evidence of underlying disease. Erythroid regeneration is monitored through the use of the immature reticulocyte fraction (IRF) and is demonstrated by the presence of high and medium fluorescence reticulocytes in the peripheral blood three to five days prior to the peak in absolute reticulocytes.
1. Neutral glycosphingolipids of hamster fibroblast NIL cells have been characterized as follows: glucosylceramide, lactosylceramide (betaGall yields 4Glc yields Cer), a digalactosylceramide (alphaGall yields 4betaGal yields Cer), a trihexosylceramide (alphaGall yields 4betaGall yields 4Glc yields Cer), two kinds of ceramide tetrasaccharides (A: alphaGa1NAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 1Cer, a new type of Forssman active glycolipid; B: globoside, betaGalNAcl yields 3alphaGall yields 4betaGall yields 4betaGlc yields Cer), and a ceramide pentasaccharide having a classical structure for Forssman antigen (alphaGalNAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 4Glc yields Cer). 2. Neutral glycosphingolipids of polyoma virus-transformed NIL cells (NILpy) have been characterized as having an additional ceramide tetrasaccharide which was absent in normal NIL cells. The structure of this specific glycolipid was identified as lacto-N-neotetraosylceramide (betaGall yields 4betaGlc-NAcl yields 3betaGall yields 4Glc yields Cer). Chemical quantities of ceramide tetra- and pentasaccharides in NILpy cells were much lower than in NIL cells. 3. All of these glycolipids, except glucosylceramide and lactosylceramide, were labeled externally by galactose oxidase and tritiated borohydride according to the method previously described (GAHMBERG, C. G, and HAKOMORI, S. (1973) J. Biol. Chem. 248, 4311-4317). The specific activities of the label in glycolipid of NIHpy cells were much greater than that in NIL cells, i.e. reactivity of glycolipids with galactose oxidase in NIHpy cells was much higher than for NIL cells. The surface label in glycolipids was cell cycle-dependent in NIL cells, and a remarkable exposure of a galactosyl residue of a ceramide tetrasaccharide was demonstrated only on the surface of NILpy cells, due to the presence of lacto-N-neotetraosylceramide.