Glycosphingolipids of leukemic cells in adult T-cell leukemia-lymphoma

We analyzed lipids from leukemic cells of two patients with adult T-cell leukemia and compared them with those from T-cell lymphocytes of normal subjects. The neutral glycosphingolipids and gangliosides which were isolated were characterized by thin-layer chromatography and neuraminidase treatment. Both leukemic cells and normal lymphocytes had monoglycosylceramide and diglycosylceramide as major neutral glycosphingolipids. In one patient, diglycosylceramide was markedly increased. II3NeuAc-LacCer (GM3) and more complex gangliosides were detected in both cells. The most characteristic finding in leukemic cells was the occurrence of a disialylated ganglioside, II3(NeuAc)2-LacCer (GD3), which is not found in normal lymphocytes and neutrophils. This ganglioside may be due to the induced synthesis in association with malignant transformation.     

Gangliosides of murine T lymphocyte subpopulations

Gangliosides from murine T lymphoblasts were analyzed by high-performance thin-layer chromatography followed by in situ neuraminidase treatment and immunostaining of the resulting asialogangliosides and compared with those from thymocytes and cloned T lymphocytes with defined functions. The ganglioside IVNeuGc/Ac-GgOse5Cer (GalNAc-GM1b), a marker for T lymphoblasts [Müthing, J., Egge, H., Kniep, B., & Mühlradt, P. F. (1987) Eur. J. Biochem. 163, 407-416], was found only in small amounts as the N-acetylated species in gangliosides from thymocytes and a cytolytic T cell clone. Two helper clones expressed this ganglioside like T blasts. The structures of the two major disialogangliosides from T blasts, IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha type) with C24:0/24:1 and C16:0 fatty acids, were elucidated by neuraminidase treatment and immunostaining and by fast atom bombardment mass spectrometry. Gangliosides of this type were detected in thymocytes only in minor amounts, whereas GM1b-type gangliosides prevailed in cells from this organ. Analysis of the T lymphoblast gangliosides from six genetically unrelated mouse strains showed that terminally sialylated GgOse4Cer (GM1b), IVNeuAc-GgOse5Cer (GalNAc-GM1b), and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha) were conserved structures in all strains examined. We conclude that maturation or stimulation of T cells may be correlated with elongation of a common GM1b-type precursor structure resulting in GalNAc-GM1b or GD1 alpha-type gangliosides.     

Preferential reactivity of autoantibodies in murine lupus NZB mice to neuraminidase-treated monosialogangliosides on B cells of mouse spleen

When analyzed by flow cytometry, reactivity of IgM autoantibodies in sera from NZB mice to spleen B cells, but not to T cells, from BALB/c mice was remarkably increased after treatment of the cells with Vibrio cholerae neuraminidase. By TLC immunostaining with the antibodies, neither neutral nor acidic glycosphingolipids from both BALB/c and NZB mouse spleens were found to be reactive, but after neuraminidase treatment of the TLC plate, prior to the immunostaining, three components became reactive. All of the reactive glycosphingolipids were found to carry a single sialic acid residue and were at a concentration less than 1.3% of the total lipid-bound sialic acids. Their mobilities on TLC plate were close to those of IV3 NeuAcnLc4Cer, IV3 NeuAcII3 NeuAcGg4Cer, and IV3 NeuAcII3 NeuAc2Gg4Cer. In addition, the monosialogangliosides, which became reactive with the autoantibodies after neuraminidase treatment, were found to be predominantly distributed on B cells from BALB/c mice spleen, but not on T cells by TLC immunostaining. These studies demonstrate that the majority of IgM autoantibodies to spleen lymphocytes in sera from NZB mice might react preferentially to terminal sugar residues of three new glycosphingolipids masked by a single sialic acid on B cells, but not on T cells.     

Specific staining on thin-layer chromatograms of glycosphingolipids of neolacto series and gangliosides with a terminal N-acetylneuraminyl residue by different procedures with wheat germ agglutinin

Sensitive staining methods with wheat germ agglutinin were developed for the detection of glycosphingolipids of neolacto series (A) and gangliosides with a terminal N-acetylneuraminyl residue (B) on thin-layer chromatograms. (A) Neolacto series glycosphingolipids were treated by beta-galactosidase on the chromatograms in the presence of taurodeoxycholate. Then the chromatograms were incubated with biotinated wheat germ agglutinin followed by incubation with a complex of avidin and biotinated horseradish peroxidase, and the reaction was detected by 4-chloro-1-naphthol. In the case of gangliosides, sialidase treatment on the chromatograms was performed before the beta-galactosidase treatment. The sensitivity of the method for Lc3Cer, nLc4Cer, sialyl-nLc4Cer, and sialyl-nLc6Cer was 4 pmol, 7.6 pmol, 2.9 pmol and 1.4 pmol, respectively. (B) The gangliosides on the chromatograms were oxidized by periodic acid and reduced by NaBH4. Then the chromatograms were stained with wheat germ agglutinin as mentioned above. As little as 0.5 pmol of GM3, NeuAc-nLc4Cer, and NeuAc-nLc6Cer was detected by this method, whereas the detected limits for these gangliosides were 10 pmol, 10 pmol and 2 pmol, respectively, when periodate oxidation was omitted. GM4, GD3 and GD1a were an order less reactive than GM3, GM2, GM1 or GD1b were not stained under the same condition. In contrast to NeuAc-containing gangliosides, any gangliosides with N-glycolylneuraminic acid were not stained by the method in (B).     

A new monoclonal antibody directed to sialyl alpha 2-3lactoneotetraosylceramide and its application for detection of human gastrointestinal neoplasms

A new monoclonal antibody (NS24) directed to the N-acetylneuraminyl alpha 2-3Gal beta 1-4GlcNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3(NeuAc)nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3(NeuAc)nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl alpha 2-3lactoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3(NeuAc)nLc6Cer], I-active ganglioside [VIII3(NeuAc)-VI3(NeuAc)IV6kladoLc8Cer], GM4(NeuAc), GM3(NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Synthetic N-acetylneuraminyl alpha 2-3lactotetraosylceramide [IV3(NeuAc)Lc4Cer] and its asialo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3(NeuAc)nLc4Cer was detected dose-dependently by a thin-layer chromatography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.     

Selective terminal alpha 2-3 and alpha 2-6 sialylation of glycosphingolipids with lacto-series type 1 and 2 chains in human meconium

Human meconium was found to contain two kinds of gangliosides with the same carbohydrate sequence belonging to the lacto-series. They were detected by TLC-immunostaining with monoclonal antibodies directed to the NeuAc alpha 2-6Gal and Lc4Cer structures. One of these two gangliosides, a major one, which migrated on TLC to a position below that of standard IV3NeuAcnLc4Cer from human erythrocytes, reacted with the antibody to NeuAc alpha 2-6Gal. The other minor one, which migrated on TLC to a position corresponding to standard IV3NeuAcnLc4Cer, was detected with the antibody to Lc4Cer only when the plate, on which the individual gangliosides were separated, was subjected to prior treatment with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation anaylsis with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation anaylsis and enzyme treatment after isolation with antibody monitoring, were shown to be IV6NeuAcnLc4Cer for the former and IV3NeuAcLc4Cer for the latter, indicating that the lacto-series type 2 (nLc4Cer) and 1 (Lc4Cer) chains are sialylated at different linkages, alpha 2-6 and alpha 2-3, respectively. IV6NeuAcLc4Cer and IV3NeuAcnLc4Cer were not detected, even in trace amounts, on TLC-immunostaining with the monoclonal antibodies. The concentrations of IV6NeuAcnLc4Cer and IV3NeuAcLc4Cer were 448 and 18 nmol/g dry wt of human meconium.     

Rabbit thymus gangliosides: species- and organ-specific distribution, age-dependence and their cell biological significance

Gangliosides of thymuses from rabbit, mouse, rat, calf, and man were analyzed. The ganglioside compositions of the thymuses showed species specificities, and the compositions of the species other than the rabbit were found to be markedly different from that of the rabbit, which contained characteristically substantial amounts of IV3NeuGc-nLc4Cer and VI3NeuGc-nLc6Cer (Iwamori, M. & Nagai, Y. (1981) Biochim. Biophys. Acta 665, 214-220). The inter-species differences in the thymus ganglioside compositions were not remarkable in the 8 mouse and 2 rabbit strains examined. Rabbit thymocytes, but not those of mouse and rat, were lysed with human Hanganutziu-Deicher serum in the presence of guinea pig complement, reflecting the high content of gangliosides containing N-glycolylneuraminic acid in rabbit thymus. As to age-dependent changes of gangliosides in rabbit thymus and spleen, the concentrations gradually decreased with age, while the molar ratio of total gangliosides to total phospholipids was constant in the spleen throughout life and in the thymus at 3, 4, and 6 weeks of age. It was noted that old (180 weeks of age) rabbit thymus, which is occupied largely by fat tissue, still contained a significant amount of neolactoseries gangliosides.     

The human H3N2 influenza viruses A/Victoria/3/75 and A/Hiroshima/52/2005 preferentially bind to α2-3-sialylated monosialogangliosides with fucosylated poly-N-acetyllactosaminyl chains

Among influenza A viruses, subtype H3N2 is the major cause of human influenza morbidity and is associated with seasonal epidemics causing annually half million deaths worldwide. Influenza A virus infection is initiated via hemagglutinin that binds to terminally sialylated glycoconjugates exposed on the surface of target cells. Gangliosides from human granulocytes were probed using thin-layer chromatography overlay assays for their binding potential to H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. Highly polar gangliosides with poly-N-acetyllactosaminyl chains showing low chromatographic mobility exhibited strong virus adhesion which was entirely abolished by sialidase treatment. Auxiliary overlay assays using anti-sialyl Lewis(x) (sLe(x)) monoclonal antibodies showed identical binding patterns compared with those performed with the viruses. A comprehensive structural analysis of fractionated gangliosides by electrospray ionization quadrupole time-of-flight mass spectrometry revealed sLe(x) gangliosides with terminal Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc epitope and extended neolacto (nLc)-series core structures as the preferential virus binding gangliosides. More precisely, sLe(x) gangliosides with nLc8, nLc10 and nLc12Cer cores, carrying sphingosine (d18:1) and a fatty acid with variable chain length (mostly C24:0, C24:1 or C16:0) in the ceramide moiety and one or two additional internal fucose residues in the oligosaccharide portion, were identified as the preferred receptors recognized by H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. This study describes glycan-binding requirements of hemagglutinin beyond binding to glycans with a specific sialic acid linkage of as yet undefined neutrophil receptors acting as ligands for H3N2 viruses. In addition, our results pose new questions on the biological and clinical relevance of this unexpected specificity of a subtype of influenza A viruses.     

Glycosphingolipid biosynthesis in early chick embryos

Early chick embryos at the age of 22 h (neurula stage) and 48 h (20-25 somite stage) were explanted from eggs and cultured in vitro in the presence of radioactive sugar precursors. Metabolically labelled glycosphingolipids were isolated. Amongst these, neutral and acidic components, the latter including sulfatide and gangliosides, were identified. Cleavage by exoglycosidases, as well as immunostaining with antibodies on thin-layer plates, showed that at both embryonic stages glycosphingolipids were synthesized that belong to the globo series (globoside, Forssman glycolipid), the lacto series (lactoneotetraosylceramide, nLc4Cer, and two nLc4Cer-based gangliosides, a monosialo and a disialo species), and the ganglio series (ganglioside Gtet1a and higher sialylated derivatives).     

Expression of Mouse Sialic Acid on Gangliosides of a Human Glioma Grown as a Xenograft in SCID Mice

Ganglioside sialic acid content was examined in the U87-MG human glioma grown as cultured cells and as a xenograft in severe combined immunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N-glycolylneuraminic acid (NeuGc)-containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N-acetylneuraminic acid (NeuAc)-containing gangliosides, whereas mouse cells express both NeuAc- and NeuGc-containing gangliosides. Small amounts of NeuGc ganglioside sialic acid (2-3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87-MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87-MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87-MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo-GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.     

ON THE COMPOSITION AND STRUCTURE OF INDIVIDUAL GANGLIOSIDES FROM THE BRAIN OF ELASMOBRANCHES

Gangliosides with a short carbohydrate chain: II³(NeuAc)₂-LacCer, II³(NeuAc)₂-GgOse₃Cer, II₃(NeuAc)₃-LacCer, II³NeuAc-LacCer, and II³NeuAC-GgOse₃ Cer, were found to be predominant in the brain of 8 species of cartilaginous fish, elasmobranches. N-acetylneuraminic acid was the only sialic acid found in these gangliosides, the N-glycolyl derivative being practically absent. 4-Sphingenine was shown to be the predominant sphingoid in elasmobranch brain gangliosides. The sequential enzymatic hydrolysis of II³(NeuAc)₂ -LacCer from shark and ray brain by acylneur-aminyl hydrolase (EC 3.2.1.18) and β-D-galactoside-galactohydrolase (EC 3.2.1.23), as well as permethylation studies, provide further evidence for the following structure of this major elasmobranch brain ganglioside:     

A simple and specific assay of glycosyltransferase and glycosidase activities by an enzyme-linked immunosorbent assay method, and its application to assay of galactosyltransferase activity in sera from patients with cancer.

A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.     

Structural characterization of gangliosides from murine T lymphocytes

Mouse spleen cells were prepared from CBA/J mice, and T lymphocytes were selectively stimulated with the T cell mitogen concanavalin A and further propagated in the presence of the T cell growth factor interleukin-2. The T cells were metabolically labeled with D-[1-14C]galactose and D[1-14C]glucosamine, and the gangliosides were extracted and purified by DEAE-Sepharose column chromatography. Carbohydrate backbone structures of the asialogangliosides, prepared by mild acid hydrolysis, were determined by high-performance liquid chromatography, treatment with exoglycosidases and immunostaining. Monosialylated gangliosides were isolated by gradient elution from DEAE-Sepharose and further separated by preparative high-performance thin-layer chromatography in two solvent systems. Isolated fractions were characterized by preparation of asialogangliosides by mild acid hydrolysis, the action of Vibrio cholerae neuraminidase, and fast-atombombardment mass spectrometry. The following structures were identified: IVNeuAc-GgOse4Cer; IVNeuGc-GgOse4Cer; IVNeuAc-GgOse5Cer; and IVNeu-Gc-GgOse5Cer. The latter two gangliosides were not detected on B lymphoblasts and may be T-cell-specific structures. All gangliosides were heterogeneous in their ceramide moieties, being substituted with C16:0, C24:0, and C24:1 fatty acids. A preliminary study of several other mouse strains showed no strain-specific genetic variations in the T cell gangliosides. The possible role of these gangliosides is discussed.     

High-performance liquid chromatography of long-chain neutral glycosphingolipids and gangliosides

We describe a method for analyzing the perbenzoyl derivatives of both neutral glycosphingolipids and gangliosides with a single high-performance liquid chromatography system. Use of this system, combined with endo- and/or exoglycosidase treatment of glycosphingolipids, provides a sensitive method for obtaining structural information on these compounds. This system has two advantages over previously published chromatography procedures: (i) it uses a commercially available column, and (ii) this single column can be used to analyze gangliosides and their neutral glycosphingolipid products generated by neuraminidase treatment. With this method, we have studied 24 different glycosphingolipids, containing one to ten sugars and one or two sialic acid residues, and have demonstrated its usefulness in evaluating the gangliosides present in human leukocytes.     

Neutral glycosphingolipids and gangliosides from spleen T lymphoblasts of genetically different inbred mouse strains

The gangliosides GM1b, GalNAc-GM1b and GD1α are typical compounds of concanavalin A stimulated splenic T lymphoblasts of CBA/J inbred mice. Their structural characterization has been described in previous studies. The intention of this work was the comparative TLC immunostaining analysis of the glycosphingolipid composition of lectin stimulated splenic T lymphoblasts obtained from six genetically different inbred mouse strains. The strains examined were AKR, BALB/c, C57BL/6, CBA/J, DBA/2 and WHT/Ht, which are commonly used for biochemical and immunological studies. The neutral glycosphingolipid GgOse4Cer, the precursor for GM1b-type gangliosides, was expressed by all six strains investigated. AKR, C57BL/6 and DBA/2 showed high and BALB/c, CBA/J and WHT/Ht diminished expression in T lymphoblasts, based on single cell calculation. The gangliosides GM1b and GalNAc-GM1b, elongation products of GgOse4Cer, displayed strain-specific differences in their intensities, which were found to correlate with the intensities of GgOse4Cer expression of the same strains. Concerning sialic acid substitution of gangliosides, GM1b and GalNAc-GM1b predominantly carry N-acetylneuraminic acid, whereas choleragenoid receptors GM1a and Gal-GalNAc-GM1b, which are also expressed by all six strains, are characterized by dominance of N-glycolylneuraminic acid. Two highly polar gangliosides, designated with X and Y, which have not been previously recognized in murine lymphoid tissue, were detected by positive anti-GalNAc-GM1b antibody and choleragenoid binding, respectively. Both gangliosides were restricted to AKR, DBA/2 and C57BL/6 mice. The other three strains BALB/c, CBA/J and WHT/Ht are lacking these structures. In summary, the GM1b-type pathway is quite active in all six strains analysed in this study. Strain-specific genetic variations in T lymphoblast gangliosides were observed with the occurrence of gangliosides X and Y. This study and data from other groups strongly indicate for GM1b-type gangliosides a functional association with T cell activation and leukocyte mediated reactions. Abbreviations: ConA, concanavalin A; GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) [48] and the ganglioside nomenclature system of Svennerholm [49] for GM1a-type gangliosides. Glucosylceramide or GlcCer, Glcβ1-1Cer; lactosylceramide or LacCer, Galβ1-4Glcβ1-1Cer; gangliotriaosylceramide or GgOse3Cer or Gg3, GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliotetraosylceramide or GgOse4Cer or Gg4, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliopentaosylceramide or GgOse5Cer, GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliohexaosylceramide or GgOse6Cer, Galβ1-3GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer. GM3, II3NeuAc-LacCer; GM1 or GM1a, II3NeuAc-GgOse4Cer; GM1b, IV3NeuAc-GgOse4Cer; GalNAc-GM1b, IV3NeuAc-GgOse5Cer; GD1a, IV3NeuAc, II3NeuAc-GgOse4Cer; GD1b, II3(NeuAc)2-GgOse4Cer; GD1c, IV3(NeuAc)2-GgOse4Cer; GD1α, IV3NeuAc, III6NeuAc-GgOse4Cer. Only NeuAc-substituted gangliosides are presented in this list of abbreviations This revised version was published online in November 2006 with corrections to the Cover Date.     

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

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

Mistletoe lectin I is a sialic acid-specific lectin with strict preference to gangliosides and glycoproteins with terminal Neu5Ac alpha 2-6Gal beta 1-4GlcNAc residues

Mistletoe lectin I (ML-I) is a type II ribosome-inactivating protein, which inhibits the protein biosynthesis at the ribosomal level. ML-I is composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding specificities. Using comparative solid-phase binding assays along with electrospray ionization tandem mass spectrometry, ML-I was shown to preferentially bind to terminally alpha2-6-sialylated neolacto series gangliosides from human granulocytes. IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer were identified as ML-I receptors, whereas the isomeric alpha2-3-sialylated neolacto series gangliosides were not recognized. Only marginal binding of ML-I to terminal galactose residues of neutral glycosphingolipids with a Galbeta1-4Glc or Galbeta1-4GlcNAc sequence was determined, whereas a distal Galalpha1-4Gal, GalNAcbeta1-3Gal, or GalNAcbeta1-4Gal disaccharide did not bind at all. Among the glycoproteins investigated in Western blot and microwell adsorption assays, only those carrying Neu5Acalpha2-6Galbeta1-4GlcNAc residues, exclusively, predominantly, or even as less abundant constituents in an assembly with Neu5Acalpha2-3Galbeta1-4GlcNAc-terminated glycans, displayed high ML-I binding capacity. From our data we conclude that (i) ML-I has to be considered as a sialic acid- and not a galactose-specific lectin and (ii) neolacto series gangliosides and sialoglycoproteins with type II glycans, which share the Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, are true ML-I receptors. This strict preference might help to explain the immunostimulatory potential of ML-I toward certain leukocyte subpopulations and its therapeutic success as a cytotoxic anticancer drug.     

Characteristic binding of human plasma apolipoprotein B to gangliotetraosylceramide and gangliotriaosylceramide.

The binding of human plasma low-density lipoproteins (LDL), freshly prepared by discontinuous ultracentrifugation, to several neutral and acidic glycosphingolipids was examined by TLC immunostaining with the anti [apolipoprotein B (apoB)] antibody. ApoB was found to bind characteristically to the asialogangliosides, gangliotetraosylceramide (Gg4Cer) and gangliotriaosylceramide (Gg3Cer), the former being a more potent receptor than the latter, indicating that the sequences Gal beta 1-3GalNAc beta 1-4Gal and GalNAc beta 1-4Gal are involved in the binding of apoB. A weak positive reaction with fucosylgangliotetraosylceramide (IV2Fuc-Gg4Cer), which has the same internal recognition sequences, was also observed (the binding ability was only 1/7 of that in the case of Gg4Cer). No binding to other neutral glycosphingolipids, or glycosphingolipid sulfates (I3-SO3-GalCer) and gangliosides, was detected, and therefore substitution of the receptor glycolipid with sialic acid was thought to inhibit the binding. The results indicate that, along with the binding of apoB to the LDL-binding domain of the receptor glycoprotein, interaction with some carbohydrate chains in the receptor, or with glycolipids coexisting on the plasma membrane, may be important for the binding of apoB to cells.     

Glycosphingolipid-binding specificity of the mannose-binding protein from human sera

Mannose-binding protein was purified from human serum to apparent homogeneity by affinity chromatography on mannose-Sepharose, followed by affinity chromatography on underivatized Sepharose. Approximately 0.4 mg protein was obtained from 1 liter serum. The glycosphingolipid-binding specificity of the purified protein was examined by chromatogram overlay and solid phase assays. It binds with high affinity to Lc-3Cer (GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide) and n-Lc5Cer (GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide). It does not bind to many other glycosphingolipids without terminal N-acetylglucosamine residues that were tested. Thus, these data suggest that N-acetylglucosamine-terminated glycosphingolipids may serve as cell-surface attachment sites for mannose-binding protein in vivo. In addition, the binding specificity of the protein can be used as a sensitive probe for determining the levels of Lc3Cer and nLc5Cer in tissues, as it exhibits half-maximal binding to about 10 pmol of these lipids in solid phase assays, and detects less than 20 pmol of Lc3Cer in chromatogram overlay assays. This technique was utilized to demonstrate that one sample of chronic myeloid leukemia cells contains both Lc3Cer and nLc5Cer.