Sialosylparagloboside Research Articles

Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes.

Clostridium botulinum type C 16S progenitor toxin consists of a neurotoxin (NTX), a non-toxic non-HA (NTNH), and a haemagglutinin (HA). The HA acts as an adhesin, allowing the 16S toxin to bind to intestinal epithelial cells and erythrocytes. In type C, these bindings are dependent on sialic acid. The HA consists of four distinct subcomponents designated HA1, HA2, HA3a and HA3b. To identify the binding subcomponent(s) of HA of type C 16S toxin, all of the HA-subcomponents and some of their precursor forms were produced as recombinant proteins fused to glutathione S-transferase (GST). These proteins were evaluated for their capacity to adhere to intestinal epithelial cells of guinea pig and human erythrocytes. GST-HA1, GST-HA3b and GST-HA3 (a precursor form of HA3a and HA3b) bound intestinal epithelial cells and erythrocytes, whereas GST alone, GST-HA2 and GST-HA3a did not. GST-HA3b and GST-HA3 showed neuraminidase-sensitive binding to the intestinal epithelial cells and erythrocytes, whereas GST-HA1 showed neuraminidase-insensitive binding. TLC binding assay revealed that GST-HA3b and GST-HA3 recognized sialosylparagloboside (SPG) and GM3 in the ganglioside fraction of the erythrocytes, like native type C 16S toxin [Inoue, K. et al. (1999). Microbiology 145, 2533-2542]. On the other hand, GST-HA1 recognized paragloboside (PG; an asialo- derivative of SPG) in addition to SPG and GM3. Deletion mutant analyses of GST-HA3b showed that the C-terminal region of HA3b is important for its binding activity. Based on these data, it is concluded that the HA component contains two distinct carbohydrate-binding subcomponents, HA1 and HA3b, which recognize carbohydrates in different specificities.

Autoantibodies to peripheral nerve glycosphingolipids SPG, SLPG, and SGPG in Guillain–Barré syndrome and chronic inflammatory demyelinating polyneuropathy

Unlike CNS myelin, human peripheral nerve myelin has the acidic glycosphingolipids sialosyl paragloboside (SPG), sialosyl lactosaminyl paragloboside (SLPG), and sulfated glucuronyl paragloboside(SGPG). To elucidate the pathogenesis of Guillain–Barré syndrome (GBS) and chronic inflammatory demyelinating neuropathy (CIDP), we investigated the autoantibodies to peripheral nerve molecules in patients with these diseases and compared the frequency of the autoantibodies with that of autoantibody to GM1 which is present in both the CNS and PNS. The report of Sheikh et al. (Ann. Neurol. 1995; 38: 350) that Campylobacter jejuni bears the SGPG epitope led us to study whether sera from patients with GBS subsequent to C. jejuni enteritis have anti-SGPG antibody; but, high anti-SGPG antibody titers were not found in the GBS patients from whom C. jejuni was isolated. Although the frequency of the anti-SPG, anti-SLPG, and anti-SGPG antibodies were lower than that of the anti-GM1 antibody in GBS, 5 patients with demyelinating GBS had high IgG anti-SPG antibody titers. IgG anti-SPG antibody may function in the development of demyelinating GBS. We found that 6 CIDP patients had elevated IgM anti-SGPG antibody titers. Immunoelectrophoresis failed to detect IgM M-protein in 3 of the patients. IgM anti-SGPG antibody could be a diagnostic marker for a subgroup of CIDP with or without paraprotein.

Isolation and characterization of gangliosides from Theileria sergenti.

The gangliosides of Theileria sergenti piroplasms were isolated and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated on TLC. G-1, G-2, G-3, and G-4 ganglioside showed the same mobility as GM3, sialosylparagloboside (SPG), i-active ganglioside, and I-active ganglioside on the TLC plate, respectively. In order to characterize the molecular species of gangliosides from T. sergenti, G-1, G-2, G-3, and G-4 gangliosides were purified and tested by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside had reactivity to anti-GM3 monoclonal antibody. G-2 gave reaction with monoclonal antibody to SPG containing N-glycolylneuraminic acid (NeuGc). G-3 showed reactivity to the anti-i-active ganglioside (NeuGc) monoclonal antibody. G-4 was recognized by the monoclonal antibody which reacts with I-active ganglioside (NeuGc). In addition, sialic acid moiety of the gangliosides from T. sergenti piroplasms was also analyzed. N-acetylneuraminic acid-containing gangliosides were hardly detectable in T. sergenti piroplasms. Gangliosides from T. sergenti (G-1, G-2, G-3, and G-4) carried only NeuGc as their sialic acid moiety. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuGc) [NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer], SPG (NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], i-active ganglioside (NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], and I-active ganglioside(NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3 (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6) Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], respectively.

Production of monoclonal antibodies directed to Hanganutziu-Deicher active gangliosides, N-glycolylneuraminic acid-containing gangliosides.

We have established three kinds of monoclonal antibodies against gangliosides containing N-glycolylneuraminic acid (NeuGc) by immunization of BALB/c mice with the purified gangliosides inserted into liposomes comprising Salmonella minnesota R595 lipopolysaccharides, and fusion of spleen cells with a mouse myeloma cell line. One monoclonal antibody, SHS-1, which was generated by immunizing mice with purified i-active ganglioside(NeuGc), reacted specifically with the i-active ganglioside(NeuGc) used as an immunogen. Structurally related gangliosides, such as GM3(NeuGc), sialosylparagloboside (SPG) (NeuGc), or I-active ganglioside(NeuGc), corresponding gangliosides [GM3 containing N-acetylneuraminic acid (NeuAc), SPG(NeuAc), i-active ganglioside(NeuAc), and I-active ganglioside(NeuAc)], other gangliosides, or neutral glycosphingolipid (GSL) were not recognized by the monoclonal antibody. These findings indicate that the SHS-1 monoclonal antibody may be specific for NeuGc-containing i-active ganglioside. On the other hand, the other two monoclonal antibodies, MSG-1 and SPS-20, which were generated by immunizing mice with purified ganglioside GM3(NeuGc) and SPG(NeuGc), respectively, showed cross-reactivity to structurally related gangliosides. The MSG-1 monoclonal antibody exhibited reactivity to ganglioside GM3(NeuAc). The SPS-20 monoclonal antibody also cross-reacted with SPG(NeuAc), i-active ganglioside(NeuGc), and i-active ganglioside(NeuAc). Neither MSG-1 nor SPS-20 reacted with corresponding gangliosides, other gangliosides, or neutral GSLs tested. Using the SHS-1 antibody specific for i-active ganglioside(NeuGc), we studied the expression of NeuGc-containing antigen in human colon cancer tissue. An NeuGc-containing glycoconjugate was detected in the colon cancer tissue.

Gangliosides as a possible receptor on the bovine erythrocytes for Theileria sergenti.

To elucidate whether or not gangliosides on the bovine erythrocytes serve as a receptor for Theileria sergenti merozoites, the reactivities of the T. sergenti piroplasms with gangliosides were studied by the liposome agglutination test. The parasites reacted weakly with I-active ganglioside containing N-acetylneuraminic acid (NeuAc) and strongly with I-active ganglioside containing N-glycolylneuraminic acid (NeuGc). However, none of the other gangliosides expressed on the bovine erythrocytes, such as GM3 (NeuAc), GM3 (NeuGc), sialosylparagloboside (SPG) (NeuAc), SPG (NeuGc), i-active ganglioside (NeuAc), and i-active ganglioside (NeuGc), were recognized. After infection with T. sergenti, furthermore, the content of I-active ganglioside (NeuAc) was less (p < 0.05), and I-active ganglioside (NeuGc) content was much less in the erythrocytes (p < 0.01), though the contents of other NeuAc- and NeuGc-containing gangliosides did not so vary with T. sergenti infection. These results suggest that the parasites recognize the I-active ganglioside as their receptor and bind preferentially to NeuGc-carrying I-active ganglioside rather than to NeuAc-type in the target cell membranes, and that the reduction of the contents of I-active gangliosides (NeuAc and NeuGc) on the erythrocytes was related to T. sergenti infection.

Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase α activity

The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase α were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase α, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 μM of CSE, 50 μM of SPG or GM3, and 80 μM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, the ceramide backbone was also found to be necessary for maximal inhibition since the inhibition was largely abolished by substituting the lipid backbone with cholesterol. Increasing the number of sialic acid moieties per molecule further enhanced the inhibition, while elongating the sugar chain diminished it. It was clearly shown that the N-acetyl residue of the sialic acid moiety is particularly essential for inhibition by both SPG and GM3 because the loss of this residue or substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase α activity.

Alteration of ganglioside composition in the erythrocytes associated with Theileria sergenti infection.

The changes in ganglioside composition of bovine erythrocytes associated with Theileria sergenti infection were investigated using the erythrocytes before and after the infection. The erythrocytes before infection with T. sergenti had GM3, sialosylparagloboside (SPG), i-active, and I-active ganglioside as predominant gangliosides. After infection with T. sergenti merozoites, the contents of SPG and i-active ganglioside were slightly less, and I-active ganglioside content was much less in the erythrocytes, though GM3 content did not so vary. The decreased I-active ganglioside content showed a recovery as the parasitemia waned to low level in the infected cattle. The total amount of lipid-bound sialic acid also decreased in the erythrocytes after the infection. Similar changes were also caused by the incubation of liposomes containing ganglioside fraction obtained from bovine erythrocytes with T. sergenti piroplasms. These results suggest that the reduction of the contents of SPG, i-active, and I-active ganglioside on the erythrocytes was related to the T. sergenti infection.

Regional distribution of sulfatide in human kidney, and anti-sulfatide antibodies in sera from patients with nephritis detected by TLC immunostaining

Glycolipids were isolated from lipid extract of human kidney. The major neutral glycolipids have been identified as ceramide monohexoside (CMH), ceramide dihexoside (CDH), ceramide trihexoside (CTH), and globoside. As the major acidic glycolipids, Gal Cer sulfate (sulfatide), Lac Cer sulfate, GM3, sialosyl paragloboside, GD3, and disialosyl paragloboside were identified and the most abundant component was sulfatide. Sulfatide was 2 times more concentrated in medulla compared to cortex. In addition, the localization of sulfatide antigen was determined in renal sections by immunoperoxidase staining method. Strong positive staining with sulfatide was observed in distal tubules, limbs of Henle's loop and collecting tubules of normal tissue, whereas glomeruli were negative of staining. However, positive results of glomerular epithelial cells occurred in FSGS and IgA nephropathy so far. Acidic fraction of lipid extract were chromatographed and then tested for antigen by immunostaining. Sera from patients with nephritis contain antibodies to the sulfatides of human kidney as determined by the direct binding of antibody to thin-layer chromatograms. These results suggest that sulfatide antigen may play important role in the occurrence and aggravation of glomerular diseases.

Erythrocyte gangliosides act as receptors for Neisseria subflava: identification of the Sia-1 adhesin

Neisseria gonorrhoeae was recently shown to bind to a subset of lactose-containing glycolipids (N. Strömberg, C. Deal, G. Nyberg, S. Normark, M. So, and K.-A. Karlsson, Proc. Natl. Acad. Sci. USA 85:4902-4906, 1988). A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAc alpha 2-3Gal beta 1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein profile identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subflava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GN01. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was nonfimbrial in nature, with aggregates of the adhesin extended out from the cells in a patchy fashion.

Glycosphingolipid Patterns of Peripheral Blood Lymphocytes, Monocytes, and Granulocytes Are Cell Specific1

We analyzed the amounts and types of glycosphingolipids (GSLs) from peripheral blood lymphocytes, monocytes, and granulocytes isolated by counter-current elutriation. The three cell types contained different amounts of neutral and acidic GSLs. The highest amount of neutral GSLs (109 micrograms/10(8) cells) was found in granulocytes, with considerably less found in monocytes (11 micrograms/10(8) cells) and lymphocytes (4 micrograms/10(8) cells). The neutral GSLs were composed of four types of lipids, GL1 through GL4 (mono-, di-, tri-, and tetraosylceramide). The highest percentage of GL1 was detected in lymphocytes and the lowest percentage in granulocytes, with the reverse order observed for GL2. GL3 and GL4, which were minor components of the neutral GSLs, were highly cell specific, with lymphocytes containing GL3 and GL4 of the globo series, granulocytes containing GL3 and GL4 of the lacto or neolacto series, and monocytes containing GL3 and GL4 of both types. The acidic GSL, sialosyl hexaosylceramide (lacto-series), was abundant in granulocytes but not in monocytes or lymphocytes. Another ganglioside, GM3, although present in all three cell types, was most abundant in monocytes and lymphocytes, whereas sialosyl paragloboside was higher in granulocytes than in lymphocytes and monocytes. These results indicate that peripheral blood lymphocytes, monocytes, and granulocytes have distinct "GSL fingerprints."

Detection of GD3 ganglioside in childhood acute lymphoblastic leukemia with monoclonal antibody to GD3: Restriction to immunophenotypically defined T‐cell disease

We have recently reported that the disialoganglioside GD3 is found in cellular lipid extracts of T-cell acute lymphoblastic malignancies (T-ALL) but is not detectable by resorcinol staining in extracts of non-T acute lymphoblastic leukemia blasts (non-T-ALL). We have now extended this study to assess the detectability of GD3 in T-ALL vs non-T-ALL utilizing an anti-GD3 antibody, R24. Gangliosides isolated from T-ALL and non-T-ALL blasts by two different methods were separated by thin-layer chromatography and stained with anti-GD3 and a control antibody specific for GM3 and sialosylparagloboside (SPG). Anti-GD3 reactivity was observed in extracts from T-ALL cells in all cases, whereas GD3 was not detected in any of the non-T-ALL samples. The anti-GM3/SPG antibody stained GM3 in all of the leukemic samples analyzed as well as SPG in the non-T-ALL samples. Indirect immunofluorescence was used to assess the expression of GD3 at the surface of leukemic blasts. Fluorescence-activated cell sorting analysis with R24 showed that whereas T-ALL blasts were highly reactive with this antibody, non-T-ALL blasts were totally unreactive. In an analysis of a larger number of leukemia patients by fluorescence microscopy, 20 out of 28 samples with the T-ALL phenotype were positive for R24, whereas zero out of 11 non-T-ALL samples were reactive. These results confirm our earlier finding of the specificity of GD3 to the T-ALL subclass of childhood leukemias and furthermore suggest the potential value of anti-GD3 as an immunological tool for the diagnosis and therapy of T-cell ALL.

Biosynthesis of the cancer-associated sialyl-Lex determinant in human amniotic fluid.

Biosynthesis of the sialyl-Lex determinant (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)-GlcNAc beta 1-3-R) in human amniotic fluid has been shown to proceed via the same sequence of glycosylation steps established previously for lung carcinoma PC 9 cells (Holmes, E. H., Ostrander, G.K. & Hakomori, S. (1986) J. Biol. Chem. 261, 3737-3743): sialylation of type-2-chain-precursor substrates (paragloboside) by an amniotic alpha 2-3-sialyltransferase precedes fucosylation of sialylated intermediates (sialosyl paragloboside) by an organ-characteristic alpha 1-3-L-fucosyltransferase.