g(m3)-ganglioside and sialosylparagloboside

The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase alpha 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 alpha, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 microM of CSE, 50 microM of SPG or GM3, and 80 microM 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 alpha activity.

Topics: Animals; Carbohydrate Sequence; Cattle; DNA Polymerase II; G(M1) Ganglioside; G(M3) Ganglioside; Globosides; Glycolipids; Kinetics; Molecular Sequence Data; N-Acetylneuraminic Acid; Sialic Acids; Sulfates; Thymus Gland

Gangliosides of hepatomas have been analyzed by using a monoclonal antibody directed to N-acetylneuraminosyl(alpha 2-6)lactoneotetraosylceramide (sialyl(alpha 2-6)paragloboside), which was prepared by injecting the monosialoganglioside fraction of human meconium into BALB/c mice. The monoclonal antibody, named MSG-15, was found to bind sialyl(alpha 2-6)paragloboside, but it failed to react with other gangliosides, including N-acetylneuraminosyl(alpha 2-3)lactoneotetraosylceramide (sialyl (alpha 2-3)paragloboside) and "Ii"-type gangliosides. MSG-15 was found to recognize NeuAc alpha 2-6Gal beta structure of the ganglioside. Gangliosides obtained from human hepatomas were analyzed by immunostaining on high-performance thin-layer chromatography plates using the monoclonal antibody MSG-15. All primary hepatoma samples used in this study (nine samples) were found to contain sialyl(alpha 2-6)paragloboside, which accounted for 13-31% of the monosialoganglioside fractions in the hepatomas. Furthermore, MSG-15 recognized several monosialogangliosides in addition to sialyl(alpha 2-6)paragloboside. These gangliosides apparently also contain a terminal NeuAc alpha 2-6Gal beta structure. Other ganglioside fractions obtained from hepatoma and meconium were immunostained on thin layer chromatography plates with MSG-15. Additionally, another monoclonal antibody (H-11), which recognizes terminal lactosamine structure, was used to immunostain these fractions after sialidase treatment. Bands stained with both monoclonal antibodies showed similar mobilities to each other in the di- and trisialoganglioside fractions as well as monosialoganglioside fraction. In control liver, GM3 ganglioside accounted for 92% of monosialoganglioside fraction, and sialyl(alpha 2-6)paragloboside accounted for less than 1% of the fraction. Immunohistochemical study by using MSG-15 in tissue sections from hepatocellular carcinoma and normal liver tissues demonstrated that only hepatocellular carcinoma cells gave a positive reaction. These results suggest that the biosynthetic pathway of gangliosides containing NeuAc alpha 2-6Gal beta 1-4GlcNAc beta structure is activated in hepatoma cells.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Hepatocellular; Chemical Phenomena; Chemistry; G(M3) Ganglioside; Gangliosides; Globosides; Glycosphingolipids; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Oligosaccharides

The precise specificities H-D antibodies and newly obtained avian antisera to the H-D antigen-active glycosphingolipids. N-glycolylneuraminyl (alpha 2-3) lactosyl ceramide (hematoside) and N-glycolylneuraminyl (alpha 2-3) lactoneotetraosyl ceramide (sialosylparagloboside) were compared by gel precipitation and hemagglutination inhibition using other gangliosides with a related structure and chemically modified hematoside derivatives and the oligosaccharides from both the antigenic glycosphingolipids. Human H-D antibodies were found to have the same affinity for both antigenic glycosphingolipids; however, the avian anti-hematoside or anti-sialosylparaglobosides showed a higher affinity for the homologous antigen. Human H-D antibodies were directed more exactly toward the terminal sialic acid residue and more roughly to the hydrophobic portion of both compounds; however, the two avian antibodies were directed more specificity to the whole structure of the homologous antigen glycosphingolipid.

Topics: Animals; Blood Group Antigens; Chickens; Epitopes; G(M3) Ganglioside; Globosides; Glycosphingolipids; Humans; Isoantibodies; Structure-Activity Relationship