Developmental and topographic expression of neutral glycosphingolipids in vertebrate brain.

We have examined neutral glycosphingolipid (Ngsl) expression in embryonic (E), post-natal (P) and adult rodent brain employing digoxigenin immunostaining (DIG-IS) and anti-Ngsl antisera (both monospecific polyclonal and monoclonal) directed toward specific carbohydrates. Several previously unknown long-chain (-CHO = or > 4) Ngsls have been identified. Four Ngsls have been purified and characterized as GgOse4Cer or GA1, Galactosyl beta 1-3globoside, Fuc alpha 1-3nLcOse4Cer or Lewis X (Le(x)) and a novel GalNAc beta 1-4GA1. A few transient bands appear at different developmental ages. Several fast migrating cerebrosides have also been identified during the early phase of active myelination and tentatively characterized as derivatives of galactosylceramide. Immunohistochemical localization of GA1, Le(x) and GalNAc-GA1 in adult rodent brain shows unique and specific cellular topographies of these carbohydrate antigens.

Structures of four oligosaccharides derived from the glycoinositolphospholipid of Leishmania adleri

Glycoinositolphospholipids (GIPLs) were extracted from the trypanosomatid Leishmania adleri by hot phenol extraction and the carbohydrate moieties isolated after base cleavage. Purification of the crude oligosaccharides by high performance anion exchange (HPAE) chromatography yielded four fractions whose structures were determined by a combination of methylation analysis, fast atom bombardment (FAB) mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy

The use of NMR spectroscopy in the structure determination of a Leptomonas samueli glycosylphosphosphingolipid-derived oligosaccharide

Nuclear magnetic resonance (NMR) spectroscopy provides an extremely powerful technique to determine the structure of oligosaccharides, particularly when used in conjuction with other physical techniques such as methylation analysis and fast atom bombardment mass spectroscopy (FAB-MS). This brief review describes the application of NMR to the determination of the structure of an oligosaccharide isolated from the glycophosphosphingolipid (GPS) from the monogenetic trypanosomatid Leptomonas samueli. Where ambiguities arise in the NMR interpretation, the use of other data will be discussed

Free and protein-linked glycoinositolphospholipids in Trypanosoma cruzi

Two glycoinositol phospholipids (GIPL A and GIPL B) have been purified from epimastigotes of Trypanosoma cruzi at the logarithmic phase of growth (2 days). The GIPLs differ mainly in the lipid moiety and are similar to the lipopeptidophosphoglycan (LPPG) previously isolated from epimastigotes at the stationary phase (4-5 days). [3H]-palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in GIPL A and into a sphinganine ceramide with palmitic acid and lignoceric acid as the fatty acids in GIPL B. The lipids could be released by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) or glycosylphosphatidylinositol phospholipase D (GPI-PLD) from rat serum. The oligosaccharides share the common core structure of the glycosylphosphatidilinositol (GPI) membrane anchors. Microheterogeneity was demonstrated, as well as substitution by galactose, which is mainly in the furanose configuration as was previously described for the LPPG. However, methylation analysis indicated that 20 percent of the galactose is present as terminal pyranose units. In infective trypomastigotes, [3H]-palmitic acid was incorporated into the anchor of the Tc-85 glycoprotein. The lipid cleaved by phospholipase C digestion was identified as 1-O-hexadecylglycerol and the main oligosaccharide has the structure of the conserved core of all GPI anchors. [3H]-palmitic acid-labelled Tc-85 released into the culture medium as membrane vesicles showed 80 percent resistance to the action of PI-PLC. However, after mild alkaline hydrolysis, part of the radioactivity was released by the enzyme

Molecular characterization of gangliotetraosylceramide (GA1) in normal human brain and its developmental change.

Several neutral glycosphingolipids have recently been purified from normal human brain to the criterion of migration as homogeneous bands in two different solvent systems. One of these has been permethylated and analyzed by gas chromatography. Stepwise specific exoglycosidase hydrolysis confirms its structure as GAl or Gal beta 1-->3GalNAc beta 1-->4Gal beta 1-->4Glc beta 1-->1Cer. The neutral glycosphingolipids of rat CNS have been examined in order to investigate changes in GA1 during critical ages of brain development. Employing the highly sensitive techniques of digoxigenin (DIG) immunostaining and Fluorescence Assisted Carbohydrate Electrophoresis (FACE), we have identified several previously uncharacterized long chain neutral glycosphingolipids in brain and myelin. A major band with Rf close to that of nLcOse5Cer purified from bovine erythrocytes has been identified as GA1 by immuno-TLC using mono-specific polyclonal anti-GA1 antisera. It appears in 5 day-postpartum (P5) developing brain, increases until 21 days (P21) and subsequently declines. This phasic change along with changes in other nonsialylated glycosphingolipids of the developing brain strongly suggests that GA1 and other neutral glycosphingolipids may play a mediator role(s) in brain development and/or myelinogenesis.