De-N-acetyllactotriaosylceramide as a novel cationic glycosphingolipid of bovine brain white matter: isolation and characterization.

A novel cationic lipid was separated from bovine brain white matter by a series of chromatographies on carboxymethyl-Sephadex and silica gel in chloroform and methanol. Its structure was identified unambiguously as de-N-acetyllactotriaosylceramide (deNAcLc(3)Cer) by mass spectrometry and (1)H NMR. The natural occurrence of this glycolipid in white matter extract was detected by immunostaining of thin-layer chromatography with monoclonal antibody 5F5, which is directed to deNAcLc(3)Cer and recognizes the terminal beta-glucosaminyl (GlcNH(2)) residue, having a free NH(2) group. A de-N-acetylase capable of hydrolyzing the N-acetyl group of Lc(3)Cer was detected in bovine brain extract using N-[(14)C]acetyl-labeled Lc(3)Cer as a substrate. The biogenesis and possible functional significance of deNAcLc(3)Cer are discussed.

Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry.

Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w'') and fatty acyl chains (Y0'-w'') was identified by MS3 as their [M+H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophage-lineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species.

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking.

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are all enriched in the detergent-insoluble complex which has been suggested to be purified caveolae. I studied the relationship of the molecules with caveolae in cultured cells by immunocytochemical methods. In cells reacted with antibodies to various membrane proteins and lipids on ice and fixed before applying secondary antibodies, labeling did not show concentration in caveolae. In contrast, when cells were incubated with the primary and secondary antibodies on ice and then transferred to 37 degrees C without fixation, labeled Thy-1.2, beta 2-microglobulin, lactosyl ceramide, ceramide tetrahexose, Forssman antigen, and sphingomyelin became concentrated in caveolae, whereas labeled transferrin receptor did not. Thy-1.2 and sphingolipids formed common patches and were sequestered in the same caveolae when crosslinked with two primary antibodies simultaneously. On the other hand, when either Thy-1.2 alone or lactosyl ceramide alone was crosslinked and sequestered to caveolae, the other antigen remained evenly distributed. Caveolar sequestration of the antigens occurred in the presence of cytochalasin D, nocodazole, or a mixture of the two reagents. The results show that not only GPI-anchored proteins but also glycosphingolipids and sphingomyelin are sequestered in caveolae only after crosslinking, and that the sequestration does not require the intact cytoskeleton.

Isolation and characterization of a trisialyllactosylceramide, GT3, containing an O-acetylated sialic acid in cod fish brain.

An O-acetylated ganglioside that generated a trisialyllactosylceramide or GT3 by base treatment was found for the first time in cod fish brain. This ganglioside was isolated by high-performance liquid chromatography, and characterized by fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy in addition to chemical analysis. The structure was identified as a modified GT3 in which the external sialic acid is O-acetylated at the C-9 position. The chemical structure is as follows: II3(9-O-Ac-NeuAc2-8NeuAc2-8NeuAc2-3)Lac Cer.

Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer) and to its 9-O-acetylated derivative.

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other unidentified gangliosides. In order to characterize further the epitope of A2B5, two new ganglioside antigens isolated from chicken brain are identified in this study. One is the ganglioside NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide (GT3) and the other is a 9-O-acetylated derivative of GT3). This derivative was purified from 10-day embryonic chicken brain. Acetyl groups substituted on sialic acid were removed either by alkali treatment or by incubation with influenza virus C, which contains receptor-destroying enzyme (a neuraminidate 9-O-acetyl esterase). The product of alkali treatment or viral action was detected by the antibody 18B8 which is specific for GT3. The deacetylated product still reacts with A2B5. These data and the results of mild oxidation of the antigen with sodium periodate suggest that the epitope recognized by antibody A2B5 contains the trisialyl structure found in GT3 but does not include the polyalcohol chain of the terminal sialic acid which can be oxidized by periodate or acetylated without modifying the affinity for the antibody. The epitope recognized by A2B5 is different from the epitope recognized by the antibody 18B8 in that 18B8 requires the three sialic acids with an intact and unsubstituted polyalcohol chain. Antibody 18B8 does not bind to 9-O-acetylated GT3 or GT3 oxidized by sodium periodate.

Antibodies against ganglioside GT3 in the sera of patients with type I diabetes mellitus.

Clinical and experimental data support the concept that type I diabetes mellitus results from autoimmune destruction of pancreatic beta cells. Although both proteins and glycolipids are targets of anti-islet cell antibodies, the Ag have not been purified or characterized. Previously, we observed that rat insulinoma (RIN) cell lines varied in their reactivity with both human antibodies and murine mAb A2B5, which binds to polysialo gangliosides. To determine the chemical basis of the varied immunoreactivity, we analyzed the glycosphingolipids of 5 RIN lines. Glycolipids bound by two mAb and by antibodies in the sera of type I diabetics were identified. The more immunoreactive RIN lines contained a much higher content of gangliosides and a higher proportion of complex gangliosides. The major gangliosides were GM3, GD3, and GT3. By high performance TLC immunostaining, we demonstrated that A2B5 and R2D6, an anti-beta cell murine mAb, bound most strongly to ganglioside GT3. The binding of human sera to gangliosides was analyzed by an ELISA assay. Although both normal and diabetic sera contained antibodies to various glycolipids, binding to GT3 was significantly elevated in 31 new-onset type I diabetics (p less than 0.001). The presence of the GT3 trisialosyl epitope on human islet cells was shown by immunofluorescent staining by both R2D6 and A2B5. These findings support previous suggestions that gangliosides play an important role in the immunopathology of type I diabetes, and identify for the first time a specific ganglioside Ag that is the target for autoantibodies in a subset of diabetic patients.

Trisialosyllactosylceramide (GT3) is a ganglioside of human lung.

A trisialoganglioside not found before in human tissue was isolated from showed this to be II3(NeuAc)3-LacCer or GT3. The ganglioside constituted 40% of the trisialogangliosides of human lung or 0.2 nmol ganglioside per g tissue.

Isolation and partial characterization of fucose- and N-acetylglucosamine-containing neutral glycosphingolipids from human senile cataracts.

Five neutral glycosphingolipids were isolated from human cataracts using silicic acid column chromatography and preparative thin-layer chromatography. Three of these glycolipids were partially identified by gas-liquid chromatography as glucosylceramide, lactosylceramide, and trihexosylceramide. Two glucosamine-containing glycosphingolipids (one of which contained fucose) were also detected. One of these two lipids contained galactose, glucose, N-acetylglucosamine in the molar ratio of 2:1:1, while the other contained fucose, galactose, glucose, and N-acetylglucosamine with the molar ratio of 1:2:1:1. Dihydrosphingosine (sphinganine) was the major long-chain base detected in all these fractions. The fatty acids of these neutral glycosphingolipids were variable in chain length, although the majority of them were greater than 20 carbons. This represents the first time whereby a family of neutral glycosphingolipids has been detected in human cataracts. This is also the first demonstration of the existence of a neutral fucolipid in the lenses of any species.

Monosialogangliosides of rabbit skeletal muscle. Characterization of N-acetylneuraminosyl lacto-N-noroctaosyl ceramide.

Rabbit skeletal muscle contained 28.4 nanomol/g wet weight of lipid-bound sialic acid, and 73.4% of the total lipid-bound sialic acid was recovered in a monosialoganglioside fraction. Monosialoganglioside components were isolated and the structures were determined by exoglycosidase treatment and permethylation analysis. The major monosialoganglioside was GM3 (73.2% of monosialogangliosides), and gangliosides of the lacto-series with at least three repeating units of lactosamine, Gal(beta, 1-4)GlcNAc(beta, 1-3), were identified. A newly found ganglioside was N-acetylneuraminosyl lacto-N-noroctaosyl ceramide, NeuAc(alpha, 2-3)Gal(beta, 1-4)GlcNAc (beta, 1-3)Gal(beta, 1-4)GlcNAc(beta, 1-3)Gal(beta, 1-4)GlcNAc(beta, 1-3)Gal(beta, 1-4)Glc(beta, 1-1)ceramide. The amounts of lacto-series gangliosides in the monosialoganglioside fraction were as follows: sialosyl lacto-N-neotetraosyl ceramide (9.8%), sialosyl lacto-N-norhexaosyl ceramide (11.9%) and sialosyl lacto-N-noroctaosyl ceramide (5.1%). N-Glycolylneuraminic acid was found in sialosyl lacto-N-neotetraosyl ceramide and sialosyl lacto-N-norhexaosyl ceramide but the amount was less than 0.2%. The mobilities of lacto-series gangliosides were compared with those of ganglio-series gangliosides. Fatty acid and long chain base compositions were also determined.

An improved technique for separation of neutral glycosphingolipids by high-performance liquid chromatography.

We have developed a high-performance liquid chromatographic (HPLC) procedure for separation of O-acetyl-N-p-nitrobenzoyl derivatives of six neutral glycosphingolipids: glucosylceramide, lactosylceramide, globotriaosylceramide, lactotriaosylceramide, globotetraosylceramide, and neolactotetraosylceramide. The recoveries of glucosylceramide and globotetraosylceramide for the derivatization procedure and HPLC analysis were approximately 75%, and one nanomole of glycolipid could be detected. The procedure was used for analysis of human erythrocyte neutral glycolipids.

Structural characterization of lactotetraosylceramide, a novel glycosphingolipid isolated from human meconium.

In the course of work on a systematic structural mapping of nonacid glycosphingolipids of human meconia, special attention was given to a major component preliminarily identified as an isomer of neolactotetraosylceramide (paragloboside). This component was isolated in its pure form from meconium of a blood group O individual and subjected to detailed structural analyses, using mass spectrometry and proton NMR spectroscopy on intact permethylated and permethylated-reduced (LiAlH4) derivatives, and gas liquid chromatography on degradational products of native, permethylated, and permethylated-reduced derivatives. The isolated compound was conclusively shown to have the structure Galp beta 1 yields 3GlcNAcp beta 1 yields 3Galp beta 1 yields 4Glcp beta 1 yields 1Cer, and is thus identified as lactotetraosylceramide. The major fatty acids were 2-hydroxy fatty acids with 16 and 20 to 24 carbon atoms, and the bases were sphingosine and phytosphingosine. This glycolipid, although not isolated and structurally characterized before, has long been thought of as a precursor substance of the Lewis active glycolipids and of ABH-active glycolipids with a type 1 saccharide chain.