Immunochemistry of I/i-active oligo- and polyglycosylceramides from rabbit erythrocyte membranes. Determination of branching patterns of a ceramide pentadecasaccharide by 1H nuclear magnetic resonance

1H NMR spectra of the ceramide hexasaccharide obtained after the removal of the terminal alpha-Gal and subterminal beta-Gal residues from the ceramide decasaccharide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc (beta 1-6)]Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer, showed that terminal and internal GlcNAc residues are differentiated by their chemical shifts. This finding enabled us to determine the primary structure of the title compound as Gal(alpha 1-3)Gal(beta 1-4)GlcNAc (beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-6)]Gal(beta 1-4)GlcNAc (beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-6)]Gal(beta 1-4)GlcNAc (beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer. Alternative branching of this oligosaccharide chain was excluded since the removal of all terminal alpha-Gal and penultimate beta-Gal residues yielded a ceramide nonasaccharide containing one terminal and two internal 1----3-linked GlcNAc residues, as well as two terminal 1----6-linked GlcNAc units. The intermediate degradation products of the ceramide deca- and pentadecasaccharides , viz. the ceramide octa- and dodecasaccharide , obtained by the removal of alpha-Gal residues only, as well as the linear ceramide heptasaccharide, Gal(alpha 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)Cer, and ceramide hexasaccharide, Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc (beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer, were also investigated. The usefulness of the glycosylation-induced chemical shifts is discussed.     

Syngeneic monoclonal antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma

It has previously been reported that a mouse (C57BL/6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to react only with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). In the present study, the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAc alpha 2-3Gal beta 1-4Glc (or -GlcNAc)) of gangliosides by three different assay systems including enzyme immunostaining on thin layer plates, sandwich radioimmunoassay, and enzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosides having NeuGc terminus, including NeuGc alpha 2-3Gal beta 1-4Glc-ceramide and NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide, nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was isolated from B16 melanoma cells, and its structure was determined to be NeuAc alpha 2-3Gal beta 1-4Glc-ceramide by fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:1 as its long-chain base and C16:0, C24:1, and C24:0 as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.     

Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus

A sensitive assay system for receptor activity of gangliosides to paramyxovirus was developed. This system involves incorporation of gangliosides into neuraminidase-treated chicken erythrocytes (asialoerythrocytes) followed by estimation of virus-mediated agglutination and hemolysis. The asialoerythrocytes coated with I-active ganglioside (Sia 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-Cer) were effectively agglutinated by hemagglutinating virus of Japan (HVJ, Sendai virus). The hemolysis of the asialoerythrocytes mediated by HVJ was restored to the highest level by labeling the cells with gangliosides possessing lacto-series oligosaccharide chains, i.e., I-active ganglioside, N-acetylneuraminosylparagloboside (SiaPG(NeuAc)), and i-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer). The specific receptor activity of ganglioside GD1a possessing a gangliotetraose chain was lower than those of the gangliosides described above. Gangliosides GM3, GD3, GM1a, GD1b, SiaPG(NeuGc) showed little effect on the restoration of HVJ-mediated hemolysis. On infection with Newcastle disease virus (NDV), the highest specific restoration of lysis was found in chicken asialoerythrocytes coated with SiaPG(NeuAc or NeuGc) and GM3(NeuAc or NeuGc), whereas those coated with I-active ganglioside, GD3, GM1a, and GD1b showed very low NDV-mediated hemolysis. The above results indicate that the determinants of receptor for HVJ contain sialylated branched and/or linear lacto-series oligosaccharides carried by I,i-active gangliosides and SiaPG(NeuAc) and sialosylgangliotetraose chain carried by GD1a. The determinants for NDV are carried by SiaPG(NeuAc or NeuGc) containing linear lacto-series oligosaccharide and GM3(NeuAc or NeuGc). The absence of detectable binding of free oligosaccharides obtained from I-active ganglioside and sialoglycoprotein GP-2 isolated from bovine erythrocyte membranes as HVJ receptor (Suzuki, Y., et al. J. Biochem. (1983) 93, 1621-1633; (1984) 95, 1193-1200) indicates that HVJ recognizes the sialooligosaccharides oriented out of the lipid bilayer in the cell membranes where the hydrophobic ceramide or peptide backbone of the receptor is integrated.     

A new ganglioside in human meconium detected by antiserum against the human milk sialyloligosaccharide, LS-tetrasaccharide b

Antibodies directed against human milk sialyloligosaccharides [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59] are used to identify human meconium gangliosides by radioimmuneoverlay-thin-layer chromatography or by direct binding on nitrocellulose filters of sialyl[3H]oligosaccharide alditols obtained from gangliosides after ozonolysis and alkali-fragmentation. Thin-layer chromatograms of meconium monosialylgangliosides immunostained with rabbit antisera specific for LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) or LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) reveal their corresponding gangliosides, 6'-LM1 and a previously undescribed ceramide derivative of LS-tetrasaccharide b, respectively. The sialyl[3H]oligosaccharides derived from the monosialylganglioside fraction of meconium are separated by paper chromatography and assayed for binding to specific anti-sialyloligosaccharide sera. Antisera specific for LS-tetrasaccharide c and 3'-sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) identify their corresponding 3H-labeled haptens released from the major meconium gangliosides 6'-LM1 and GM3, respectively. Binding of a ganglioside-derived sialyl[3H]oligosaccharide by anti-LS-tetrasaccharide b serum is consistent with the presence in meconium of a monosialylganglioside with the following proposed structure: (formula; see text)     

A new ganglioside of the lactotetraose series, GalNAc-3'-isoLM1, detected in human meconium

A monoclonal antibody produced by immunization with cells of the human glioma cell line D-54 MG reacted with ganglioside GM2. The binding epitope of the antibody was found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal. Immunological detection of glycolipid antigens on thin-layer plates with this monoclonal antibody, DMAb-1, revealed the presence of a new ganglioside. This ganglioside, co-migrating with NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer(6'-LM1) and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GalNAc beta 1-4Gla beta 1-4Glc beta 1-1Cer (GalNAc-isoGM1) at chromatographic separation was isolated from human meconium. Its structure was determined by permethylation and fast atom bombardment-mass spectometry analyses. The new ganglioside was found to be a combination of the lacto and ganglio series gangliosides, and the structure found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GlcNAc alpha 1-3Gal beta 1-4Glc beta 1-1Cer(GalNAc-3'-isoLM1).     

Identification of novel gangliosides containing lactosaminyl-GM1 structure from rat spleen

Two novel monosialogangliosides were isolated from rat spleen. The structures of the gangliosides (shown below, where NeuNGc is N-glycolylneuraminic acid) were characterized by compositional analysis, methylation analysis, hydrolyses with exoglycosidases, direct probe fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectrometry. A novel structure common to both gangliosides was N-acetyllactosaminyl-GM1 LacN-GM1; where GM1 is II3NeuAc-GgOse4Cer), and this is the first paper to report the occurrence of a new group of gangliosides. [formula: see text] Furthermore, in a monosialoganglioside fraction of rat spleen, the occurrence of a ganglioside having two lactosamine units (Gal alpha 1-3(Gal beta 1-4GlcNAc beta 1-3)2-GM1(NeuAc) (alpha Gal-(LacN)2-GM1] was suggested. These gangliosides have a unique structure, which includes the ganglio series ganglioside core and the extended modification characteristic of the lacto series.     

Identification of N-glycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. 252Cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2----8NeuGc alpha 2----3Gal beta I----4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.     

Primary structure of human milk nona- and decasaccharides determined by a combination of fast atom bombardment mass spectrometry and1H-/13C-nuclear magnetic resonance spectroscopy. Evidence for a new core structure,iso-lacto-N-octaose

The structure of a nonasaccharide and of two decasaccharides isolated from human milk has been investigated by using methylation, fast atom bombardment mass spectrometry and 1H-/13C-nuclear magnetic resonance spectroscopy. The structures of these oligosaccharides were: trifucosyllacto-N-hexaose; Fuc alpha 1-2Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3[Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4Glc, difucosyllacto-N-octaoses; Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6[Gal beta 1-3GlcNAc beta 1-3]Gal beta 1-4Glc and Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6[Fuc alpha 1-3 Gal beta 1-3GlcNAc beta 1-3]Gal beta 1-4Glc. The two decasaccharides possess a new type of core structure proposed to be named iso-lacto-N-octaose.     

Isolation and characterization of a heptaglycosylceramide from bovine erythrocyte membranes.

A heptaglycosylceramide was isolated from bovine erythrocyte membranes. The structure was characterized to be Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta1-3)Gal(beta 1-4)Glc-NAc(beta 1-4)al(beta 1-4)GlcCer. A hexaglycosylceramide that has the same sequence except for the terminal alpha-galactosyl unit has also been isolated. We have previously found that gangliosides isolated from bovine erythrocyte membranes contain a keratan sulfate type repeating unit --[3Gal(beta 1-4)-GlcNAc beta]--n. This study shows that the keratan sulfate type repeating unit is also present in the neutral glycosphingolipids of bovine erythrocyte membranes.     

Immunochemistry of I/i-active oligo- and polyglycosylceramides from rabbit erythrocyte membranes. Characterization of linear, di-, and triantennary neolactoglycosphingolipids

A triantennary ceramide pentadecasaccharide (BIrab-2) with blood group I and B-like activity and an unbranched ceramide heptasaccharide (Birab) with i- and B-like activity were isolated in high yield from rabbit erythrocyte membranes. The structures of the native substances and the products obtained after treatment with alpha-galactosidase (BIrab-2 alpha, Birab alpha) and subsequent Smith degradation (BIrab-2 alpha SD) were determined by sugar analysis, methylation analysis, and fast atom bombardment-mass spectrometry of the permethylated derivatives. Together with the results of 1H NMR analysis (Dabrowski, U., Hanfland, P., Egge, H., Kuhn, S., and Dabrowski, J. (1984) J. Biol. Chem. 259, 7649-7651), the following structures were established for the native substances: (formula; see text) and Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer. Both compounds exhibit strong blood group B-like activity. BIrab-2 alpha is a strong receptor for human anti-I cold agglutinin and Birab for anti-i cold agglutinin.     

Structure elucidation of sulphated oligosaccharides from recombinant human tissue plasminogen activator expressed in mouse epithelial cells.

Sulphated N-linked carbohydrate chains isolated from recombinant human tissue plasminogen activator expressed in mouse epithelial (C127) cells were analysed as oligosaccharide alditols by methylation analysis, liquid secondary ion mass spectrometry, and one- and two-dimensional 1H-NMR spectroscopy. The results demonstrate that the major component has the following novel structure: NeuAc-alpha 2-6Gal beta 1-4GlcNAc beta 1-2[NeuAc alpha 2-3Gal beta 1- 4GlcNAc beta 1-4]-Man alpha 1-3[NeuAc alpha 2-3(SO4-6)Gal beta 1- 4-GlcNAc beta 1-2Man alpha 1-6]-Man beta 1-4GlcNAc beta 1- 4[Fuc alpha 1-6]GlcNAc-o1.     

Characterization of the binding of propionibacterium granulosum to glycosphingolipids adsorbed on surfaces. An apparent recognition of lactose which is dependent on the ceramide structure

The binding properties of a strain of Propionibacterium granulosum derived from human skin was investigated with reference to glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells using externally (125I) and metabolically [( 35S]methionine) labeled bacteria. Binding was found to lactosylceramide (LacCer; Gal beta 1-4Glc beta 1-Cer) but not to glycolipids lacking the lactose sequence (i.e. Glc beta 1-Cer, Gal beta 1-Cer or Gal alpha 1-4Gal beta 1-Cer). In microtiter wells, binding occurred at 50 ng and became half-maximal at the theoretical value for a monomolecular layer of LacCer (i.e. 100-200 ng/well). The bacteria also bound to glycolipids with various substitutions (e.g. GalNAc beta 1-4, Gal beta 1-3GalNAc beta 1-4, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4, Gal alpha 1-3, GlcNAc beta 1-3, Gal beta 1-3GlcNAc beta 1-3, Gal beta 1-4GlcNAc beta 1-3, and Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3) at the Gal of LacCer, although only those species with GalNAc beta 1-4 or Gal beta 1-3GalNAc beta 1-4 were as active as LacCer itself. Glycolipids with other additions (e.g. Gal alpha 1-4 and NeuAc alpha 2-3) were negative. For unsubstituted LacCer, the binding required either a trihydroxy base or 2-hydroxy fatty acid, specifying the epithelial type of ceramide; LacCer composed of a dihydroxy base and nonhydroxy fatty acid was negative. This is interpreted as indicating that the proper presentation of the binding epitope depends on the ceramide structure. The relevance of this to biological membranes has not yet been established. Neither free lactose (up to 20 mg/ml) nor lactose-bovine serum albumin (5 mg/ml) prevented the binding of bacteria to LacCer, two facts that support the solid-phase binding data demonstrating a low affinity binding and the crucial importance of a particular lactose epitope.     

The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant)

One of the monoclonal (AH-6) antibodies prepared by hybridoma technique against human gastric cancer cell line MKN74 was found to react with a series of glycolipids having the Y determinant (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc). The structure of one such glycolipid isolated from human colonic cancer and from dog intestine was identified as lactodifucohexaosyl-ceramide (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide; IV3,III3Fuc2nLc4Cer). The hapten glycolipid did not react with monoclonal antibodies directed to Lea, Leb, and X-hapten structures, and the AH-6 antibody did not react with the X-hapten ceramide pentasaccharide (Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), H1 glycolipid (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), nor with glycolipids having the Leb (Fuc alpha 1 leads to 2Gal beta 1 leads to 3[Fuc alpha 1 leads 4]GlcNAc beta 1 leads to R) determinant. The antibody reacted with blood group O erythrocytes, but not with A erythrocytes. Immunostaining of thin layer chromatography with the monoclonal antibody AH-6 indicated that a series of glycolipids with the Y determinant is present in tumors and in O erythrocytes.     

Specific inhibition of macrophage migration inhibition factor by fucosylated glycolipid RM.

The effects of glycolipids on the interaction of the MIF (migration inhibition factor) with rat macrophages were examined using a migration inhibition assay system. MIF activity was specifically blocked by fucosylated Glycolipid RM [Gal alpha 1-3Gal(2-1 alpha Fuc) beta 1-3GalNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide, (1978) J. Biochem. 83, 85-90], but not by Cytolipin R, hematoside, or blood group B active glycolipid [Gal alpha 1-3Gal(2-1 alpha Fuc) beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide]. Inhibition of MIF activity was proportional to the concentration of Glycolipid RM. These findings suggest that Glycolipid RM acts as a receptor for MIF.     

Biosynthesis in vitro of SA-Lex and SA-diLex by alpha 1-3 fucosyltransferases from colon carcinoma cells and embryonic brain tissues.

The sialyl-fucosyl-lactosamine-epitope present in sialyl (SA)-Lex (NeuAc alpha 2-3Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta 1-3Gal beta 1-4Glc-Cer), a carcinoembryonic antigen, has been recognized recently as a ligand for the binding of leukocyte-endothelial cell adhesion molecule 1 (LECAM-1) to myeloid and tumour cell surfaces. We have recently detected the presence of an alpha 1-3 fucosyltransferase (FucT-3) activity in both embryonic chicken brain (ECB) and human colon carcinoma cells (Colo-205) which catalyses the biosynthesis in vitro of SA-Lex and SA-diLex. Fucosyltransferase activities from both sources are stimulated in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Co2+ and Fe2+), although absolute metal requirement is not observed. Substrate specificity studies with this partially purified (ECB, 3000-fold; Colo-205, 100-fold) novel FucT-3 indicate the preference for terminally sialyl-substituted glycolipid acceptors, as observed by the lower Km values when sialyl-neolactotetraosyl ceramide, LM1, (Neu-Gc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4 Glc-Cer; Km = 0.048 mM) and sialyl-norhexaosylceramide, NeuGc-nLc6, (Neu-Gc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer; Km = 0.032 mM) were used as substrates. Fucosyltransferase from Colo-205 requires the presence of the acyl group of the ceramide moiety and an acetyl group on glucosamine in the acceptor glycolipid since lyso-LM1 was found to be completely inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

A GM1b-derived disialoganglioside GD1c is the predominant ganglioside of rat thymocytes.

The thin-layer chromatographic (TLC) pattern of gangliosides of rat thymocytes showed a profile characterized by the occurrence of a predominant ganglioside which did not correspond to any reference gangliosides of rat brain. The ganglioside was isolated from rat thymus, and characterized by compositional analysis, methylation analysis, sialidase treatment, negative-ion fast atom bombardment (FAB) mass spectrometry, and proton NMR spectroscopy. The structure was elucidated to be NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-3GalNac beta 1-4Gal beta 1-4Glc beta 1-1Cer. This is the major ganglioside of rat thymus lymphoid cells and is one of the GM1b-derived gangliosides, GD1c, having two N-glycolylneuraminic acids. This is the first report on the occurrence of GD1c in normal animal cells.     

Chemical characterization of milk oligosaccharides of the Japanese black bear, Ursus thibetanus japonicus

Two trisaccharides, two tetrasaccharides, one penta-, one hexa-, two hepta-, one deca- and two undeca-saccharides were isolated from several Japanese black bear milk samples by chloroform/methanol extraction, gel filtration and preparative thin-layer chromatography. The oligosaccharides were characterized by 1H-NMR as follows: Gal(alpha 1-3)Gal(beta 1-4)Glc (alpha 3'-galactosyllactose), Fuc(alpha 1-2)Gal(beta 1-4)Glc (2'-fucosyllactose), Gal(alpha 1-3)(Fuc(alpha 1-2))Gal(beta 1-4)Glc (B-tetrasaccharide), Gal(alpha 1-3)Gal(beta 1-4)(Fuc(alpha 1-3))Glc, Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]Glc (B-pentasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)Glc (monofucosylhexasaccharide), Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)Glc (difucosylheptasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]Glc (difucosylheptasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)]Gal(beta 1-4)Glc (difucosyldecasaccharide), Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)[Gal(alpha 1-3) Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)]Gal(beta 1-4)Glc (trifucosylundecasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)[Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)]Gal(beta 1-4)Glc (trifucosylundecasaccharide). Lactose was present only in trace amounts. B-pentasaccharide was a dominant saccharide in early lactation milk, while alpha 3'-galactosyllactose was dominant in milk, later. The milk oligosaccharides of the Japanese black bear were compared with those of the Ezo brown bear.     

Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy

The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.     

Characterization of a novel type of chain-terminator Gal beta 1-6Gal beta 1-4)GlcNAc in an oligosaccharide related to N-glycosylated protein glycans isolated from GM1 the urine of patients with gangliosidosis

Two new oligosaccharides were isolated from the urine of a patient with GM1 gangliosidosis. Final purification of the oligosaccharides was accomplished by capillary supercritical fluid chromatography. Structural analysis was by chemical analysis, chemical-ionization mass spectrometry and 400-MHz 1H-NMR spectroscopy, leading to two primary structures. The first is derived from a classical triantennary N-acetyllactosamine-type glycan: Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-3Man beta 1-4GlcNAc. The second is unusual with a terminal disaccharide Gal beta 1-6Gal, which had not yet been described for glycans of the N-acetyllactosamine type: Gal beta 1-6Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6Man beta 1-4GlcNAc.