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.     

Novel phosphonoglycosphingolipids containing pyruvylated galactose from the nervous system of Aplysia kurodai

We have reported the existence of a phosphonoglycosphingolipid containing a pyruvylated galactose, FGL-IIb, in nerve fibers of Aplysia kurodai (Araki, S., Abe, S., Ando, S., Kon, K., Fujiwara, N. & Satake, M. (1989) J. Biol. Chem. 264, 19922-19927). We have now isolated two other pyruvylated galactose-containing phosphonoglycosphingolipids, named FGL-V and FGL-IIa, from the nervous tissue of Aplysia, and characterized them as [3,4-O-(S-1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3[6'-O-(2- aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6) Gal beta 1----4Glc beta 1----1 ceramide and [3,4,O-(S-1-carboxyethylidene)] Gal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 11----ceramide, respectively. Their major aliphatic components are palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, the nervous system of Aplysia contains several pyruvylated phosphonoglycolipids.     

Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue.     

Immunochemical studies on blood groups. Purification and characterization of radioactive 3H-reduced di- to hexasaccharides produced by alkaline beta-elimination-borohydride 3H reduction of Smith degraded blood group A active glycoproteins

Treatment of blood group A active glycoprotein from human ovarian cyst fluid by one stage of Smith degradation followed by alkaline beta-elimination in the presence of NaB[ 3H4 ] (Carlson degradation) liberated tritiated oligosaccharide alditols. The carbohydrate mixture was fractionated by gel filtration, elution from charcoal, paper chromatography, and high pressure liquid chromatography. Structures were established based on sugar composition, periodate oxidation, methylation analysis, and analysis of oligosaccharide alditols as permethylated and N-trifluoroacetylated derivatives by gas-liquid chromatography-mass spectrometry. The following structures have been deduced: Gal beta 1----3GalNAc-ol, GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1---- 3GlcNAc beta 1----6(3-deoxy)GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1----3[GlcNAc beta 1----6]GalNAc-ol, Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3[Gal beta 1----4GlcNAc beta 1----6]Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol. The smaller structures represent pieces of the larger structures. Together they provide direct evidence for the core structure of the carbohydrate side chains in the blood group substances as proposed by K. O. Lloyd and E. A. Kabat [1968) Proc. Natl. Acad. Sci. U.S.A. 61, 1470-1477). Oligosaccharides previously isolated after Carlson degradation of intact human ovarian cyst fluid HLeb , Lea, and B substances and from human and horse B substances contained various alpha-linked L- fucopyranose and alpha-linked Gal substitutions on the composite structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three saponins from Oxytropis species.

Three flavonoids and three saponins have been isolated from Oxytropis species. Their structures were determined as isorhamnetin-3-O-beta-D-glucoside, rhamnetin-3-O-beta-D-galactoside, apigenin, 3-O-[alpha-L-rhamnopyranosyl (1----2)-beta-D-glucopyranosyl(1----4)-beta-D-glucuronopyranosyl]+ ++soyasapogenol B, 3-O-[beta-D-glucopyranosyl(1----2)-beta-D-glucuronopyranosyl] azukisapogenol and a new saponin 3-O-[beta-D-glucopyranosyl(1----2)-beta-D-glucopyranosyl]-25-O-alpha-L- rhamnopyranosyl-(20S,24S)-3 beta,16 beta, 20,24,25-pentahydroxy-9,19-cycloanostane.     

Immunochemical studies on the interaction between synthetic glycoconjugates and alpha-L-fucosyl binding lectins

Evonymus europaea lectin precipitated with alpha DGal(1----3) beta DGal(1----4)beta DGlcNAc-bovine serum albumin (BSA), alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA, alpha LFuc(1----2)beta DGal(1----4)DGlcNAc, and alpha DGal(1----3)[alpha LFuc(1----2)]beta DGal-BSA. However, the lectin neither precipitated with alpha LFuc(1----2)-beta DGal-BSA, alpha DGal(1----3)beta DGal-BSA, or beta DGal(1----4)beta DGlcNAc-BSA nor agglutinated erythrocytes of Oh phenotype having multiple terminal beta DGal(1----4)beta DGlcNAc residues. These results indicate that the minimal structural requirement for glycoprotein precipitation or cell agglutination by the lectin includes any of the three trisaccharides (fucosylated or nonfucosylated) derived from the blood group B tetrasaccharide. The monosaccharides linked to the beta-D-galactosyl residue in the blood group B tetrasaccharide, namely, alpha-D-galactose, alpha-L-fucose, and N-acetyl-beta-D-glucosamine, participate almost equally in binding to the lectin in as much as removal of any one of these sugars reduces the inhibiting potency of the resulting trisaccharide. alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA (H type 1) and alpha LFuc(1----2)beta DGal(1----4)beta DGlcNAc (H type 2) were precipitated to the same extent. The E. europaea lectin neither precipitated alpha DGal(1----4)-beta DGal(1----4)beta DGlcNAc-BSA, Lea-BSA, Leb-BSA, or beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA nor agglutinated Oh,Lea and Oh,Leb erythrocytes, demonstrating that terminal D-galactose linked alpha-(1----4) to subterminal beta-D-galactose, or alpha-L-fucose linked to N-acetylglucosamine, prevents lectin binding. Corey-Pauling-Koltun molecular models, built on the basis of data from 1H NMR and hard-sphere exo-anomeric (HSEA) calculations provided by Lemieux and co-workers [Lemieux, R. U., Bock, K., Delbaere, L. T. J., Koto, S., & Rao, V. S. (1980) Can. J. Chem. 58, 631-653], show that these alpha-D-galactosyl and alpha-L-fucosyl groups act to sterically hinder lectin binding to these oligosaccharides; these observations also suggest that the lectin binds to the beta-side of these oligosaccharides. These sides, on both blood group H type 1 and blood group H type 2 oligosaccharides, provide a similar contour which can fully account for their equal reactivity with E. europaea lectin. The only difference found between Lotus and Ulex I lectins in precipitating ability was that only Lotus precipitated with beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-beta-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man alpha 1----6(Man alpha 1----3)Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man alpha 1----6(Man alpha 1----3)Man alpha 1----6 and Man alpha 1----3Man alpha 1----6 and Man alpha 1----3Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were found at Asn12, as previously published [Schlüter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc alpha 2----3 or Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(NeuAc alpha 2----6 or NeuAc alpha 2----3Gal-beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNac beta 1----4GlcNAc beta 1---- with some bifurcation at ----6Man alpha 1----6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal beta 1----3GalNAc alpha 1----, monosialylated or desialylated at position 3 of Gal or/and position 6 of GalNAc.     

Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of oligosaccharide fragments of the repeating unit of Salmonella kentucky O-specific polysaccharide and conversion of the oligosaccharides into the glycosyl phosphates

alpha-D-Man-(1----2)-alpha-D-Man-(1----3)-D-Gal, a structural fragment of the main chain of Salmonella serogroups C2 and C3 O-specific polysaccharides, and the isomer with the central residue beta have been synthesised, as have some oligosaccharides related to the structure of the O-specific polysaccharide of S. kentucky (serogroup C3), namely, alpha-D-Glc-(1----4)-D-Gal, alpha-D-Man-(1----3)-[alpha-D-Glc-(1----4)]-D-Gal, and alpha-D-Man-(1----2)-alpha-D-Man-(1----3)-[alpha-D-Glc-(1----4)]-D-Gal, and the isomers with the D-Glc unit beta. Each oligosaccharide was converted into the alpha-glycosyl phosphate.     

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.     

Structures of oligosaccharides cleaved by base-borohydride from an I, H and Lea active ovarian cyst glycoprotein

Oligosaccharides were cleaved by base-borohydride from an I, H and Lea active ovarian cyst glycoprotein and purified by Bio-Gel P-6 and paper chromatography. The structures of five oligosaccharides, determined by compositional analyses, quantitative periodate oxidation, chronic acid oxidation, methylation analyses and enzymatic degradations, were as follows: oligosaccharide I, beta DGal1----3DGalNAc-ol; II, beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGalNAc-ol; III, alpha LFuc1----2 beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGalNAc-ol; IV, beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGal1NAcol; and V, beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3 beta DGal1----4 beta DGlcNAc1----6[beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3 beta DGal1----3 beta DGal1----3]DGalNAc-ol. Of the oligosaccharides 60% had a molecular size of a decasaccharide or smaller, the tetra- and pentasaccharides II and III predominating. Oligosaccharides I through IV have been previously isolated from several glycoproteins by other laboratories; the decasaccharide, V, is a new structure.     

Total synthesis of globotriaosyl-E and Z-ceramides and isoglobotriaosyl-E-ceramide

Stereoselective, total synthesis of O-alpha-D-galactopyranosyl-(1----4) -O-beta-D-galactopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-N -tetracosanoyl-[2S,3R,4E (and 4Z)]-sphingenine and O-alpha-D -galactopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-O-beta-D -glucopyranosyl-(1----1)-N-tetracosanoyl-(2S,3R,4E)-sphin gen ine was achieved by using O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl) -(1----4)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyranosyl)-(1----4)-2,3,6- tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate, O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl) -(1----4)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyranosyl)-(1----4)-2,3,6- tri-O-acetyl-alpha (and beta)-D-glucopyranosyl fluoride, and O-(2,3,4,6-tetra-O-acetyl-alpha-D -galactopyranosyl)-(1----3)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyran osyl)-(1----4)-2,3,6-tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate.     

Chemical structure of neutral sugar chains isolated from human mature milk kappa-casein

The carbohydrate chains linked to human kappa-casein from mature milk were released by alkaline borohydride treatment as reduced oligosaccharides. The neutral oligosaccharides of lower molecular weight were fractionated and purified by gel filtration and preparative thin layer chromatographies. Seven neutral oligosaccharides (a di- (0.5%), two tetra- (30.5%), two penta- (5.4%) and two hexasaccharide alditols (10.9%] were obtained in homogeneity, and followed by methylation analysis with gas-liquid chromatography-mass spectrometry and by anomer analysis with 13C nuclear magnetic resonance. Their chemical structures were identified to be Gal beta 1----3GalNAc-ol (I), Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (II), Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (III), GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (IV), GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (V), Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (VI) and Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (VII). Five oligosaccharide alditols (III-VII) were the novel carbohydrate chains of kappa-casein from mammalian milk.     

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.     

The structure of the O-glycosidic oligosaccharide chains of the major Zajdela hepatoma ascites-cell-membrane glycoprotein

Glycoprotein MII2, the major cell surface glycoprotein (molecular mass 110 kDa) of Zajdela hepatoma ascites cells, contains about 25 O-glycosidic oligosaccharide chains per molecule. They were released as oligosaccharide-alditols by alkaline borohydride treatment of MII2, and purified by gel filtration on Bio-Gel P-6 followed by high-voltage paper electrophoresis. Four oligosaccharide-alditol fractions (A-D) were obtained in relative yields of 8:6:3:3. The structure of the components of fractions A-C was determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis, to be as follows. (A) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B1) NeuAc alpha(2----3)Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B2) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (C) NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol. On the basis of sugar composition and characteristics on Bio-Gel P-6 filtration, paper electrophoresis and thin-layer chromatography, the structure of the carbohydrate component of fraction D is proposed to be as follows. (D) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol     

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.     

Steroidal saponins from the rhizomes of Smilax sieboldii.

Six new steroidal saponins were isolated from the rhizomes of Smilax sieboldii. Their structures were determined by spectroscopic analysis and hydrolysis to be 3 beta-hydroxy-(25R)-5 alpha-spirostan-6-one (laxogenin) 3-O-beta-D-glucopyranosyl-(1----4)-O-[alpha-L-arabinopyranosyl-(1- ---6)]-beta-D-glucopyranoside, laxogenin 3-O-alpha-L-arabinopyranosyl-(1----6)-beta-D-glucopyranoside, 3 beta,27-dihydroxy-(25S)-5 alpha-spirostan-6-one 3-O-beta-D-glucopyranosyl-(1----4)-O-[alpha-L-arabinopyranosyl-(1- ---6)]- beta-D-glucopyranoside, 26-O-beta-D-glucopyranosyl-3 beta,22 xi,26-trihydroxy-(25R)-5 alpha-furostan-6-one 3-O-alpha-L-arabinopyranosyl-(1----6)-beta-D-glucopyranoside, 26-O-beta-D-glucopyranosyl-3 beta,22 xi,26-trihydroxy-(25R)-5 alpha-furostan-6- one 3-O-beta-D-glucopyranosyl-(1----4)-O-[alpha-L-arabinopyranosyl-(1- ---6)]- beta-D-glucopyranoside and (25R)-5 alpha-spirostan-3 beta-ol (tigogenin) 3-O-beta-D-glucopyranosyl-(1----4)-O-[alpha-L-arabinopyranosyl- (1----6)]-beta-D-glucopyranoside. The inhibition of cAMP phosphodiesterase by the saponins was evaluated.     

New evidence of the substrate specificity of endo-beta-N-acetylglucosaminidase D

The susceptibility of a variety of oligosaccharides to endo-beta-N-acetylglucosaminidase D was investigated. The oligosaccharides having the structures of Man alpha 1----6 (GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, derived from complex type triantennary sugar chains, released +/- Fuc alpha 1----6GlcNAcOT upon incubation with the enzyme at almost the same rate as Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. When the reaction products were reduced with NaB3H4 and analyzed by Bio-Gel P-4 column chromatography, a new radioactive peak was detected in both cases. This new radioactive oligosaccharide was confirmed to be Man alpha 1----6(GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAcOT in the former case and Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAcOT in the latter. These results indicated that endo-beta-N-acetylglucosaminidase D does not require the presence of a free hydroxyl group at the C-4 position of the alpha-mannosyl residue of the trisaccharide glycon: Man alpha 1----3Man beta 1----4GlcNAc beta 1----.     

Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H). I. Isolation and chemical characterization

A series of blood group H antigens reacting with monoclonal antibody MBrl has been found in human blood group A and AB erythrocytes, but not in O or B erythrocytes. These H antigens are clearly different from the globo-H structure (Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer), which was previously isolated from O erythrocytes and is also reactive with the MBrl antibody. The new series of H antigens associated with blood group A has been characterized as having TLC mobilities which approximately coincide with those of H2, H3, and H4 glycolipids. One of these A-associated H antigens, having a similar TLC mobility as the H2 glycolipid, was isolated from A erythrocytes and was characterized by 1H NMR spectroscopy, methylation analysis, and enzymatic degradation as having the structure shown below: (formula, see text). The structure represents a precursor of the repetitive A epitope attached to type 2 chain, previously called type 3 chain A (Clausen, H., Levery, S. B., Nudelman, E., Tsuchiya, S., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1199-1203). This A-associated H structure is hereby called type 3 chain H.