alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

Chemical structures of three neutral oligosaccharides obtained from horse (thoroughbred) colostrum.

1. Three neutral oligosaccharides were obtained from horse colostrum by ion-exchange, activated charcoal column and preparative paper chromatographies. 2. The following structures were elucidated by methanolysis, methylation analysis and 75 MHz 13C-NMR spectroscopy; Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (HM-3a), Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4Glc (HM-3b) and Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc (HM-5). 3. HM-3a and HM-5 have been found in human milk, named as lacto-N-neotetraose and lacto-N-neohexaose, respectively. HM-3b has been isolated from goat milk. 4. An homology and heterogeneity were assumed among the following animal species' milk oligosaccharides: horse, human, goat and tammar wallaby.     

Identification of erythrocyte Gal alpha 1-3Gal glycosphingolipids with a mouse monoclonal antibody, Gal-13

The Gal alpha 1-3Gal structural determinant has been found to have a unique distribution in mammals. Although this determinant is abundantly expressed by erythrocytes and nucleated cells of many mammals, it has not been detected in human cells. However, our previous studies (Galili, U., Rachmilewitz, E. A., Peleg, A., and Flechner, I. (1984) J. Exp. Med. 160, 1519-1531; Galili, U., Clark, M. R., and Shohet, S. B. (1986) J. Clin. Invest. 77, 27-33) have suggested that this epitope is present in small amounts and may be involved in immune-mediated destruction of senescent human erythrocytes. To have a means for exploring this possibility and for studying the species and tissue distribution of this epitope we have raised a monoclonal antibody (Gal-13) which specifically binds to glycoconjugates with a nonreducing terminal Gal alpha 1-3Gal disaccharide. Mice were immunized with rabbit erythrocytes, which express an abundance of glycoconjugates with Gal alpha 1-3Gal epitopes. Clones were screened with a solid-phase binding assay (enzyme-linked immunosorbent assay) for antibodies which bound to ceramide pentahexoside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3-Gal beta Gal beta 1-4Glc1-1Cer) but not to ceramide trihexoside (Gal alpha 1-4Gal beta 1-4Glc1-1Cer). Gal-13 bound to a number of neutral glycosphingolipids from rabbit and bovine erythrocytes. These glycosphingolipids have previously been shown to be a family of linear and branched polylactosamine structures, which have non-reducing terminal Gal alpha 1-3Gal epitopes. The antibody did not bind to the human blood group B glycolipid, Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc1-1Cer, and, therefore, branching at the penultimate galactose blocks Gal-13 binding. However, after removal of the fucose from the B antigen Gal-13 recognized the resulting derivative. Other Gal alpha 1-3Gal glycosphingolipids with an isogloboside or globoside core structure were not recognized by Gal-13 suggesting that the antibody binds to Gal alpha 1-3Gal carried by a lactosamine core structure. Gal-13 has been used to demonstrate that the Gal alpha 1-3Gal ceramide pentahexoside has been evolutionarily conserved in red cells of animals up to the stage of New World monkeys but is not found in Old World monkey red cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical characterization of milk oligosaccharides of the polar bear, Ursus maritimus

Two trisaccharides, three tetrasaccharides, two pentasaccharides, one hexasaccharide, one heptasaccharide, one octasaccharide and one decasaccharide were isolated from polar bear milk samples by chloroform/methanol extraction, gel filtration, ion exchange chromatography and preparative thin-layer chromatography. The oligosaccharides were characterized by 1H-NMR as follows: the saccharides from one animal: Gal(alpha1-3)Gal(beta1-4)Glc (alpha3'-galactosyllactose), Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose), Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (B-tetrasaccharide), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (A-tetrasaccharide), Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)Gal(beta1-4)Glc NAc(beta1-6)]Gal(beta1-4)Glc; the saccharides from another animal: alpha3'-galactosyllactose, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, A-tetrasaccharide, GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]Glc (A-pentasaccharide), Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[F uc(alpha1-3)]Glc (difucosylheptasaccharide) and Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)?Gal(alpha1-3) Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)?Gal(beta1-4)Glc (difucosyldecasaccharide). Lactose was present only in small amounts. Some of the milk oligosaccharides of the polar bear had alpha-Gal epitopes similar to some oligosaccharides in milk from the Ezo brown bear and the Japanese black bear. Some milk oligosaccharides had human blood group A antigens as well as B antigens; these were different from the oligosaccharides in Ezo brown and Japanese black bears.     

Structural characterization of oligosaccharides in the milk of an African elephant (Loxodonta africana africana)

The oligosaccharides present in the milk of an African elephant (Loxodonta africana africana), collected 4 days post partum, were separated by size exclusion-, anion exchange- and high-performance liquid chromatography (HPLC) before characterisation by (1)H NMR spectroscopy. Neutral and acidic oligosaccharides were identified. Neutral oligosaccharides characterised were isoglobotriose, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc and a novel oligosaccharide that has not been reported in the milk or colostrum of any other mammal: Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc. Acidic oligosaccharides that are also found in the milk of Asian elephant were Neu5Ac(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc, while Neu5Gc(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc have not been found in Asian elephant milk. The oligosaccharides characterised contained both alpha(2-3)- and alpha(2-6)-linked Neu5Ac residues. They also contain only the type II chain, as found in most non-human, eutherian mammals.     

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.     

Globotetraosylceramide is recognized by the pig edema disease toxin

The pig edema disease toxin has been shown by a tlc glycolipid binding assay to bind specifically to globotetraosylceramide (Gb4, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer.). Binding was reduced for globotriosylceramide (Gb3, Gal alpha 1-4Gal beta 1-4GlcCer) and more markedly for the Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer). Paragloboside, blood group A glycolipids, glycolipids terminating in Gal NAc beta 1-4Gal-, and glycolipids in which globoside was present as an internal sequence did not bind the toxin. Isogloboside (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4GlcCer) was efficiently recognized. This toxin is genetically related to the verotoxin (or Shiga-like) family of toxins for which Gb3 has been shown to be the receptor. The difference in susceptibility of cell lines to the cytotoxicity of the pig edema disease toxin and the Shiga and Shiga-like toxins is consistent with the difference in receptor glycolipid binding.     

Structural determination of three neutral oligosaccharides in bovine (Holstein-Friesian) colostrum, including the novel trisaccharide; GalNAc alpha 1-3Gal beta 1-4Glc

Two trisaccharides, and a pentasaccharide were obtained from bovine colostrum. Their chemical structures were determined by using methylation and 13C-NMR analyses as follows: GalNac alpha 1-3Gal beta 1-4Glc, Gal alpha-1-3Gal beta 1-4Glc, GaL beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc. GalNAc alpha 1-3Gal beta 1-4Glc, which was identified in this study, is a novel oligosaccharide from natural sources. Gal alpha 1-3Gal beta 1-4Glc and Gal beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc (lacto-N-novopentaose) have been already found in ovine colostrum, and in horse colostrum and marsupial milk, respectively.     

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.     

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.     

Chemical characterization of the oligosaccharides in beluga (Delphinapterus leucas) and Minke whale (Balaenoptera acutorostrata) milk

Carbohydrates were extracted from the milk of a beluga, Delphinopterus leucas (family Odontoceti), and two Minke whales, Balaenoptera acutorostrata (Family Mysticeti), sampled late in their respective lactation periods. Free oligosaccharides were separated by gel filtration and then neutral oligosaccharides were purified by preparative thin layer chromatography and gel filtration, while acidic oligosaccharides were purified by ion-exchange chromatography, gel filtration and high performance liquid chromatography (HPLC). Their structures were determined by 1H-NMR. In one of the Minke whale milk samples, lactose was a dominant saccharide, with Fuc(alpha1-2)Gal(beta1-4)Glc(2'-fucosyllactose), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc(lacto-N-neotetraose), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc(A-tetrasaccharide), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (para lacto-N-neohexaose), Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl lacto-N-neotetraose), Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LST c) and Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl para lacto-N-neohexaose) also being found in the milk. The second Minke whale sample contained similar amounts of lactose, 2'-fucosyllactose and A-tetrasaccharide, but no free sialyl oligosaccharides. Sialyl lacto-N-neotetraose and sialyl para lacto-N-neohexaose are novel oligosaccharides which have not been previously reported from any mammalian milk or colostrum. These and other oligosaccharides of Minke whale milk may have biological significance as anti-infection factors, protecting the suckling young against bacteria and viruses. The lactose of Minke whale milk could be a source of energy for them. The beluga whale milk contained trace amounts of Neu5Ac(alpha2-3)Gal(beta1-4)Glc(3'-N-acetylneuraminyllactose), but the question of whether it contained free lactose could not be clarified. Therefore, lactose may not be a source of energy for suckling beluga whales.     

Structural characterization of neutral oligosaccharides of human midcycle cervical mucin

It was previously shown that alkaline borohydride treatment of human midcycle cervical mucin releases a heterogeneous population of reduced neutral, sialylated, and sulfated oligosaccharides (Yurewicz, E. C., and Moghissi, K. S. (1981) J. Biol. Chem. 256, 11895-11905). Three major neutral oligosaccharides were isolated with approximate compositions of Fuc:Gal:GlcNAc:N-acetylgalactosaminitol (GalNAcol) = 0:2:1:1 (A1), 1:2:1:1 (A2), and 2:2:1:1 (A3). They comprised roughly 21%, 13%, and 8% of human cervical mucin oligosaccharide chains, respectively. In the present report, each was analyzed by periodate oxidation, methylation, and sequential degradation with glycosidases. A1 was shown to contain more than one component, but structural analyses clearly demonstrated the presence of one predominant (75%) tetrasaccharide. The proposed structure, Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3)GalNAcol, has previously been found in human gastric, submaxillary, and ovarian cyst mucins in their carbohydrate-to-protein linkage regions. beta-Galactosidase from Aspergillus niger selectively cleaved the Gal beta 1-4GlcNAc linkage in the intact tetrasaccharide. Enzymatic hydrolysis of the Gal beta 1-3GalNAcol linkage required prior removal of the Gal beta 1-4GlcNAc beta 1-unit attached to 0-6 of GalNAcol. The data for A2 indicated a mixture of two oligosaccharides, Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNAc beta 1-6(Gal beta 1-3)GalNacol and Fuc alpha 1-2Gal beta 1-4GlcNac beta 1-6(Gal beta 1-3)-GalNacol, in an approximate molar ratio of 3 to 4:1, respectively. Two structures are consistent with the data obtained for A3: Fuc alpha 1-2Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNAc beta 1-6(Gal beta 1-3)GalNAcol and/or Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNac beta 1-6(Fuc alpha 1-2Gal beta 1-3)GalNacol. The results indicate that A1 represents the "core" tetrasaccharide of the larger human cervical mucin oligosaccharides A2 and A3.     

Human serum contains a novel beta 1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo (N-acetyllactosaminoglycans).

Incubation of UDP-GlcNAc and radiolabeled GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) with human serum resulted in the formation of the branched hexasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (2) in yields of up to 22.2%. The novel reaction represents midchain branching of the linear acceptor; the previously known branching reactions of oligo-(N-acetyllactosaminoglycans) involve the nonreducing end of the growing saccharide chains. The structure of 2 was established by use of appropriate isotopic isomers of it for degradative experiments. The hexasaccharide 2 was cleaved by an exhaustive treatment with jack bean beta-N-acetylhexosaminidase, liberating two GlcNAc units and the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). Endo-beta-galactosidase from Bacteroides fragilis cleaved 2 at one site only, yielding the disaccharide GlcNAc beta 1-3Gal (4) and the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (5). The structure of 5 was established by partial acid hydrolysis and subsequent identification of the disaccharide GlcNAc beta 1-6Gal (6), together with the trisaccharides GlcNAc beta 1-6Gal beta 1-4GlcNAc (7) and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (8) among the cleavage products. Galactosylation of 2 with bovine milk beta 1,4-galactosyltransferase and UDP-[6-3H]Gal gave the octasaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4GlcNAc beta 1-3([6-3H]-Gal beta 1-4GlcNAc beta 1-6)[U-14C] Gal beta 1-4GlcNAc (17), which could be cleaved with endo-beta-galactosidase into the trisaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3Gal (18) and the branched pentasaccharide GlcNAc beta 1-3-([6-3H]Gal beta 1-4GlcNAc beta 1-6) [U-14C]Gal beta 1-4GlcNAc (19). Partial hydrolysis of 2 with jack-bean beta-N-acetylhexosaminidase gave the linear pentasaccharide 1 and the branched pentasaccharide Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (20). The serum beta 1,6-GlcNAc transferase catalyzed also the formation of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (11) from UDP-GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (10). The pentasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (16), too, served as an acceptor for the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

New galactosyllactose containing alpha-glycosidic linkage isolated from ovine (Booroola dorset) colostrum

Three trisaccharides, tetra-, penta-, hexa- and certain higher oligosaccharides were obtained from ovine colostrum as free forms. The chemical structure of the three trisaccharides were determined by methylation and 13C-NMR analyses to be as follows: Gal alpha 1-3Gal beta 1-4Glc, Gal beta 1-3Gal beta 1-4Glc (3'-galactosyllactose) and Gal beta 1-6Gal beta 1-4Glc (6'-galactosyllactose). Gal alpha 1-3Gal beta 1-4Glc, which had been confirmed as the oligosaccharide portion of a glycolipid prepared biosynthetically from rat spleen or bone marrow, has been identified for the first time from natural sources as a free form. The trisaccharide containing alpha-galactosyl unit is a novel compound in mammalian milk.     

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.     

Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal 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-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.     

Accumulation of unique globo-series glycolipids in PC 12h pheochromocytoma cells

In a previous paper, we reported the presence of a unique globo-series glycolipid as one of the major neutral glycolipid: Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1' Cer, in the subcloned PC 12h pheochromocytoma cells (Ariga, T., Yu, R. K., Scarsdale, J. N., Suzuki, M., Kuroda, Y., Kitagawa, H., and Miyatake, T. (1988) Biochemistry 27, 5335-5340). Recently we found that the subcloned PC 12h cells accumulated other unusual neutral glycolipids. In order to characterize these glycolipids, PC 12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated four minor neutral glycolipids, which were purified by droplet counter-current, Iatrobeads column, and preparative thin-layer chromatographies. These glycolipid structures were determined by fast atom bombardment-mass spectrometry, proton nuclear magnetic resonance spectroscopy, permethylation study, and sequential degradation with various exoglycosidases to be as follows: A, Fuc alpha 1-2Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; B, GalNAc beta 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; C, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; and D, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer. Glycolipids A and B were tentatively characterized in normal rat small intestine (Breimer, M. E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 557-568; Angstrom, J., Breimer, M. E., Falk, K.-E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 682-688). Glycolipids C and D have not been reported in the literature.     

Studies on the chemical structure of neutral glycosphingolipids in eggs of the sea hare, Aplysia juliana.

Six neutral glycosphingolipids (GL-1-GL-6) were obtained from eggs of the sea hare (Aplysia juliana) and were characterized by FABMS, 1H-NMR, partial acid hydrolysis, methylation studies and GC analysis of the component sugars, fatty acids and long-chain bases. The following structures were determined to be Glc beta 1-1Cer (89%) and Gal beta 1-1Cer (11%) for GL-1, Glc beta 1-1Cer (47%) and Gal beta 1-1Cer (53%) for GL-2 having hydroxy fatty acids in the ceramide moiety, Gal beta 1-4Glc beta 1-1Cer for GL-3, Fuc alpha 1-2Gal beta 1-4Glc beta 1-1Cer for GL-4, Gal alpha 1-2Gal beta 1-4Glc beta 1-1Cer for GL-5 and GalNAc alpha 1-3(Gal alpha 1-2)Gal beta 1-4Glc beta 1-1Cer for GL-6. The fatty acid composition of each glycosphingolipid, except for GL-2, which contained 2-hydroxypalmitic acid, consisted of mostly saturated C16-C20 acids, especially palmitic acid and stearic acid. The long-chain bases of all glycosphingolipids consisted mainly of branched nonadeca-4-sphingenine and octadeca-4-sphingenine. GL-6, which was one of the major glycosphingolipids, may be a precursor of a series of phosphonoglycosphingolipids which have been isolated from the skin of A. kurodai.     

Occurrence of a unique sialyl tetrasaccharide in colostrum of a bottlenose dolphin (Tursiops truncatus)

Crude oligosaccharides were recovered from bottlenose dolphin (Tursiops truncatus) colostrum after chloroform/methanol extraction of lipids and protein precipitation, and purified using gel filtration, anion exchange chromatography and high performance liquid chromatography (HPLC). Their chemical structures characterized by NMR spectroscopy were as follows: GalNAc(beta1-4)[Neu5Ac(alpha2-3)]Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)Glc and Gal(alpha1-4)Gal(beta1-4)Glc. The monosialyltetrasaccharide and neutral trisaccharide have not previously been found as free forms in any natural sources including milk or colostrum, although these structures have been found in the carbohydrate units of glycosphingolipids GM2 and Gb3.     

Isolation and NMR spectroscopic characterization of sialic acid compounds and hemofiltrate of chronic uremia patients

Eight sialyloligosaccharides have been isolated from the hemofiltrate of a patient with end stage renal disease using reverse osmosis, gel filtration, ion-exchange and high-performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H- and 13C-NMR spectroscopy: 1 NeuAc alpha 2-3Gal beta 1-4Glc; 2 NeuAc alpha 2-6Gal beta 1-4Glc; 3 NeuAc alpha 2-3Gal beta 1-4GlcNAc; 4 NeuAc-alpha 2-6Gal beta 1-4GlcNAc; 5 NeuAc alpha 2-3Gal beta 1-4-GlcNAc alpha 1-P; 6 NeuAc alpha 2-6Gal beta 1-4GlcNAc alpha 1-P; 7 NeuAc alpha 2-3Gal beta 1-3GalNAc alpha 1-P; 8 NeuAc alpha 2-8NeuAc. While compounds 1-7 are also components of normal human urine, di-N-acetyl-D-neuraminic acid (8) could be isolated for the first time from biological material. The origin and possible clinical relevance of these compounds have to be proved in further investigations.