Assignment of the1H- and13C-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series

The assignment of the 13C- and 1H-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series was obtained from homonuclear and heteronuclear correlation spectroscopy. These analyses were performed on the following compounds: 1. Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 2. NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 3. Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 4. NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 5. NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc; 6. Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 7. Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc; 8. NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc.     

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH)

The sialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc (disialyllacto-N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding (Ka greater than 10(6) M-1) requires sialic acid linked alpha 2-3 to the nonreducing terminal galactose residue of reduced lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59].     

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.     

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.     

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.     

A novel sialylfucopentaose in human milk. Presence of this oligosaccharide is not dependent on expression of the secretor or Lewis fucosyltransferases

We have identified a novel oligosaccharide in human milk that is a fucosyl derivative of sialyltetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc). This oligosaccharide was purified by affinity chromatography on a column of immobilized Ricinus communis I lectin. Structural analyses of radiolabeled oligosaccharides by exoglycosidase digestions, binding by specific anti-carbohydrate antibodies, and analysis of the 3H-labeled glucitol derivative obtained after permethylation and hydrolysis are consistent with the following proposed structure. (formula; see text) The analyses of human milk sialylpentasaccharides from donors typed as Le(a-,b+), Le(a+,b-), and Le(a-,b-) secretor confirmed the secretor gene-dependent expression of the sialylated lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and the Lewis gene-dependent expression of the sialylated lacto-N-fucopentaose II (NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-4Glc). However, the presence of this novel oligosaccharide in human milk is not dependent on the expression of either the secretor gene or the Lewis gene-specified fucosyltransferases.     

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.     

Complete analysis of the1H- and13C-NMR spectra of four blood-group A active oligosaccharides

The fully assigned 1H and 13C-NMR spectra of four group A oligosaccharides by use of multiple-relayed, coherence-transfer chemical-shift-correlated spectroscopy (multiple-RELAY-COSY) and 1H-/13C-correlation spectroscopy are reported. These analyses were performed on the following compounds: III-A; GalNAc alpha 1-3[Fuc alpha 1-2]Gal: VI-A; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal: VII-A-1; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-1Glycerol: VII-A-2; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc.     

Structural determination of the oligosaccharides in the milk of an Asian elephant (Elephas maximus)

Milk of an Asian elephant (Elephas maximus), collected at 11 days post partum, contained 91 g/L of hexose and 3 g/L of sialic acid. The dominant saccharide in this milk sample was lactose, but it also contained isoglobotriose (Glc(alpha1-3)Gal(beta1-4)Glc) as well as a variety of sialyl oligosaccharides. The sialyl oligosaccharides were separated from neutral saccharides by anion exchange chromatography on DEAE-Sephadex A-50 and successive gel chromatography on Bio Gel P-2. They were purified by high performance liquid chromatography (HPLC) using an Amide-80 column and characterized by 1H-NMR spectroscopy. Their structures were determined to be those of 3'-sialyllactose, 6'-sialyllactose, monofucosyl monosialyl lactose (Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc), sialyl lacto-N-neotetraose c (LST c), galactosyl monosialyl lacto-N-neohexaose, galactosyl monofucosyl monosialyl lacto-N-neohexaose and three novel oligosaccharides as follows: 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)GlcNAc(beta1-3)Gal(beta1-4)Glc, and Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc. The higher oligosaccharides contained only the type II chain (Gal(beta1-4)GlcNAc); this finding differed from previously published data on Asian elephant milk oligosaccharides.     

Chemical characterisation of the oligosaccharides in hooded seal (Cystophora cristata) and Australian fur seal (Arctocephalus pusillus doriferus) milk

Carbohydrates were extracted from hooded seal milk, Crystophora cristata (family Phocidae). Free oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and preparative thin layer or paper chromatography and their structures determined by 1H-NMR. The hooded seal milk was found to contain inositol and at least nine oligosaccharides, most of which had lacto-N-neotetraose or lacto-N-neohexaose as core units, similar to those in milk of other species of Carnivora such as bears (Ursidae). Their structures were as follows: Gal(beta1-4)Glc (lactose); Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose); Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (lacto-N-fucopentaose IV); Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(1-4)Glc (lacto-N-neohexaose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (monofucosyl lacto-N-neohexaose a); Gal(beta1-4)GlcNAc(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (monofucosyl lacto-N-neohexaose b); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (difucosyl lacto-N-neohexaose); Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (para lacto-N-neohexaose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (monofucosyl para lacto-N-neohexaose). Milk of the Australian fur seal, Arctophalus pusillus doriferus (family Otariidae) contained inositol but no lactose or free oligosaccharides. These results, therefore, support the hypothesis that the milk of otariids, unlike that of phocids, contains no free reducing saccharides.     

Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins

The O-linked oligosaccharides attached to human erythrocyte glycophorins were extensively characterized. In addition to the previously described disialylated tetrasaccharide, NeuNAc alpha 2----3Gal beta 1----3 (Neu-NAc alpha 2----6)GalNAcOH and monosialylated trisaccharide, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, novel trisialylated oligosaccharides were isolated. Methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation were used to elucidate the following novel structures: formula; see text: These results suggest that O-linked oligosaccharides with a disialosyl group, NeuNAc alpha 2----8NeuNAc alpha 2----, may be present in various tissues.     

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.     

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.     

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)     

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.     

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 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.     

Identification of N-acetylneuraminyl alpha 2-->3 poly-N-acetyllactosamine glycans as the receptors of sialic acid-binding Streptococcus suis strains.

Streptococcus suis is a common cause of sepsis, meningitis, and other serious infections in young piglets and also causes meningitis in humans. The cell-binding specificity of sialic acid-recognizing strains of Streptococcus suis was investigated. Treatment of human erythrocytes with sialidase or mild periodate abolished hemagglutination. Hemagglutination inhibition experiments with sialyl oligosaccharides indicated that the adhesin preferred the sequence NeuNAc alpha 2-3Gal beta 1-4Glc(NAc). Resialylation of desialylated erythrocytes with Gal beta 1-3(4)GlcNAc alpha 2-3-sialyltransferase induced a strong hemagglutination, whereas no or only weak hemagglutination was obtained with cells resialylated with two other sialyltransferases. Binding of radiolabeled bacteria to blots of erythrocyte membrane proteins revealed binding to the poly-N-acetyllactosamine-containing components Band 3, Band 4.5, and polyglycosyl ceramides and to glycophorin A. The involvement of glycophorin A as a major ligand was excluded by the strong hemagglutination of trypsin-treated erythrocytes and En(a-) erythrocytes defective in glycophorin A. Sensitivity of the hemagglutination toward endo-beta-galactosidase treatment of erythrocytes and inhibition by purified poly-N-acetyllactosaminyl glycopeptides indicated that the adhesin bound to glycans containing the following structure: NeuNAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-.     

Urinary oligosaccharides of GM1-gangliosidosis. Different excretion patterns of oligosaccharides in the urine of type 1 and type 2 subgroups

Oligosaccharide patterns obtained by gel filtration of the urine of GM1-gangliosidosis Type 1 patients are quite different from those of GM1-gangliosidosis Type 2. By studies of oligosaccharides in the four major peaks obtained from the Type 1 subgroup using sequential exoglycosidase digestion, methylation analysis, and periodate oxidation, the structures of 15 oligosaccharides: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc, Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6(Gal beta 1 leads to 4Glc NAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6, and 3(Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6)Man beta 1 leads to 4GlcNAc, (formula see text) were elucidated. The amounts of total oligosaccharides excreted in the urine of the Type 2 subgroup were approximately one-tenth of those of Type 1. Moreover, the last eight oligosaccharides shown above, which have a Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to outer chain, were completely missing in the urine of Type 2.     

Poly-N-acetyllactosaminyl O-glycans attached to leukosialin. The presence of sialyl Le(x) structures in O-glycans.

Poly-N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids. Attempts were made in HL-60 and K562 cells to determine the amount of poly-N-acetyllactosaminyl O-glycans in the major sialoglycoprotein, leukosialin. Leukosialin was immunoprecipitated from [3H]glucosamine-labeled HL-60 and K562 cells. Glycopeptides were prepared by Pronase digestion, and O-glycan-containing glycopeptides were isolated by affinity chromatography using Jacalin-agarose. The glycopeptides bound to Jacalin-agarose and those unbound were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Sequential glycosidase digestion of the O-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following conclusions. 1) Leukosialin from HL-60 cells contains about 1-2 poly-N-acetyllactosaminyl O-glycan chains/molecule. 2) About 50% of these poly-N-acetyllactosaminyl O-glycans contain sialyl Le(x) termini, NeuNAc alpha 2-->3Gal beta 1-->4 (Fuc alpha 1-->3)GlcNAc beta 1-->R. The amount of sialyl Le(x) structure in leukosialin is roughly equivalent to that on cell surfaces of HL-60 cells. 3) Leukosialin from K562 cells, on the other hand, contains no detectable amount of poly-N-acetyllactosaminyl O-glycans. 4) The presence of poly-N-acetyllactosamine in O-glycans is dependent on the core 2 beta 1,6-N-acetylglucosaminyl transferase. 5) Jacalin-agarose binds to sialylated small oligosaccharides such as NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->6) GalNAc but not the hexasaccharide NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->6) GalNAc. These results indicate that the formation of polylactosaminyl O-glycans and sialyl Le(x) structure in O-glycans is dependent on the core 2 formation.