Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le(x))-containing glycan (Gal beta 4[Fuc alpha 3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le(x)-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-labeled Le(x)-Lac-BSA, Le(a)-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le(a)-Lac did not inhibit binding of Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-ZP3 binding. Thus, Le(x) is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le(a) binding defines a different but less-abundant class of sites.     

Oligosaccharide specificity of the fucolectin from the bark of laburnum Laburnum anagyroides

A comparative study of thin carbohydrate specificity of the lectin from the bark of laburnum Laburnum anagyroides (LABA) and fucolectin from asparagus pea Tetragonolobus purpureus (TPA) was performed using inhibition of agglutination of the complex formed by H-active neoglycoprotein and nanoparticles of colloidal gold. Both lectins bound most strongly the H type 2 oligosaccharides comprising O-glycanes; however, TPA was almost unable to discriminate between them. LABA bound more weakly the H type 6 trisaccharide (Fuc alpha 1-2Gal beta 1-4Glc) and difucosyllactose (Fuc alpha 1-2Gal beta 1-4[Fuc alpha 1-3]Glc), a glucoanalogue of the Le(y) antigen, and, even more weakly, the Le(a) pentasaccharide lacto-N-fucopentaose II (Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc). However, LABA did not bind the antigens Le(b), Le(c), and Le(d), very poorly interacted with the terminal Le(x), and somewhat more strongly bound the internal Le(x). The lectin also had a hydrophobic binding site. Both lectins exhibited a cluster effect with polymeric ligands (neoglycoproteins).     

Chromatographic separation of Lewis a and Lewis x oligosaccharides as glycosynthons

Lewis a and Lewis x oligosaccharides Gal beta 3(Fuc alpha 4)GlcNAc beta 3Gal beta 4Glc and Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta 4Glc are easily isolated as a mixture from biological fluids, including human milk. However, because they behave almost identically in most chromatographic systems, it is difficult to have each of them as a pure compound. Incidentally, we found that they were easily separated by HPLC as glycosynthons [Gal beta 3(Fuc alpha 4)GlcNAc beta 3Gal beta 4Glc-Glp-beta Ala-OBzl and Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta 4Glc-Glp-beta Ala-OBzl] after substitution of the terminal reducing sugar by a short peptide (pyroglutamyl-beta alanyl-O-benzyl ester) in a one-pot two-step reaction (Carbohydr. Lett. 1 (1995) 269; Bioconjug. Chem. 9 (1998) 268). Such glycosynthons are easily either converted back to native Lewis a and Lewis x oligosaccharides upon hydrazinolysis or used to synthesize glycoconjugates, such as glycoclusters, glycopeptides, glycooligonucleotides, glycosylated polymers or glycosylated matrices for therapeutic or analytical purposes.     

Comparison of L-selectin and E-selectin ligand specificities: the L-selectin can bind the E-selectin ligands sialyl Le(x) and sialyl Le(a).

The L- and E-selectins are leukocyte and endothelial cell surface molecules which mediate leukocyte-endothelial cell adhesion by interacting with carbohydrate ligands. In the present study we find that L-selectin, like E-selectin, can interact with synthetic neoglycoproteins containing Sialyl Le(x) (Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta-R), or Sialyl Le(a) (Neu5Ac-alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta-R). Additionally, both the E-selectin and L-selectin can bind the peripheral lymph node addressin, a high endothelial venule ligand for L-selectin. Despite overlapping interactions, the L- and E-selectins discriminate between their native ligands. The peripheral lymph node addressin is a preferential ligand for L-selectin; and furthermore, L-selectin expressing cells do not interact detectably with the cutaneous lymphocyte antigen, a native glycoprotein ligand for E-selectin found on a subset of lymphocytes associated with the skin.     

Senile cataract-related accumulation of Lewis(x) glycolipid in human lens.

A glycosphingolipid that reacted positively to anti-stage-specific embryonic antigen-1 (SSEA-1) antiserum accumulated in human lens in association with aging and senile cataract formation. Since this antiserum recognizes Lewis(x) (Le(x)) structure, Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, which is a typical tumor-associated and differentiation-related saccharide chain, the lens glycolipid was predicted to be a Lex antigen. The glycolipid purified from cataractous lens tissues was indeed a Lex glycolipid, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1- 4Glc beta 1-1 ceramide. Enhanced expression of the Lex glycolipid may affect the organization of lens plasma membranes through Le(x)-Le(x) interactions, as suggested for compaction in mouse preimplantation embryos and embryonic teratocarcinomas, resulting in lens opacification, namely cataract.     

Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes.

We and others have previously described the isolation of three human alpha (1,3)fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)fucosyltransferase gene. When transfected into mammalian cells, this fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)fucosyltransferase genes and provide tools for examining fucosyltransferase gene expression.     

Free oligosaccharides with Lewis x structure expressed in the segmentation period of zebrafish embryo

We previously reported that zebrafishalpha1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Gal beta1-4(Fuc alpha1-3)GlcNAc] [Kageyama et.al, J. Biochem., 125, 838-845 (1999)]. In the current study, we attempted to detect the enzyme products of zFT1 in zebrafish embryos. Oligosaccharides were prepared from the zebrafish embryos at 12, 18 and 48 h after fertilization and labelled with a fluorophore, 2-aminopyridine, for highly sensitive detections. Pyridylamino (PA)-oligosaccharides that were alpha1-3/4fucosidase sensitive and time-dependently expressed at 18 h after fertilization were identified as candidates for the in vivo products synthesized by zFT1. Structures of these oligosaccharides were determined by a combination of exoglycosidase digestions and two-dimensional HPLC sugar mapping to be the biantennary complex-type structures with two Lewis x epitopes: (Gal beta1-4)(0,1,2)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]}Man beta1-4GlcNAc, and (Gal beta1-4)(0,1)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]} Man beta1-4GlcNAc beta1-4GlcNAc. The presence of Lewis x structure of these oligosaccharides together with their expression time suggests that they are products of zFT1. Remarkably, most of these oligosaccharides were free form. Furthermore, we detected an endo-beta-N-acetylglucosaminidase activity in the 18 h embryo. These results suggest that the oligosaccharides synthesized by zFT1 are present in the embryo at the segmentation period in free form, owing to the liberation from glycoproteins with endo-beta-N-acetylglucosaminidase(s) and/or glycoamidase(s).     

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.     

Studies of oligosaccharide specificity of perch roe fucolectin,using gold labeled neoglycoproteins

The specificity of perch (Perca fluviatilis) roe fucolectin was studied using the protein dot blot technique, followed by detection with colloidal gold-labeled neoglycoproteins bearing human milk polysaccharides. The strongest binding was noted with the H type 1 pentasaccharide lacto-N-fucopentaose (LNFP I, Fuc alpha 1-2 Gal beta 1-3 GlcNAc beta 1-3 Gal beta 1-4Glc); the interaction with the H type 6 trisaccharide 2'-fucosyllactose (2-FL, Fuc alpha 1-2 Gal beta 1-4 Glc) was weaker. Binding of the perch lectin to the Lewis antigens (associated with tumors and embryonic tissues) was also studied. It was found that the lectin weakly interacted with the hexasaccharide lacto-N-difucohexaose I, Fuc alpha 1-2 Gal beta 1-3[Fuc alpha 1-4]GlcNac beta 1-3 Gal beta 1-4 Glc), but not with Lea, Lec, Lex antigens. Thus, perch roe lectin exhibited pronounced differences in carbohydrate specificity from other fucolectins--a feature that may be used in structural studies and isolation of fucose-containing glycoconjugates.     

Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(alpha1-3)GalNAc-, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc- and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc- (Lewis X) terminal structures.

The carbohydrate moieties of glycosphingolipids from eggs of the human parasite, Schistosoma mansoni, were enzymatically released, labelled with 2-aminopyridine (PA), fractionated and analysed by linkage analysis, partial hydrolysis, enzymatic cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-electrospray ionization mass spectrometry. Apart from large, highly fucosylated structures with five to seven HexNAc residues, we found short, oligofucosylated species containing three to four HexNAc residues. Their structures have been determined as Fuc(alpha1-3)GalNAc(beta1-4)[ +/- Fuc (alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4) Glc-PA, Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-4) GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, and Fuc(alpha1-3) GalNAc(beta1-4)[ +/- Fuc(alpha1-2) +/- Fuc(alpha1-2)Fuc(alpha1-3)]Glc NAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA. The last structure exhibits a trifucosyl sidechain previously identified on the cercarial glycocalyx. These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope in schistosome glycoconjugates. To what degree these glycans contribute to the pronounced antigenicity of S. mansoni egg glycolipids remains to be determined. In addition, we have identified the compounds GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3) GalNAc (beta1-4)Glc-PA, the latter of which is a Lewis X-pentasaccharide identical to that present on cercarial glycolipids, as well as Gal(beta1-3)GalNAc(1-4)Gal(1-4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host.     

Structural convergence of antibody binding of carbohydrate determinants in Lewis Y tumor antigens

Antibodies targeting human epithelial carcinomas bearing Lewis Y (Le(y)) carbohydrate antigens provide a striking illustration of convergent immune recognition. We report a 1.9A resolution crystal structure of the Fab of a humanized antibody (hu3S193) in complex with the Le(y) tetrasaccharide, Fuc(alpha 1-->2)Gal(beta 1-->4)[Fuc(alpha 1-->3)]GlcNAc. Comparisons of the hu3S193 and BR96 antibodies bound to Le(y) tumor antigens revealed extremely similar mechanisms for recognition of the carbohydrate epitopes. Solvent plays a critical role in hu3S193 antibody binding to the Le(y) carbohydrate epitope. Specificity for Le(y) is maintained because a conserved pocket accepts an N-acetyl group of the core Gal(beta 1-->4)GlcNAc disaccharide. Closely related blood-group determinants (Le(a) and Le(b)) cannot enter the specificity pocket, making the Le(y) antibodies promising candidates for immunotherapy of epithelial cancer.     

Sulfated oligosaccharides isolated from the respiratory mucins of a secretor patient suffering from chronic bronchitis

The most acidic carbohydrate chains released by alkaline borohydride treatment of the bulk of airway mucins secreted by a patient (blood group O, secretor) suffering from a mildly infected chronic bronchitis have been fractionated using high-performance anion-exchange chromatography (HPAEC) according to a protocol already described [Lo-Guidice et al., J. Biol. Chem. 269 (1994) 18794] and were analyzed using 1H-NMR spectroscopy and matrix-assisted laser-adsorption-time-of-flight (MALDI-TOF) spectrometry. Many fractions corresponded to mixtures of oligosaccharides. This confirmed the wide diversity of the post-translational processes involved in the biosynthesis of airway mucins, which had already been observed in bronchial diseases, such as chronic bronchitis and cystic fibrosis (CF). Seven fractions were directly purified by HPAEC, allowing their structural determination. Six of them corresponded to 3-O-sulfated oligosaccharide chains terminated by a sulfated N-acetyllactosamine, a sulfated Lewis X or a sulfated Lewis A determinant, and the last one corresponded to a 6-O-sulfated chain terminated by a sulfated H-2 determinant. Three oligosaccharides had core type 2 and the other four had core type 4: IIIc2-9: Gal(beta1-3)[HSO(3)-3-Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol, IIIc2-10: Gal(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)[HSO(3)-6-]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-4: Fuc(alpha1-2)Gal(beta1-3)[HSO(3)-3-Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-8: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3)-3-Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol, IIIc2-7: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[Gal(beta1-4)[HSO(3)-6-]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-3: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3)-3-Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]GalNAc-ol, IIIc1-4: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3) -3-Gal(beta1-3)[Fuc(alpha1-4)]GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol. Like previous data concerning the airway mucins from another patient (blood group O and non-secretor) suffering from chronic bronchitis [Lo-Guidice et al., Glycoconj. J. 14 (1997) 113], no disialylated oligosaccharide and no sialylated and sulfated oligosaccharide bearing sialyl Lewis X epitope could be isolated. This is in contrast with the data obtained with the airway mucins secreted by the patient severely infected by Pseudomonas aeruginosa and suffering from CF, suggesting that important differences occur in the biosynthesis of airway mucins secreted by patients suffering from different bronchial diseases with or without severe infection.     

Novel polyfucosylated N-linked glycopeptides with blood group A, H, X, and Y determinants from human small intestinal epithelial cells

A novel type of N-linked glycopeptides representing a major part of the glycans in human small intestinal epithelial cells from blood group A and O individuals were isolated by gel filtrations and affinity chromatography on concanavalin A-Sepharose and Bandeiraea simplicifolia lectin I-Sepharose. Sugar composition, methylation analysis, 1H NMR spectroscopy of the underivatized glycopeptides and FAB-mass spectrometry and electron impact-mass spectrometry of the permethylated glycopeptides indicated a tri- and tetra-antennary structure containing an intersecting N-acetylglucosamine and an alpha (1----6)-linked fucose residue in the core unit for the majority of the glycans. In contrast to most glycopeptides of other sources, the intestinal glycopeptides were devoid of sialic acid, but contained 6-7 residues of fucose. The outer branches contained the following structures: Fuc alpha 1-2Gal beta 1-3GleNAc beta 1- (H type 1) Fuc alpha 1-2Gal beta 1-4GleNAc beta 1- (H type 2) Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1- (X) Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GleNAc beta 1- (Y) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GleNAc beta 1- (A type 1) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GleNAc beta 1- (monofucosyl A type 2) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1- (trifucosyl A type 2) The blood group determinant structures were mainly of type 2, whereas glycolipids from the same cells contained mainly type 1 determinants. The polyfucosylated glycans represent a novel type of blood group active glycopeptides. The unique properties of the small intestinal glycopeptides as compared with glycopeptides of other tissue sources may be correlated with the specialized functional properties of the small intestinal epithelial cells.     

The solution conformation of the Le chi group

The solution conformation of the non-reducing terminal Gal beta 1----4 (Fuc alpha 1----3)GlcNAc (Lewis X or Le chi) group in the oligosaccharide Lacto-N-fucopentaose (LNFP) III has been determined by high resolution 1H NMR spectroscopy and semi-empirical quantum mechanical calculations. The two methods give the same single conformer for the Le chi group showing close packing of the Gal and Fuc rings. The metal binding properties and homotypic oligomer formation of LNFP III have also been investigated by NMR spectroscopy. No evidence for metal binding or high-affinity homotypic oligomer formation has been found.     

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 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 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.     

Assessment of the two Helicobacter pylori alpha-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli

We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto-N-neotetraose Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N-acetylglucosaminyl residues and thus carrying the LewisX group (Le(X)) was also produced. We report here a fermentation process for the large-scale production of Le(X) oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better Le(X) activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli (E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of Le(X) carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc represented 70% of the total oligosaccharide amount of futA-on-driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3-fucosyllactose Gal(beta1-4)[Fuc(alpha1-3)]Glc was produced in futA-on-driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.     

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.