Fuc-TIX: a versatile alpha1,3-fucosyltransferase with a distinct acceptor- and site-specificity profile

alpha1,3-Fucosyltransferases (Fuc-Ts) convert N-acetyllactosamine (LN, Galbeta1-4GlcNAc) to Galbeta1-4(Fucalpha1-3)GlcNAc, the Lewis x (CD15, SSEA-1) epitope, which is involved in various recognition phenomena. We describe details of the acceptor specificity of alpha1,3-fucosyltransferase IX (Fuc-TIX). The unconjugated N- and O-glycan analogs LNbeta1-2Man, LNbeta1-6Manalpha1-OMe, LNbeta1-2Manalpha1-3(LNbeta1-2Manalpha1-6)Manbeta1-4GlcNAc, and Galbeta1-3(LNbeta1-6)GalNAc reacted well in vitro with Fuc-TIX present in lysates of appropriately transfected Namalwa cells. Fuc-TIX reacted well with the reducing end LN of GlcNAcbeta1-3'LN (underscored site reacted) and GlcNAcbeta1-3'LNbeta1-3'LN (both LNs reacted), but very poorly with the reducing end LN of LNbeta1-3'LN. However, Fuc-TIX reacted significantly better with the non-reducing end LN as compared to the other LN units in the glycans LNbeta1-3'LN and LNbeta1-3'LNbeta1-3'LNbeta1-3'LN, confirming our previous data on LNbeta1-3'LNbeta1-OR. In contrast, the sialylated glycan Neu5Acalpha2-3'LNbeta1-3'LNbeta1-3'LNbeta1-3'LN was fucosylated preferentially at the two most reducing end LN units. We conclude that Fuc-TIX is a versatile alpha1,3-Fuc-T, that (1) generates distal Lewis x epitopes from many different acceptors, (2) possesses inherent ability for the biosynthesis of internal Lewis x epitopes on growing polylactosamine backbones, and (3) fucosylates the remote internal LN units of alpha2,3-sialylated i-type polylactosamines.     

Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic alpha3-fucosylation of pure polylactosamines

We report that isomeric monofucosylhexasaccharides, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4 GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, and bifucosylhexasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 (Fucalpha1-3)GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4( Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc with GDP-fucose and alpha1,3-fucosyltransferases of human milk. The pure isomers were characterized in several ways;1H-NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Galbeta1-4(Fucalpha1-3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin. Isomeric monofucosyl derivatives of the pentasaccharides GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1- 4Gl cNAc and Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcN Ac and the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc were also obtained in pure form, implying that the methods used are widely applicable. The isomeric Lewis x glycans proved to be recognized in highly variable binding modes by polylactosamine-metabolizing enzymes, e.g., the midchain beta1,6-GlcNAc transferase (Lepp?nen et al., Biochemistry, 36, 13729-13735, 1997).     

Human alpha3-fucosyltransferases convert chitin oligosaccharides to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus

Human alpha3-fucosyltransferases (Fuc-Ts) are known to convert N-acetyllactosamine to Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis x antigen); some of them transfer fucose also to GalNAcbeta1-4GlcNAc, generating GalNAcbeta1-4(Fucalpha1-3)GlcNAc determinants. Here, we report that recombinant forms of Fuc-TV and Fuc-TVI as well as Fuc-Ts of human milk converted chitin oligosaccharides of 2-4 GlcNAc units efficiently to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus. The product structures were identified by mass spectrometry and nuclear magnetic resonance experiments; rotating frame nuclear Overhauser spectroscopy data suggested that the fucose and the distal N-acetylglucosamine are stacked in the same way as the fucose and the distal galactose of the Lewis x determinant. The products closely resembled a nodulation factor of Mesorhizobium loti but were distinct from nodulation signals generated by NodZ-enzyme.     

Effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the Galbeta1-terminated core saccharides on the binding of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N)

In our recent publication, we defined core aspects of the carbohydrate specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N), especially its potent interaction with the linear tetrasaccharide Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (Ibeta1-3L). The assumed role of galectin-4 as a microvillar raft stabilizer/organizer and as a malignancy-associated factor in hepatocellular and gastrointestinal carcinomas called for further refinement of its binding specificity. Thus, the effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the terminal Galbeta1-core saccharides were thoroughly examined by the enzyme-linked lectinosorbent and lectin-glycan inhibition assays. The results indicate that (a) a high-density of polyvalent Galbeta1-3/4GlcNAc (I/II), Galbeta1-3GalNAc (T) and/or GalNAcalpha1-Ser/Thr (Tn) strongly favors G4-N/glycoform binding. These glycans were up to 2.3 x 10(6), 1.4 x 10(6), 8.8 x 10(5), and 1.4 x 10(5) more active than Gal, GalNAc, monomeric I/II and T, respectively; (b) while lFuc is a poor inhibitor, its presence as alpha1-2 linked to terminal Galbeta1-containing oligosaccharides, such as H active Ibeta1-3L, markedly enhances the reactivities of these ligands; (c) when blood group A (GalNAcalpha1-) or B (Galalpha1-) determinants are attached to terminal Galbeta1-3/4GlcNAc (or Glc) oligosaccharides, the reactivities are also increased; (d) with lFucalpha1-3/4 linked to sub-terminal GlcNAc, the reactivities of these haptens are reduced; and (e) short chain Le(a)/Le(x)/Le(y) and the short chains of sialyl Le(a)/Le(x) are poor inhibitors. These distinct binding features of G4-N establish the important concept of affinity enhancement by high density polyvalencies of glycotopes (vs. multi-antennary I/II) and by introduction of an ABH key sugar to Galbeta1-terminated core glycotopes. The polyvalent ligand binding properties of G4-N may help our understanding of its crucial role for cell membrane raft stability and provide salient information for the optimal design of blocking substances such as anti-tumoral glycodendrimers.     

Several polylactosamine-modifying glycosyltransferases also use internal GalNAcbeta1-4GlcNAc units of synthetic saccharides as acceptors

The GalNAcbeta1-4GlcNAc determinant (LdN) occurs in some human and bovine glycoconjugates and also in lower vertebrates and invertebrates. It has been found in unsubstituted as well as terminally substituted forms at the distal end of conjugated glycans, but it has not been reported previously at truly internal positions of polylactosamine chains. Here, we describe enzyme-assisted conversion of LdNbeta1-OR oligosaccharides into GlcNAcbeta1-3GalNAcbeta1-4GlcNAcbeta1-OR. The extension reactions, catalyzed by human serum, were modeled after analogous beta3-GlcNAc transfer processes that generate GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-OR. The newly synthesized GlcNAcbeta1-3GalNAc linkages were unambiguously identified by nuclear magnetic resonance data, including the appropriate long-range correlations in heteronuclear multiple bond correlation spectra. The novel GlcNAcbeta1-3'LdN determinant proved to be a functional acceptor for several mammalian glycosyltransferases, suggesting that human polylactosamines may contain internal LdN units in many distinct forms. The GlcNAcbeta1-3'LdN determinant was unusually resistant toward jackbean beta-N-acetylhexosaminidase; the slow degradation should lead to a convenient method for the search of putative internal LdN determinants in natural polylactosamine chains.     

Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate Gal-6-sulfotransferase

We have previously cloned keratan sulfate Gal-6-sulfotransferase (KSGal6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of Gal residue of keratan sulfate. In this study, we examined whether KSGal6ST could transfer sulfate to sialyl N -acetyllactosamine oligosaccharides or fetuin oligo-saccharides. KSGal6ST expressed in COS-7 cells catalyzed transfer of sulfate to NeuAcalpha2-3Galbeta1-4GlcNAc (3'SLN), NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc (SL1L1), NeuAcalpha2-3Galbeta1-4(6-sulfo)GlcNAcbeta1-3(6-sulfo) Galbeta1-4(6-su lfo)GlcNAc (SL2L4), and their desialylated derivatives except for Galbeta1-4GlcNAc, but not to NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc (SLex). When the sulfated product formed from 3'SLN was degraded with neuraminidase and reduced with NaBH(4), the resulting sulfated disaccharide alditol showed the same retention time in SAX-HPLC as that of [(3)H]Gal(6SO(4))beta1-4GlcNAc-ol. KSGal6ST also catalyzed sulfation of fetuin. When the sulfated oligosaccharides released from the sulfated fetuin after sequential digestion with proteinase and neuraminidase were subjected to a reaction sequence of hydrazin-olysis, deaminative cleavage and NaBH(4)reduction, the major product was co-eluted with [(3)H]Gal(6SO(4))beta1-4anhydromannitol in SAX-HPLC. These observations show that KSGal6ST is able to sulfate position 6 of Gal residue of 3'SLN and fetuin oligosaccharides. The relative rates of the sulfation of SL2L4 was much higher than the rate of the sulfation of keratan sulfate. These results suggest that KSGal6ST may function in the sulfation of sialyl N -acetyllactosamine oligosaccharide chains attached to glycoproteins.     

Structural and thermodynamic analyses of solute-binding Protein from Bifidobacterium longum specific for core 1 disaccharide and lacto-N-biose I

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Galbeta1-3GlcNAc) and galacto-N-biose (GNB; Galbeta1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K(d) values of 0.087 and 0.010 microm, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.     

Functional expression and genomic structure of human N-acetylglucosamine-6-O-sulfotransferase that transfers sulfate to beta-N-acetylglucosamine at the nonreducing end of an N-acetyllactosamine sequence

The cDNA and gene encoding human N-acetylglucosamine-6-O-sulfotransferase (Gn6ST) have been cloned. Comparative analysis of this cDNA with the mouse Gn6ST sequence indicates 96% amino acid identity between the two sequences. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase, which transferred sulfate to the terminal GlcNAc in GlcNAcbeta1-O-CH(3), GlcNAcbeta1-3Galbeta1-O-CH(3) and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc but not in GlcNAcalpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4 Xylbeta1-O-Ser. In addition, neither Galbeta1-4GlcNAcbeta1-O-naphthalenemethanol nor GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4X ylbeta1-O-Ser were utilized as acceptors. These findings indicate that a terminal beta-linked GlcNAc residue is necessary for acceptor substrates of Gn6ST. The human Gn6ST gene spans about 7 kb, consists of two exons and exhibits an intron-less coding region.     

Identification of the sialic acid structures recognized by minute virus of mice and the role of binding affinity in virulence adaptation

Sialic acid binding is required for infectious cell surface receptor recognition by parvovirus minute virus of mice (MVM). We have utilized a glycan array consisting of approximately 180 different carbohydrate structures to identify the specific sialosides recognized by the prototype (MVMp) and immunosuppressive (MVMi) strains of MVM plus three virulent mutants of MVMp, MVMp-I362S, MVMp-K368R, and MVMp-I362S/K368R. All of the MVM capsids specifically bound to three structures with a terminal sialic acid-linked alpha2-3 to a common Galbeta1-4GlcNAc motif: Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-4Galbeta1-4GlcNAc (3'SiaLN-LN), Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (3'SiaLN-LN-LN), and Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)-GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc (sLe(x)-Le(x)-Le(x)). In addition, MVMi also recognized four multisialylated glycans with terminal alpha2-8 linkages: Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha ((Sia)(3)), Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GD3), Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GT3), and Neu5Acalpha2-8Neu5Acalpha2-3(GalNAcbeta1-4)Galbeta1-4Glc (GD2). Interestingly, the virulent MVMp-K368R mutant also recognized GT3. Analysis of the relative binding affinities using a surface plasmon resonance biospecific interaction (BIAcore) assay showed the wild-type MVMp and MVMi capsids binding with higher affinity to selected glycans compared with the virulent MVMp mutants. The reduced affinity of the virulent MVMp mutants are consistent with previous in vitro cell binding assays that had shown weaker binding to permissive cells compared with wild-type MVMp. This study identifies the sialic acid structures recognized by MVM. It also provides rationale for the tropism of MVM for malignant transformed cells that contain sLe(x) motifs and the neurotropism of MVMi, which is likely mediated via interactions with multisialylated glycans known to be tumor cell markers. Finally, the observations further implicate a decreased binding affinity for sialic acid in the in vivo adaptation of MVMp to a virulent phenotype.     

Purification and characterization of a chitinase from Amycolatopsis orientalis with N-acetyllactosamine-repeating unit releasing activity

We report a novel enzyme from the culture filtrate of Amycolatopsis orientalis, that endoglycosidically releases an N-acetyllactosamine-repeating unit (Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAc, LN2) from a synthetic chromogenic substrate Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1). The enzyme activity was purified by 80% saturated ammonium sulfate precipitation followed by gel filtration and affinity chromatography. The enzyme splits 1, Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (3), and GlcNAcbeta1,4GlcNAcbeta-pNP (4) into the corresponding oligosaccharides and p-nitrophenol. The catalytic efficiencies (k(cat)/K(m)) for compounds 1, 2, and 4 were 0.6, 0.05, and 13, respectively. Compound 4 acts as a fairly good substrate for the enzyme, and LN2-releasing activity was inhibited by 4 and GlcNAcbeta1,4GlcNAcbeta1,4GlcNAcbeta-pNP (7), indicating that this enzyme activity is derived from a kind of chitinase. The enzyme hydrolyzed 1 by a mechanism leading to retention of the anomeric configuration. This is the first report of a N-acetyllactosamine-repeating unit releasing enzyme.     

Novel poly-GalNAcbeta1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing beta1,4-N-acetylgalactosaminyltransferase and alpha1,3-fucosyltransferase

Glycans containing the GalNAcbeta1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Galbeta1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAcbeta1-4GlcNAcbeta1-)n. These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Galbeta1-4GlcNAcbeta1-)n. We also stably expressed human alpha1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with alpha1,3-fucosylated LDN (LDNF) GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-R as well as novel "poly-LDNF" structures (-3GalNAcbeta1-4(Fucalpha 1-3)GlcNAcbeta1-)n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common alpha-subunit in L8-GalNAcT cells. The alpha-subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAcbeta1-4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

First evidence for occurrence of Galbeta1-3GlcNAcbeta1-4Man unit in N-glycans of insect glycoprotein: beta1-3Gal and beta1-4GlcNAc transferases are involved in N-glycan processing of royal jelly glycoproteins

On a way of structural analysis of total N-glycans linked to glycoproteins in royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985-1989 (2002)), we found that some complex type N-glycans containing a beta1-3galactose residue occur on the insect glycoproteins. Up to date, it has been considered that naturally occurring insect glycoproteins do not bear the galactose-containing N-glycans, therefore, in this report we describe the structural analysis of the complex type N-glycans of royal jelly glycoproteins.By a combination of endo- and exo-glycosidase digestions, IS-MS analysis, and 1H-NMR spectroscopy, the structures of the beta1-3 galactose-containing N-glycan were identified as the following; GlcNAcbeta1-2Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, Manalpha1-3Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this is the first report showing that the Galbeta1-3GlcNAcbeta1-4Man unit occurs in N-glycans of insect glycoproteins, indicating a beta1-3 galactosyl transferase and beta1-4GlcNAc transferase (GNT-IV) are expressed in the honeybee cells.     

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.     

Urinary oligosaccharides of fucosidosis. Evidence of the occurrence of X-antigenic determinant in serum-type sugar chains of glycoproteins.

Urine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucose-rich glycopeptides. Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucalpha1 leads to 6GlcNAc, Fucalpha1 leads to 2Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 2Manalpha1 leads to 3Manbeta1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to3)GlcNAcbeta1 leads to 2Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc, and Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 6Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc. In additon, the structure of a minor decasaccharide was found to be Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to [Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to]Manbeta1 leads to 4GlcNAc.     

Interactions of the fucose-specific Pseudomonas aeruginosa lectin, PA-IIL, with mammalian glycoconjugates bearing polyvalent Lewis(a) and ABH blood group glycotopes

Pseudomonas aeruginosa Fuc > Man specific lectin, PA-IIL, is an important microbial agglutinin that might be involved in P. aeruginosa infections in humans. In order to delineate the structures of these lectin receptors, its detailed carbohydrate recognition profile was studied both by microtiter plate biotin/avidin-mediated enzyme-lectin-glycan binding assay (ELLSA) and by inhibition of the lectin-glycan interaction. Among 40 glycans tested for binding, PA-IIL reacted well with all human blood group ABH and Le(a)/Le(b) active glycoproteins (gps), but weakly or not at all with their precursor gps and N-linked gps. Among the sugar ligands tested by the inhibition assay, the Le(a) pentasaccharide lacto-N-fucopentaose II (LNFP II, Galbeta1-3[Fucalpha1-4]GlcNAcbeta1-3Galbeta1-4Glc) was the most potent one, being 10 and 38 times more active than the Le(x) pentasaccharide (LNFP III, Galbeta1-4 [Fucalpha1-3]GlcNAcbeta1-3Galbeta1-4Glc) and sialyl Le(x) (Neu5Acalpha2-3Galbeta1-4[Fucalpha1-3] GlcNAc), respectively. It was 120 times more active than Man, while Gal and GalNAc were inactive. The decreasing order of PA-IIL affinity for the oligosaccharides tested was: Le(a) pentaose > or = sialyl Le(a) tetraose > methyl alphaFuc > Fuc and Fucalpha1-2Gal (H disaccharide)>2'-fucosyllactose (H trisaccharide), Le(x) pentaose, Le(b) hexaose (LNDFH I) and gluco-analogue of Le(y) tetraose (LDFT)>H type I determinant (LNFP I)>Le(x) trisaccharide (Galbeta1-4[Fucalpha1-3]GlcNAc) > sialyl Le(x) trisaccharide > Man > Gal, GalNAc, and Glc (inactive). The results presented here, in accordance with the crystal 3D structural data, imply that the combining site of PA-IIL is a small cavity-type best fitting Fucalpha1- with a specific shallow groove subsite for the remainder part of the Le(a) saccharides, and that polyvalent glycotopes enhance the reactivity. The Fuc > Man Ralstonia solanacearum lectin RSL, which resembles PA-IIL in sugar specificity, differs from it in it's better fit to the B and A followed by H oligosaccharides vs. Fuc, whereas, the second R. solanacearum lectin RS-IIL (the structural homologue of PA-IIL) binds Man > Fuc. These results provide a valuable information on PA-IIL interactions with mammalian glycoforms and the possible spectrum of attachment sites for the homing of this aggressive bacterium onto the target molecules. Such information might be useful for the antiadhesive therapy of P. aeruginosa infections.     

Studies on Galalpha3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

The carbohydrate binding preferences of the Galalpha3Galbeta4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Galalpha3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) was preferred over the B5 glycosphingolipid (Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), suggesting that the alpha2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fucalpha2Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), devoid of a terminal alpha3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the alpha2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. The A6 type 2 glycosphingolipid (GalNAcalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GalNAcalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Galalpha3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.     

Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of I-extension enzyme and beta1,4-galactosyltransferase I.

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->4GlcNAcbeta1 -->2)Manalpha1-->6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and beta1, 4galactosyltransferase I (beta4Gal-TI). First, N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain than in Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R, due to preferential action of iGnT on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain. On the other hand, galactosylation was much more efficient on beta1,6-linked GlcNAc than beta1,2-linked GlcNAc, preferentially forming Galbeta1-->4GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalph a1-->6Manbeta -->R. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R and Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and beta4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalpha1-->6Manbet a-->R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galbeta1-->4GlcNAcbeta1-->6 side chain on Galbeta1-->4GlcNAcbeta1-->2Man-->R side chain converts the acceptor to one that is much more favorable for iGnT and beta4Gal-TI.     

Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose

Lacto-N-tetraose (Galbeta1 -3GlcNAcbeta1-3Galbeta1-4Glc, LNT) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, LNnT) were enzymatically synthesized by consecutive additions of GlcNAc and Gal residues to lactose. Lacto-N-triose II (GlcNAcbeta1-3Galbeta1-4Glc) was prepared first by the transfer of GlcNAc from UDP-GlcNAc to lactose by beta-1,3-N-acetylglucosaminyltransferase from bovine serum. The resulting lacto-N-triose II was converted into LNT and LNnT utilizing two kinds of beta-D-galactosidase-mediated transglycosylations. Thus, beta-D-galactosidase from Bacillus circulans ATCC31382 induced regioselective galactosyl transfer from o-nitrophenyl beta-D-galactoside to the OH-3" position of lacto-N-triose II, and commercially available beta-D-galactosidase from B. circulans to the OH-4" position of lacto-N-triose II. These convenient processes are suitable for large-scale preparations of LNT and LNnT. As another method, LNT was directly synthesized from lactose as an initial substance, utilizing lacto-N-biosidase (Aureobacterium sp. L-101)-mediated transglycosylation with Galbeta1-3GlcNAcbeta-pNP donor.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.