Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1

As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Galbeta1,4GlcNAcbeta1,3]nwere synthesized at the 10-100 micromol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc and Galbeta1, 4GlcNAcbeta1,3[1-13C]Galbeta1,4GlcNAcbeta1,3Galbeta 1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAcbeta1,3Galbeta1, 4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk beta1,4 galactosyltransferase and human serum beta1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by1H and13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues.     

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.     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

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.     

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.     

Anti-alpha-galactosyl antibodies recognizing epitopes terminating with alpha1,4-linked galactose: human natural and mouse monoclonal anti-NOR and anti-P1 antibodies

The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Galalpha1-4GalNAcbeta1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), Galalpha1-4GalNAc (NOR-di), Galalpha1-4Galbeta1-3Galbeta1-4Glc ((Gal)3Glc), and Galalpha1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Galalpha1-4Gal/GalNAcbeta1-3Gal. Type 2 antibodies were specific to Galalpha1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Galalpha1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Galalpha1-4Galbeta1-4Glc-Cer) present in all normal erythrocytes and P1 (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Galalpha1-4Galbeta1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-alpha-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-alpha-galactosyl antibodies still awaits elucidation.     

Determination by electrospray mass spectrometry and 1H-NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso-lacto-N-octaose core.

We have isolated a nonfucosylated and three variously fucosylated neutral oligosaccharides from human milk that are based on the iso-lacto-N-octaose core. Their structures were characterized by the combined use of electrospray mass spectrometry (ES-MS) and NMR spectroscopy. The branching pattern and blood group-related Lewis determinants, together with partial sequences and linkages of these oligosaccharides, were initially elucidated by high-sensitivity ES-MS/MS analysis, and then their full structure assignment was completed by methylation analysis and 1H-NMR. Three new structures were identified. The nonfucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc, has not previously been reported as an individual oligosaccharide. The monofucosylated and trifucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc and Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6[Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3]Galbeta1-4Glc, both containing an internal Lex epitope, are also novel structures.     

Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.     

A novel variant form of murine beta-1, 6-N-acetylglucosaminyltransferase forming branches in poly-N-acetyllactosamines

A novel form of murine beta-1,6-N:-acetylglucosaminyltransferase that forms branches in poly-N:-acetyllactosamines (designated as IGnT B) was cloned based on sequence homology to the known IGnT (designated as IGnT A). When expressed as proteins, IGnT B showed higher specific activity than IGnT A. The C-terminal 1/4 of IGnT B was identical to that of IGnT A, while the rest of the predicted sequences showed 63% identity. Genomic analysis indicated that IGnT A and IGnT B were derived by alternative splicing; the unique portion was encoded by exon 1, and the common portion was encoded by exons 2 and 3. IGnT B showed an expression profile closely related to that of IGnT A and was strongly expressed in the liver, kidney and intestine, and moderately in the mammary gland, submaxially gland, embryonic stem cells, and embryonal carcinoma cells. The specificity of IGnT B examined using various substrates was indistinguishable from that of IGnT A, which is classified as the central acting IGnT (cIGnT). Thus, IGnT B acted on Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, but not on GlcNAcbeta1-3Galbeta1-4Glc. It formed branches in both of the internal galactosyl residues of Galbeta1-4Glc-NAcbeta1-3Galbeta1-4GlcNAcbeta1-++ +3Galbeta1-4Glc, and prolonged incubation resulted in production of the di-branched oligosaccharide. Although addition of sialic acid to the terminal galactosyl residue did not abolish the acceptor activity, alpha2-6 sialylation was a preferred one as compared to alpha2-3 sialylation.     

A novel difucosylated neutral glycosphingolipid from the eggs of the sea urchin, Hemicentrotus pulcherrimus: I. Purification and structural determination of the glycolipid.

A novel fucose-containing neutral glycosphingolipid (GL-5) was purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. The chemical structure was determined to be Fuc alpha 1-3GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-4Glc beta 1-1Cer by methylation analysis, partial acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The unique characteristics of GL-5 are that: the reducing terminal disaccharide portion is not Gal beta 1-4Glc but GlcNAc beta 1-4Glc; it includes a GalNAc beta 1-4GlcNAc sequence and a Fuc-GalNAc linkage; the defucosylated core is a novel trisaccharide chain; and the sugar structure is one of the smallest ever characterized for a difucosylated glycolipid. The major fatty acids were 22:1 and 22h:1, and about 30% of the total acids was 2-hydroxylated. All the long-chain bases were phytosphingosines, of which about 90% was n-t18:0. The similarity of the ceramide moiety to that of glucosylceramide from the same eggs [Kubo, H. et al. (1992) J. Biochem. 111, 726-731] suggests a close biosynthetic relationship between GL-5 and the glucosylceramide.     

Carbohydrate dynamics at a micellar surface: GD1a headgroup transformations revealed by NMR spectroscopy.

The conformational dynamics of the carbohydrate headgroup of ganglioside GD1a, NeuAc alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer, anchored in a perdeuterated dodecylphosphocholine micelle in aqueous solution, were probed by high resolution NMR spectroscopy. The observed 1H/1H NOE interactions revealed conformational averaging of the terminal NeuAc alpha 2-->3Gal and Gal beta 1-->3GalNAc glycosidic linkages. The pronounced flexibility of this trisaccharide moiety was substantiated further by two-dimensional proton-detected 13C T1, T1 rho and 1H/13C NOE measurements. The anchoring effect of the micelle allowed the detection of conformational fluctuations of the headgroup on the time scale of a few hundred picoseconds. NMR experiments performed on the GD1a/DPC micelles in H2O at low temperatures permitted the observation of hydroxyl proton resonances, contributing valuable conformational information.     

Characterization of neutral and acidic glycosphingolipids from the lectin-producing mushroom, Polyporus squamosus

The polypore mushroom Polyporus squamosus is the source of a lectin that exhibits a general affinity for terminal beta-galactosides, but appears to have an extended carbohydrate-binding site with high affinity and strict specificity for the nonreducing terminal trisaccharide sequence NeuAcalpha2 --> 6Galbeta1 --> 4Glc/GlcNAc. In considering the possibility that the lectin's in vivo function could involve interaction with an endogenous glycoconjugate, it would clearly be helpful to identify candidate ligands among various classes of carbohydrate-containing materials expressed by P. squamosus. Since evidence has been accumulating that glycosphingolipids (GSLs) may serve as key ligands for some endogenous lectins in animal species, possible similar roles for fungal GSLs could be considered. For this study, total lipids were extracted from mature fruiting body of P. squamosus. Multistep fractionation yielded a major monohexosylceramide (CMH) component and three major glycosylinositol phosphorylceramides (GIPCs) from the neutral and acidic lipids, respectively. These were characterized by a variety of techniques as required, including one- and two-dimensional (1)H- and (13)C-nuclear magnetic resonance (NMR) spectroscopy; electrospray ionization-mass spectrometry (ESI-MS, tandem-MS/collision-induced decay-MS, and ion trap-MS(n)); and component and methylation linkage analysis by gas chromatography-mass spectrometry. The CMH was determined to be glucosylceramide having a typical ceramide consisting of 2-hydroxy fatty-N-acylated (4E,8E)-9-methyl-sphinga-4,8-dienine. The GIPCs were identified as Manalpha1 --> 2Ins1-P-1Cer (Ps-1), Galbeta1 --> 6Manalpha1 --> 2Ins1-P-1Cer (Ps-2), and Manalpha1 --> 3Fucalpha1 --> 2Galalpha1 --> 6Galbeta1 --> 6Manalpha1 -->2Ins1-P-1Cer (Ps-5), respectively (where Ins = myo-inositol, P = phosphodiester, and Cer = ceramide consisting mainly of long-chain 2-hydroxy and 2,3-dihydroxy fatty-N-acylated 4-hydroxy-sphinganines). Of these GSLs, Ps-2 could potentially interact with P. squamosus lectin, and further investigations will focus on determining the binding affinity, if any, of the lectin for the GIPCs isolated from this fungus.     

Caenorhabditis elegans galectins LEC-1-LEC-11: structural features and sugar-binding properties

Galectins form a large family of beta-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2-5 and 8-11) from a lambdaZAP cDNA library. Among them, lec-2-5 were found to encode 31-35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8-11 were found to encode 16-27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1-4, -6, and 8-10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various beta-galactosides, i.e., N-acetyllactosamine (Galbeta1-4GlcNAc), lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), and asialofetuin, demonstrated that LEC-1-4, -6, and -10 have a significant affinity for beta-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galbeta1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.     

Carbohydrate specificity of an agglutinin isolated from the root of Trichosanthes kirilowii

The root of Trichosanthes kirilowii, which has been used as Chinese folk medicine for more than two thousand years, contains a Gal specific lectin (TKA). In order to elucidate its binding roles, the carbohydrate specificities of TKA were studied by enzyme linked lectinosorbent assay (ELLSA) and by inhibition of lectin-glycoform binding. Among glycoproteins (gp) tested, TKA reacted strongly with complex carbohydrates with Galbeta1-->4GlcNAc clusters as internal or core structures (human blood group ABH active glycoproteins from human ovarian cyst fluids, hog gastric mucin, and fetuin), porcine salivary glycoprotein and its asialo product, but it was inactive with heparin and mannan (negative control). Of the sugar inhibitors tested for inhibition of binding, Neu5Ac alpha2-->3/6Galbeta1-->4Glc was the best and about 4, 14.6 and 27.7 times more active than Galbeta1-->4GlcNAc(II), Galbeta1-->3GalNAc(T) and Gal, respectively. From these results, it is suggested that this agglutinin is specific for terminal or internal polyvalent Galbeta1-->4GlcNAcbeta1-->, terminal Neu5Ac alpha2-->3/6Galbeta1-->4Glc and cluster forms of Galbeta1-->3GalNAc alpha residues. The unusual affinity of TKA for terminal and internal Galbeta1-->glycotopes may be used to explain the possible attachment roles of this agglutinin in this folk medicine to target cells.     

A remodeling system of the 3'-sulfo-Lewis a and 3'-sulfo-Lewis x epitopes

It has been reported that the chemically synthesized 3'-sulfo-Le(a) and 3'-sulfo-Le(x) epitopes have a high potential as a ligand for selectins. To elucidate the physiological functions of 3'-sulfated Lewis epitopes, a remodeling system was developed using a combination of a betaGal-3-O-sulfotransferase GP3ST, hitherto known alpha1,3/1,4-fucosyltransferases (FucT-III, IV, V, VI, VII, and IX) and arylsulfatase A. The pyridylaminated (PA) lacto-N-tetraose (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc) was first converted to 3'-sulfolacto-N-fucopentaose II (sulfo-3Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc)-PA by sequential reactions with GP3ST and FucT-III. The 3'-sulfolacto-N-fucopentaose III (sulfo-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc)-PA was then synthesized from lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc)-PA by GP3ST and FucT-III, -IV, -V, -VI, -VII, or -IX in a similar manner. The substrate specificity for the 3'-sulfated acceptor of the alpha1,3-fucosyltransferases was considerably different from that for the non-substituted and 3'-sialylated varieties. When the GP3ST gene was introduced into A549 and Chinese hamster ovary cells expressing FucT-III, they began to express 3'-sulfo-Le(a) and 3'-sulfo-Le(x) epitopes, respectively, suggesting that GP3ST is responsible for their biosynthesis in vivo. The expression of the 3'-sialyl-Le(x) epitope on Chinese hamster ovary cells was attenuated by the introduction of GP3ST gene, indicating that GP3ST and alpha2,3-sialyltransferase compete for the common Galbeta1-4GlcNAc-R oligosaccharides. Last, arylsulfatase A, which is a lysosomal hydrolase that catalyzes the desulfation of 3-O-sulfogalactosyl residues in glycolipids, was found to hydrolyze the sulfate ester bond on the 3'-sulfo-Le(x) (type 2 chain) but not that on the 3'-sulfo-Le(a) (type 1 chain). The present remodeling system might be of potential use as a tool for the study of the physiological roles of 3'-sulfated Lewis epitopes, including interaction with selectins.     

Structural determination of novel lacto-N-decaose and its monofucosylated analogue from human milk by electrospray tandem mass spectrometry and 1H NMR spectroscopy

We have isolated and characterised two neutral oligosaccharides, one nonfucosylated and the other monofucosylated, from human milk that are based on the doubly branched lacto-N-decaose core. Their structures have been determined by a combined use of electrospray tandem mass spectrometry (ES-MS/MS) and NMR spectroscopy. The sequences of the three branches resulted from the double-branching, including the identity and location of the blood-group-related Lewis determinant and partial linkages, were elucidated by the unique method of high sensitivity negative-ion ES-MS/MS analysis. Their full structure assignment was completed by methylation analysis and 1H NMR. The monofucosylated lacto-N-decaose, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc is a novel sequence, whereas the nonfucosylated lacto-N-decaose, Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc, has not been isolated and identified as an individual oligosaccharide.     

Structure and solution conformations of a cyclic trisaccharide from high-resolution n.m.r. spectroscopy and molecular modelling.

The conformational properties of a cyclic trisaccharide: [O-beta-D-glucopyranosyl-(1----6)]3 1,6"-anhydride nonacetate (C36H48O24, 1) have been established by high-resolution 1H- and 13C-n.m.r. spectroscopy in conjunction with potential-energy and molecular-mechanics calculations. The n.m.r. parameters used were nuclear Overhauser enhancements (n.O.e.) and coupling constants. From theoretical models of the trisaccharide, a statistical-mechanics approach was used to compute an ensemble average-relaxation matrix from which the n.O.e. were calculated. The observed nuclear Overhauser enhancements as measured by n.m.r. spectroscopy may be satisfactorily modelled if averaging over two conformational states is considered. In solution, both conformations of the molecule exhibit three-fold symmetry; the beta-linked glucopyranose rings have the 4C1 conformation. In one conformer, the orientation about the (1----6) linkage is characterized by torsion angles phi = 79.5 degrees, psi = 143.5, and omega = -64.3. For the other conformer, these values are phi = -137.7, psi = 68.2, and omega = 45.6. The existence of such a conformer shows that solution behaviour is not dominated by the stabilizing influence of the exoanomeric effect.     

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

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

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-alphaGal antibody

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6' OH of Galbeta(1 --> 4)Glcbeta-OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). UDP-[6-(3)H]Gal studies indicated that alpha1,3-galactosyltransferase recognized the C-6' modified Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and alpha1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15-22. Galalpha(1 --> 3)Galbeta(1 --> 4)Glcbeta-OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6" modified derivatives (23-30). An ELISA bioassay was performed utilizing human anti-alphaGal antibodies to study the binding affinity of the derivatized epitopes (6, 15-30). Modifications made at the C-6' position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6" position resulted in significant or complete abrogation of recognition. The results indicate that the C-6' OH of the alphaGal trisaccharide epitope is not mandatory for antibody recognition.     

Molecular cloning and characterization of the Caenorhabditis elegans alpha1,3-fucosyltransferase family

The genome of Caenorhabditis elegans encodes five genes with homology to known alpha1,3 fucosyltransferases (alpha1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans alpha1,3FTs (CEFT-1 through -5). The open-reading frame for each enzyme predicts a type II transmembrane protein and multiple potential N-glycosylation sites. We prepared recombinant epitope-tagged forms of each CEFT and found that they had unusual acceptor specificity, cation requirements, and temperature sensitivity. CEFT-1 acted on the N-glycan pentasaccharide core acceptor to generate Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-Asn. In contrast, CEFT-2 did not act on the pentasaccharide acceptor, but instead utilized a LacdiNAc acceptor to generate GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc, which is a novel activity. CEFT-3 utilized a LacNAc acceptor to generate Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc without requiring cations. CEFT-4 was similar to CEFT-3, but its activity was enhanced by some divalent cations. Recombinant CEFT-5 was well expressed, but did not act on available acceptors. Each CEFT was optimally active at room temperature and rapidly lost activity at 37 degrees C. Promoter analysis showed that CEFT-1 is expressed in C. elegans eggs and adults, but its expression was restricted to a few neuronal cells at the head and tail. We prepared deletion mutants for each enzyme for phenotypic analysis. While loss of CEFT-1 correlated with loss of pentasaccharide core activity and core alpha1,3-fucosylated glycans in worms, loss of other enzymes did not correlate with any phenotypic changes. These results suggest that each of the alpha1,3FTs in C. elegans has unique specificity and expression patterns.