Enzyme-aided construction of medium-sized alditols of complete O-linked saccharides. The constructed hexasaccharide alditol Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3)GalNAc-ol resists the action of endo-beta-galactosidase from Bacteroides fragilis.

We have constructed by enzyme-aided in vitro synthesis a hexasaccharide alditol Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3) GalNAc-ol and shown that it resists the action of endo-beta-galactosidase from Bacteroides fragilis under conditions where a related pentasaccharide alditol, GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3)GalNAc-ol, was completely cleaved. Together with earlier results from this laboratory, our present data imply that endo-beta-galactosidase from B. fragilis, apparently, can be used to distinguish between GlcNAc beta 1-6Gal and GlcNAc beta 1-3Gal units within linear backbone sequences of all known types of oligo-(N-acetyllactosamino)glycans.

The beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone.

We find that the beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes converts the linear pentasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) in a site-specific way to the branch-bearing hexasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (2). The product is a positional isomer of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (3), reportedly formed from 1 by another polylactosamine beta 1,6-GlcNAc transferase activity present in human serum (Leppänen et al., Biochemistry, 30 (1991) 9287). Combined use of the two kinds of activities gave in the present experiments the heptasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), in which one of the branches occupies the position of the branch in 2 and the other the position of the branch in 3.

A novel beta-N-acetylglucosaminidase activity in hog gastric mucosal microsomes: preferential hydrolysis of terminal GlcNAc beta 1-3 linkages in GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, but GlcNAc beta 1-6 linkages in GlcNAc beta 1-3(GlcNAc beta 1-6)Gal.

Hog gastric mucosal microsomes contain beta-N-acetylglucosaminidase activity which cleaves GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc at the terminal GlcNAc beta 1-3Gal linkage faster than at the GlcNAc beta 1-6Gal bond, producing mainly GlcNAc beta 1-6Gal beta 1-4GlcNAc. In a marked contrast, GlcNAc beta 1-3(GlcNAc beta 1-6)Gal is cleaved primarily at the GlcNAc beta 1-6Gal bond, while partial hydrolysis of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc reveals similar rates of cleavage for the (1-3) and (1-6) linkages. Our data support the notion that the terminal beta 1,6-linked GlcNAc unit of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc may interact with the reducing end GlcNAc unit intramolecularly in water solution.

Improved enzymatic synthesis of a highly potent oligosaccharide antagonist of L-selectin.

The polylactosamine sLex beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc (7) (where sLex is Neu5Ac alpha2-3Gal beta1-4(Fuc alpha1-3)GlcNAc and LacNAc is Gal beta1-4GlcNAc) is a nanomolar L-selectin antagonist and therefore a potential anti-inflammatory agent (Renkonen et al. (1997) Glycobiology, 7, 453). Here we describe an improved synthesis of 7. The octasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc beta1-3'LacNAc (4) was converted into the triply branched undecasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (5) by incubation with UDP-GlcNAc and the midchain beta1,6-GlcNAc transferase activity of rat serum. Glycan 5 was enzymatically beta1,4-galactosylated to LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (6). Combined with the enzymatic conversion of 6 to 7 (Renkonen et al., loc. cit.) and the available chemical synthesis of 4, our data improve the availability of 7 for full assessment of its anti-inflammatory properties.

Enzyme-assisted synthesis of a bivalent high-affinity dodecasaccharide inhibitor of mouse gamete adhesion. The length of the chains carrying distal alpha 1,3-bonded galactose residues is critical.

Proposing to study the molecular mechanisms of mouse gamete adhesion with the aid of high affinity adhesion inhibitors of saccharide nature, we report here the enzymatic synthesis of a bivalent oligosaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), consisting of two long arms that link together two distal alpha 1,3-galactose residues. Binding data reported elsewhere (E. Litscher et al., Biochemistry, 1995, 34, 4662-4669) show that 4 is a high affinity inhibitor of mouse gamete adhesion in vitro (IC50 = 9 microM), while a related octasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two alpha-galactose residues of 4, and the length of the sugar chains joining them.

1H and 13C NMR analysis of the pentasaccharide Gal beta (1-->4)GlcNAc beta (1-->3)-[GlcNAc beta (1-->6)]Gal beta (1-->4)GlcNAc synthesized by the mid-chain beta-(1-->6)-D-N-acetylglucosaminyltransferase of rat serum.

Chemical shifts and coupling constants of completely assigned 1H and 13C NMR spectra at 500 MHz, as well as ROESY and HMBC connectivities were used to establish the structure of the pentasaccharide Gal beta (1-->4)GlcNAc beta (1-->3)[GlcNAc beta (1-->6)]Gal beta (1-->4)GlcNAc, synthesized by the action of the mid-chain beta-(1-->6)-D-N-acetylglucosaminyltransferase of rat serum from UDP-GlcNAc and the linear tetrasaccharide Gal beta (1-->4)GlcNAc beta (1-->3)Gal beta (1-->4)GlcNAc.

Stepwise transfer of alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc sequences to 3-OH and 6-OH of distal galactose residues in bi-, tri-, and tetra-antennary asialo-glycans of N-linked complex type.

The hydroxyl groups 3 and 6 of distal galactose units in bi-, tri-, and tetra-antennary asialo-glycans of N-linked complex type were substituted stepwise by transferase reactions with the sequence alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc. The products of each transferase reaction were purified chromatographically and the structures were confirmed by 1H NMR spectroscopy. Molecular weights of the final products were determined by matrix-assisted laser-desorption mass spectrometry (MALDI-MS).

Enzymatic synthesis of site-specifically (alpha 1-3)-fucosylated polylactosamines containing either a sialyl Lewis (x), a VIM-2, or a sialylated and internally difucosylated sequence.

By using two different reaction pathways, we generated enzymatically three sialylated and site-specifically alpha 1-3-fucosylated polylactosamines. Two of these are isomeric hexasaccharides Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc and Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4) GlcNAc, containing epitopes that correspond to VIM-2 and sialyl Lewis (x), respectively. The third one, nonasaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, is a sialylated and internally difucosylated derivative of a trimeric N-acetyllactosamine. All three oligosaccharides have one fucose-free N-acetyllactosaminyl unit and can be used as acceptors for recombinant alpha 1-3-fucosyltransferases in determining the biosynthesis pathways leading to polyfucosylated selectin ligands.

N-acetyllactosaminooligosaccharides that contain the beta-D-GlcpNAc-(1----6)-D-Gal or beta-D-GlcpNAc-(1----6)-D-GalNAc sequences reveal reduction-sensitive affinities for wheat germ agglutinin.

Affinity chromatography of unreduced oligosaccharides on a small column of immobilized wheat germ agglutinin (WGA) revealed high-binding affinities for several radiolabeled molecules containing at the reducing end either beta-D-GlcpNAc-(1----6)-D-Gal, beta-D-GlcpNAc-(1----6)-beta- D-Galp-(1----4)-D-GlcNAc, beta-D-GlcpNAc-(1----6)-beta-D-Galp-(1----4)DGlc, D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----6)]-D-Gal, beta-D-GlcpNAc-(1----6)- D-GalNAc, or beta-D-Galp-(1----3)-[beta-D-GlcpNAc-(1----6)]-D-GalNAc sequences. Reduction changed the binding affinities remarkably: The sequences carrying a D-galactose or 2-acetamido-2-deoxy-D-galactose residue at the reducing end lost most of their affinities, but the sequences containing a D-glucose or 2-acetamido-2-deoxy-D-glucose residue at the reducing end gained additional affinity upon reduction. These findings emphasize the role of the unreduced, 6-o-substituted D-galactose and 2-acetamido-2-deoxy-D-galactose residues for the binding of saccharides to WGA, which has been recognized previously as a lectin specific for oligosaccharides containing a 2-acetamido-2-deoxy-D-glucose or sialic acid unit. The results suggested also that WGA-agarose chromatography of alditols may become a valuable method for the fractionation of oligo-N-acetyllactosaminoglycans and related saccharides.

Elongation of both branches of biantennary backbones of oligo-(N-acetyllactosamino)glycans by human serum (1----3)-N-acetyl-beta-D- glucosaminyltransferase.

Partial reactions catalyzed by a (1----3)-N-acetyl-beta-D- glucosaminyltransferase (EC2.4.1.149), known to be present in human serum, were studied by use of biantennary "backbone" saccharides of oligo-N-acetyllactosamine-type as acceptors. Incubation of the radiolabeled blood-group I-active hexasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp- (1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-GlcNAc (1) and UDP-GlcNAc with serum gave first a transient 1:1 mixture of two isomeric heptasaccharides, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D- Galp-(1----4)-D-GlcNAc (2) and beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----3)- beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-Glc NAc (3), showing that both branches of 1 react equally well. The two heptasaccharides reacted further in the incubation mixture to form the radiolabeled octasaccharide, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[be ta-D- GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Ga lp- (1----4)-D-GlcNAc (4); during this second reaction, the composition of the heptasaccharide mixture remained unchanged, indicating that 2 and 3 reacted at approximately equal rates. The heptasaccharides 2 and 3 could not be separated from each other, but they could be detected, identified, and quantitatively determined by stepwise enzymic degradations. Partial (1----3)-N-acetyl-beta-D-glucosaminylation reactions, carried out with another acceptor, the branched pentasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta- D- GlcpNAc-(1----6)]-beta-D-Gal (11), revealed that it reacted also equally well at both branches.(ABSTRACT TRUNCATED AT 400 WORDS)

UDP-galactose:lactosylceramide alpha-galactosyltransferase activity in human placenta.

The activity of UDP-Gal:LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn2+ at pH 6.3, and the reaction product was susceptible to alpha-galactosidase.

Biosynthesis of branched polylactosaminoglycans. Embryonal carcinoma cells express midchain beta1,6-N-acetylglucosaminyltransferase activity that generates branches to preformed linear backbones.

Two types of beta1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-3Galbeta1-+ ++4GlcNAcbeta1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc from the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3(+ ++GlcNAcbeta1-6)Galbeta1 -4GlcNAc and Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3Galbeta1 -4GlcNAc from the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.

Construction of linear GlcNAc beta 1-6Gal beta 1-OR type oligosaccharides by partial cleavage of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-OR sequences with jack bean beta-N-acetylhexosaminidase.

Radiolabelled GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (1), GlcNAc beta 1-3)GlcNAc beta 1-6)Gal beta 1-OCH3 (4), GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (7), and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (10) were cleaved partially with jack bean beta-N-acetylhexosaminidase (EC 3.2.1.30), and the digests were analysed chromatographically. All four oligosaccharides were hydrolysed faster at the (1-6) branch, than at the (1-3) branch, but a high branch specificity was observed only with the glycan 4. The saccharides 1 and 7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducing N-acetylglucosamine units, but the glycan 10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAc beta 1-6Gal, GlcNAc beta 1-6Gal beta 1-OCH3, GlcNAc beta 1-6Gal beta 1-4Glc, and, in particular, GlcNAc beta 1-6Gal beta 1-4GlcNAc.

Mapping the binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharide to antibodies with NMR.

A NMR study of the binding of the synthetic disaccharides alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl 1 (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl 2, representing partial structures of the lipopolysaccharide epitope of the intracellular bacteria Chlamydia, to corresponding monoclonal antibodies (mAbs) S23-24, S25-39, and S25-2 is presented. The conformations of 1 bound to mAbs S25-39 and of 2 bound to mAbs S23-24 and S25-39 were analyzed by employing transfer-NOESY (trNOESY) and QUIET-trNOESY experiments. A quantitative analysis of QUIET-trNOESY buildup curves clearly showed that S25-39 recognized a conformation of 1 that was similar to the global energy minimum of 1, and significantly deviated from the conformation of 1 bound to mAb S25-2. For disaccharide 2, only a qualitative analysis was possible because of severe spectral overlap. Nevertheless, the analysis showed that all mAbs most likely bound to only one conformational family of 2. Saturation transfer difference (STD) NMR experiments were then employed to analyze the binding epitopes of the disaccharide ligands 1 and 2 when binding to mAbs S23-24, S25-39, and S25-2. It was found that the nonreducing pyranose unit was the major binding epitope, irrespective of the mAb and the disaccharide that were employed. Individual differences were related to the engagement of other portions of the disaccharide ligands.

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.

Synthesis of a divalent sialyl Lewis x O-glycan, a potent inhibitor of lymphocyte-endothelium adhesion. Evidence that multivalency enhances the saccharide binding to L-selectin.

The recognition of cell-surface L-selectin by its carbohydrate ligands causes lymphocytes to roll on capillary endothelium at sites of inflammation. As this primary contact is a prerequisite for extravasation of the leukocytes to the tissue, its inhibition by free oligosaccharides capable of competing with the natural L-selectin ligands in an attractive therapeutic possibility. The exact structures of the biological ligands of L-selectin are not yet known, but the principal carbohydrate epitopes share some structural features: they are O-glycosidically linked mucin-type oligosaccharides with N-acetyllactosamine backbone, which is 3'-sialylated or 3'-sulfated, 3-fucosylated and sometimes 6- or 6'-sulfated at the distal N-acetyllactosamine termini. Multivalency of the ligand, which is believed to enhance the binding, is achieved by a branched polylactosamine backbone or by a clustered array of O-glycans. We report here enzymic synthesis of a large oligosaccharide fulfilling several of the features characteristic to the L-selectin ligands: it is a dodecameric O-glycosidic core-2-type oligosaccharide alditol with a branched polylactosamine backbone carrying two distal alpha-2,3'-sialylated and alpha-1,3-fucosylated N-acetyl-lactosamine groups (sialyl Lewis x, sialyl Le(x)). The structure of each saccharide on the synthesis route from disaccharide Gal beta 1-3GalNAc to the dodecasaccharide alditol was established by several methods including one- and two-dimensional 1H-NMR spectroscopy. The last step of the synthesis, the alpha-1,3-fucosylation of the 6-linked arm proceeded sluggishly, and was associated with a noticeable shift in H1 resonance of the GlcNAc residue of the branch-bearing N-acetyllactosamine unit. The final synthesis product and its analogs lacking one or both of the fucose residues were tested as inhibitors of L-selectin-mediated lymphocyte-endothelium interaction in vitro in rejecting rat kidney transplant. While the non-fucosylated O-glycosidic oligosaccharide alditol did not possess any inhibitory activity, the mono-fucosylated one (i.e. monovalent sialyl Le(x)) prevented the binding significantly and the difucosylated dodecasaccharide alditol (i.e. divalent sialyl Le(x)) was a very potent inhibitor (IC50, inhibitory concentration preventing 50% of binding = 0.15 microM). Besides the multivalency, also the Gal beta 1-3GalNAc-ol sequence of the O-glycosidic core appeared to increase the affinity of the glycan to L-selectin. This was indicated by parallel inhibition experiments, where a disialylated and difucosylated branched polylactosamine decasaccharide, similar to the divalent dodecasaccharide alditol, but lacking the reduced O-glycosidic core, was a less effective inhibitor (IC50 = 0.5 microM) than the O-glycosidic dodecasaccharide alditol.

Enzymatic synthesis of octadecameric saccharides of multiply branched blood group I-type, carrying four distal alpha 1,3-galactose or beta 1,3-GlcNAc residues.

Radiolabeled oligosaccharide constructs were prepared to evaluate carbohydrate determinants involved in gamete adhesion in mice. The octasaccharide primer GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (1) was incubated with UDP-GlcNAc and beta 1,6-GlcNAc-transferase of hog gastric microsomes, producing the tetraantennary decasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (2). The decasaccharide was then incubated with UDP-Gal and beta 1,4-galactosyltransferase from bovine milk, yielding the tetradecasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (3). Incubation of the tetradecasaccharide 3 with UDP-Gal and alpha 1,3-galactosyltransferase from bovine thymus gave the octadecameric glycan Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (4).(ABSTRACT TRUNCATED AT 250 WORDS)

Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids.

Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional (13)C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1-metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional (13)C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.

In vitro biosynthesis of a decasaccharide prototype of multiply branched polylactosaminoglycan backbones.

Multiply branched polylactosaminoglycans are expressed in glycoproteins and glycolipids of many cells. Interest in their biology stems from their abundant expression in early embryonal cells and from their ability to carry multiple lectin-binding determinants, which makes them prominent ligands and antagonists of cell adhesion proteins. A prototype of their backbones is represented by the decasaccharide LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (5), where LacNAc is the disaccharide Gal beta1-4GlcNAc. Here, we describe in vitro biosynthesis of glycan 5. Incubation of the linear hexasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc (1) with UDP-GlcNAc and alpha midchain beta1,6-GlcNAc transferase activity (GlcNAc to Gal), present in rat serum [Gu, J., Nishikawa, A., Fujii, S., Gasa, S., & Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999], gave the doubly branched octasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (4). The latter was converted to 5 by enzymatic beta1,4-galactosylation. In the initial branching reaction of 1, two isomeric heptasaccharide intermediates, LacNAc beta1-3'LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (2) and LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'LacNAc (3), were formed first at comparable rates. Later, both intermediates were converted to 4, revealing two distinct pathways of the reaction: 1 --> 2 --> 4 and 1 --> 3 --> 4. These data suggest that, regardless of their chain length, linear polylactosamines similar to 1 contain potential branching sites at each of the internal galactoses. The enzyme-binding epitope of 1 is probably LacNAc beta1-3'LacNAc, because the trisaccharides GlcNAc beta1-3'LacNAc and LacNAc beta1-3Gal as well as the tetrasaccharide GlcNAc beta1-3'LacNAc beta1-3Gal were poor acceptors, while LacNAc beta1-3'LacNAc was a good one. Midchain beta1,6-GlcNAc transferase activities present in serum of several mammalian species, including man, resembled closely the rat serum activity in their mode of action and in their acceptor specificity. We suggest that analogous membrane-bound Golgi enzymes are involved in the biosynthesis of multiply branched polylactosamines in vivo.