Ritva Niemelä

The linear tetrasaccharide, Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc, isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAcβ1-6Galβ1-4GlcNAc, resisted the action of endo-β-galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc were cleaved in the expected manner.

The Centrally Acting β1,6N-Acetylglucosaminyltransferase (GlcNAc to Gal) FUNCTIONAL EXPRESSION, PURIFICATION, AND ACCEPTOR SPECIFICITY OF A HUMAN ENZYME INVOLVED IN MIDCHAIN BRANCHING OF LINEAR POLY-N-ACETYLLACTOSAMINES

In the present experiments the cDNA coding for a truncated form of the β1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67–74-kDa fusion protein. Typically a yield of 750 μg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, converting the acceptor to the branched pentasaccharide Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc. Accordingly, we call the recombinant β1,6-GlcNAc transferase cIGnT6 to emphasize its action atcentral rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, β1,4galactosyltransferase and β1,3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.

Enzymatic synthesis of site-specifically (α 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.

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo-β-galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml−1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAcβ1-6Galβ1-4GlcNAc, or those of the tetrasaccharides Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc and Galβ1-4GlcNAcβ1-6Galβ1-4Glc. The isomeric glycans which contained the GlcNAcβ1-3Galβ1-4GlcNAc/Glc sequence were readily cleaved.

α1,3-Fucosylation of branched blood group I-type oligo-(N-acetyllactosamino)glycans by human milk transferases is restricted to distalN-acetyllactosamine units: The resulting isomers are separated by WGA-agarose chromatography

A partially purified preparation of α1,3-fucosyltransferase(s) from human milk was used to [14C]fucosylate oligosac-charides containing Galβ1-4GlcNAc units. Substitution ofN-acetyllactosamine at position 3′ with a β-linkedN-acetylglucosamine enhanced the reactivity of the acceptor, whereas similar substitution at position 6′ was inhibitory. Thus, the trisaccharide GlcNAcβl-6Galβ1-4GlcNAc (5), the branched tetrasaccharide GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc (11) and the triply branched decasaccharide GlcNAcβ1-3(GlcNAcβ1-6)Galβl-4GlcNAcβ1-3[GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4GlcNAc (26) gave remarkably poor yields of α1,3-fucosylated products in comparison to GlcNAcβ1-3Galβ1-4GlcNAc (3). β1,4-Galactosyl derivatives of5 and11, however, gave good yields of α1,3-fucosylated products, but the fucosylation was restricted to the distalN-acetyllactosamine units of Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc (16), Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc (18) and also in Galα1-3Galβ1-4GlcNAcβ1-3(Galα1-3Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc (22). Immobilized wheat germ agglutinin (WGA), possessing high affinity for16 [1], revealed no affinity for the fucosylated derivative Galβ1-4(Fucα1-3)GlcNAcβ1-6Galβ1-4GlcNAc (17). The isomeric heptasaccharides Galβ1-4(Fucα1-3)GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc (19) and Galβ1-4GlcNAcβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]Galβ1-4GlcNAc (20) were readily separated from each other on WGA-agarose, and so were the isomeric nonasaccharides Galα1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3(Galα1-3Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc (23) and Galα1-3Galβ1-4GlcNAcβ1-3[Galα1-3Galβ1-4(Fucα1-3)GlcNAcβ1-6]Galβ1-4GlcNAc (24).

Enzyme-assisted synthesis of a bivalent high-affinity dodecasaccharide inhibitor of mouse gamete adhesion The length of the chains carrying distal α1,3-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 Galα1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3(Galα1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐6)Galβ1‐4GlcNAc (4), consisting of two long arms that link together two distal α1,3‐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 μM), while a related octasaccharide Galα1‐3Galβ1‐4GlcNAcβ1‐3(Galα1‐3Galβ1‐4GlcNAcβ1‐6)Galβ1‐4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two α‐galactose residues of 4, and the length of the sugar chains joining them.

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

The polylactosamine sLexβ1–3′(sLexβ1–6′)LacNAcβ1–3′(sLexβ1–6′)LacNAcβ1–3′(sLexβ1–6′)LacNAc (7) (where sLex is Neu5Acα2–3Galβ1–4(Fucα1–3)GlcNAc and LacNAc is Galβ1–4GlcNAc) 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 LacNAcβ1–3′LacNAcβ1–3′LacNAcβ1–3′LacNAc (4) was converted into the triply branched undecasaccharide LacNAcβ1–3′(GlcNAcβ1–6′)LacNAcβ1–3′(GlcNAcβ1–6′)LacNAcβ1–3′(GlcNAcβ1–6′)LacNAc (5) by incubation with UDP‐GlcNAc and the midchain β1,6‐GlcNAc transferase activity of rat serum. Glycan 5 was enzymatically β1,4‐galactosylated to LacNAcβ1–3′(LacNAcβ1–6′)LacNAcβ1–3′(LacNAcβ1–6′)LacNAcβ1–3′(LacNAcβ1–6′)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.

Oligo-N-acetyllactosaminoglycans bearing Galβ1-4(Fucα1-3)GlcNAc sequences reveal lower affinities than their nonfucosylated, or α(1-2) fucosylated counterparts for immobilized wheat germ agglutinin

Relative affinities of several fucosylated and nonfucosylated oligo-N-acetyllactosaminoglycans for immobilized wheat germ agglutinin (WGA) were studied using a chromatographic technique. α(1-3) Fucosylation of theN-acetylglucosamine unit(s) in mono- and biantennary saccharides of the Galβ1-4GlcNAc-R type strongly reduced the WGA-affinity. In contrast, α(1-2) fucosylation of the nonreducing galactose unit(s) of the saccharides did not reduce the affinity.

Construction of linear GlcNAcβ1-6Galβ1-OR type oligosaccharides by partial cleavage of GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OR sequences with jack bean β-N-acetylhexosaminidase

Radiolabelled GlcNAcβ1-3(GlcNAcβ1-6)Gal (1), GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OCH3 (4), GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4Glc (7), and GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc (10) were cleaved partially with jack bean β-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 glycan4. The saccharides1 and7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducingN-acetylglucosamine units, but the glycan10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAcβ1-6Gal, GlcNAcβ1-6Galβ1-OCH3, GlcNAcβ1-6Galβ1-4Glc, and, in particular, GlcNAcβ1-6Galβ1-4GlcNAc.

N-Acetyllactosaminooligosaccharides that contain the β-d-GlcpNAc-(1→6)-d-Gal or β-d-GlcpNAc-(1→6)-d-GalNAc sequences reveal reduction-sensitive affinites for wheat germ agglutinin

Abstract Affinity chromatography of unreduced oligosaccharides on a small column of immobilized wheat germ agglutinin (WGA) revealed high-binding affinites for several radiolabeled molecules containing at the reducing end either β- d -Glc p NAc-((1→6)- d -Glc p -(1→6)-β- d -Gal p -(1→4)- d -GlcNAc, β- d -Glc p NAc-(1→6)- β- d -Gal p (1→4) d Glc, d -Glc p Nac-(→3)-[β- d -Glc p NAc-(1→6)- d -GalNAc, or β- d -Gal p -(→3)-[β- d -Glc p NAc-(1→6)- d -GalNAc sequences. Reduction changed the binding affinites 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.