Leena Penttilä

Analysis of nucleotide sugars from cell lysates by ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography

Analysis of nucleotide sugar metabolism is essential in studying glycosylation in cells. Here we describe practical methods for both extraction of nucleotide sugars from cell lysates and for their analytical separation. Solid-phase extraction cartridges containing graphitized carbon can be used for the purification of nucleotide sugars by using triethylammonium acetate buffer as a ion-pairing reagent for decreasing retention. After that they are separated by high-performance liquid chromatography using a C18 reversed-phase column and the same ion-pairing reagent for increasing retention. These new sample preparation and analysis methods enable good separation of structurally similar sugar nucleotides, compatibility with rapid evaporative concentration, and possibility to automation. Monitoring the production of GDP-deoxyhexoses in genetically engineered yeast and native bacterial cells are described here as specific applications.

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.

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.

The β1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone

We find that the β1,6‐GlcNAc transferase activity present in hog gastric mucosal microsomes converts the linear pentasaccharide GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAc (1) in a site‐specific way to the branch‐bearing hexasaccharide GlcNAcβ1‐3(GlcNAcβ1‐6)Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAc (2). The product is a positional isomer of GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3(GlcNAcβ1‐6)Galβ1‐4GlcNAc (3), reportedly formed from 1 by another polylactosamine β1,6‐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 GlcNAcβ1‐3(GlcNAcβ1‐6)Galβ1‐4GlcNAcβ1‐3(GlcNAcβ1‐6)Galβ1‐4GlcNAc (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.

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.

Enzyme‐aided construction of medium‐sized alditols of complete O‐linked saccharides

We have constructed by enzyme‐aided in vitro synthesis a hexasaccharide alditol Galβ1—4GlcNAcβ1—6Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc‐ol and shown that it resists the action of endo‐β‐galactosidase from Bacteroides fragilis under conditions where a related pentasaccharide alditol, GlcNAcβ1—3Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc‐ol, was completely cleaved. Together with earlier results from this laboratory, our present data imply that endo‐β‐galactosidase from B. fragilis, apparently, can be used to distinguish between GlcNAcβ1—6Gal and GlcNAcβ1—3Gal units within linear backbone sequences of all known types of oligo‐(N‐acetyllactosamino)glycans.

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.