Jari Helin

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.

Use of Natural and Synthetic Oligosaccharide, Neoglycolipid and Glycolipid Libraries in Defining Lectins from Pathogens

Publisher Summary This chapter focuses on analytical methods that may be useful in fields related to pathogen—host carbohydrate interactions. One of the basic approaches referred in this review is thin layer chromatography (TLC) overlay assay developed by Professor Karl-Anders Karlsson and colleagues. In this technique the glycans are presented on artificial TLC surfaces as unimolecular epitopes in form of natural glycolipids or neoglycolipids. Labeled pathogens or their components are incubated with the separated glycolipids to reveal the binding structures. The TLC overlay method has been proven to be useful in defining specificities of pathogen lectins. Therefore, many glycan epitopes recognized by microbes have been identified in this way. The specific natural epitopes are usually present in tissues among a great variety of glycans. After a binding glycan has been identified, a library of similar glycolipids can further define the binding. In pathogen adhesion the actual biological role of glycolipid sequences among other glycans such as protein-linked glycans has been emphasized by studies on specific inhibition of glycolipid biosynthesis.

Utilization of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for structural studies related to biology and disease

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), because of its high sensitivity and relatively straightforward requirements for sample preparation, is contributing to the solution of structural problems in biology and to the development of therapeutic approaches through increased understanding of pharmacology and enhanced capabilities for quality control of pharmaceuticals. We are using a reflectron TOF- MS for the determination of molecular weights of individual compounds and the components of mixtures that are naturally occurring or are generated through enzymic digests, and employing the post-source decay mode to elucidate structural details. To maximize the sensitivity and information content of the spectra, varied matrices, derivative, and stepwise degradation procedures are being explored. Present studies include investigations of oligosaccharides, neutral glycolipids, gangliosides, glycoproteins, neuropeptides and proteins. Rules for fragmentation are being developed with model compounds and used for the structural elucidation of unknowns. When adequate sample amounts are available, the results are compared with low- and high-energy collision-induced decomposition spectra obtained with tandem MS in order to provide a data base for the correlation of spectral features and guidance in selection of approaches for scarce biological samples. Current projects include biophysical studies of glycoplipids, glycoproteins and oligosaccharides and investigations of the substance P receptor, transthyretin genetic variants and cisplatin-DNA interactions.